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https://openalex.org/W3110890573	https://zenodo.org/records/4475979/files/diagnostics-10-01078-v3.pdf	English	null	Retrospective Definition of Clostridioides difficile PCR Ribotypes on the Basis of Whole Genome Polymorphisms: A Proof of Principle Study	Diagnostics	2,020	cc-by	9,313	Manisha Goyal 1, Lysiane Hauben 2, Hannes Pouseele 3, Magali Jaillard 4, Katrien De Bruyne Alex van Belkum 1,* and Richard Goering 5 Received: 17 November 2020; Accepted: 10 December 2020; Published: 12 December 2020 Abstract: Clostridioides diﬃcile is a cause of health care-associated infections. The epidemiological study of C. diﬃcile infection (CDI) traditionally involves PCR ribotyping. However, ribotyping will be increasingly replaced by whole genome sequencing (WGS). This implies that WGS types need correlation with classical ribotypes (RTs) in order to perform retrospective clinical studies. Here, we selected genomes of hyper-virulent C. diﬃcile strains of RT001, RT017, RT027, RT078, and RT106 to try and identify new discriminatory markers using in silico ribotyping PCR and De Bruijn graph-based Genome Wide Association Studies (DBGWAS). First, in silico ribotyping PCR was performed using reference primer sequences and 30 C. diﬃcile genomes of the ﬁve diﬀerent RTs identiﬁed above. Second, discriminatory genomic markers were sought with DBGWAS using a set of 160 independent C. diﬃcile genomes (14 ribotypes). RT-speciﬁc genetic polymorphisms were annotated and validated for their speciﬁcity and sensitivity against a larger dataset of 2425 C. diﬃcile genomes covering 132 diﬀerent RTs. In silico PCR ribotyping was unsuccessful due to non-speciﬁc or missing theoretical RT PCR fragments. More successfully, DBGWAS discovered a total of 47 new markers (13 in RT017, 12 in RT078, 9 in RT106, 7 in RT027, and 6 in RT001) with minimum q-values of 0 to 7.40 × 10−5, indicating excellent marker selectivity. The speciﬁcity and sensitivity of individual markers ranged between 0.92 and 1.0 but increased to 1 by combining two markers, hence providing undisputed RT identiﬁcation based on a single genome sequence. Markers were scattered throughout the C. diﬃcile genome in intra- and intergenic regions. We propose here a set of new genomic polymorphisms that eﬃciently identify ﬁve hyper-virulent RTs utilizing WGS data only. Further studies need to show whether this initial proof-of-principle observation can be extended to all 600 existing RTs. Keywords: ribotyping; C. diﬃcile; unitigs; markers; WGS; GWAS; CDI; in silico PCR diagnostics diagnostics diagnostics diagnostics www.mdpi.com/journal/diagnostics Diagnostics 2020, 10, 1078; doi:10.3390/diagnostics10121078 diagnostics diagnostics 1. Introduction Clostridioides diﬃcile (C. diﬃcile), formerly known as Clostridium diﬃcile, is an anaerobic, spore-forming Gram-positive bacterial species that can survive in harsh environments. It can withstand high temperatures, exposure to ultraviolet light, toxic chemicals, and exposure to antibiotics. Colonization by C. diﬃcile is asymptomatic. The development of disease is mostly driven by host factors Diagnostics 2020, 10, 1078; doi:10.3390/diagnostics10121078 www.mdpi.com/journal/diagnostics www.mdpi.com/journal/diagnostics 2 of 14 Diagnostics 2020, 10, 1078 and disruption of the gut microbiome by frequent consumption of antibiotics [1–3]. Toxigenic strains of C. diﬃcile can be a lethal cause of C. diﬃcile infection (CDI), which is commonly associated with post antibiotic diarrhea [4,5]. C. diﬃcile is present in the environment and can be transmitted to patients or healthcare workers through contact with contaminated surfaces. Inter-human spread mainly occurs through the fecal–oral route. C. diﬃcile spores are intrinsically resistant to antibiotics and remain viable during antibiotic treatment. Clindamycin, cephalosporins, and ﬂuoroquinolones are considered as major antibiotics associated with CDI [6]. Food or water contamination, gastric acid-suppression, and asymptomatic carriage in the community are the potential risk factors of community acquired CDI [7]. One-third of the total CDI burden occurring in the USA in 2011 was community-associated [8]. CDI caused half a million hospital-acquired infections and 29,000 deaths in 2012 in the United States [8] and approximately 40,000 cases of CDI in Europe [9]. Only a limited number of studies reported emerging CDI in Asia [10]. The increasing incidence of CDI and rapid evolution of antibiotic resistance in C. diﬃcile has become a global threat to public health [11–13]. CDI diagnosis allows early pathogen isolation and treatment of infection, thereby reducing the potential of CDI transmission. Various diagnostic procedures for CDI are available, including toxigenic culture, cell cytotoxic neutralization assay, glutamate dehydrogenase assay, the detection of toxins by enzyme immunoassays, nucleic acid ampliﬁcation-based molecular tests, etc. [14–19]. Still, epidemiological C. diﬃcile strain typing is necessary to identify outbreaks within a hospital or the wider community and facilitates understanding of the dissemination of infections. Ribotyping is a classical technique for C. diﬃcile typing initially based on hybridization patterns of conserved ribosomal RNA probe sequences [20,21]. Ribotype (RT) analysis has also been extremely important in the long-term surveillance of CDI [22]. While traditional typing methods such as restriction endonuclease analysis (REA) and pulsed-ﬁeld gel electrophoresis (PFGE) were widely used in the past, PCR-based ribotyping is the current method of choice for C. 1. Introduction diﬃcile typing [23,24]. PCR ribotyping is dependent on the ampliﬁcation of the intergenic spacer region (ISR) between 16S and 23S rRNA genes [25–28]. Since most bacterial species encode multiple ribosomal alleles in their genomes, multiple fragments of diﬀerent lengths are ampliﬁed when diﬀerent species but also diﬀerent strains are considered [25,26]. There are still considerable constraints on PCR ribotyping including elevated costs, a higher probability of false-positive results, and a lack of interlaboratory portability [10,29,30]. Bacterial whole genome sequencing (WGS) has the potential to provide more detailed epidemiological information than classical PCR ribotyping [23,31]. To further explore a WGS-based approach to C. diﬃcile typing, backward compatibility with PCR ribotyping is essential [32]. Previous studies reported the association of RT001, RT017, and RT027 with lethal CDI and considered those isolates as hyper-virulent [33,34]. A survey conducted in the North East of England concluded that RT001, RT027, and RT106 were among the most prevalent and dangerous clones [35]. In the United States and Europe, RT001, RT014, RT020, RT027, and RT078 have been identiﬁed as predominant [36,37]. RT017 is a globally emerging toxigenic RT and can be found on almost every continent [38,39]. Thus, here, we tested both in silico PCR ribotyping and the De Bruijn graph-based Genome Wide Association Study (DBGWAS) [40] for their capacity to perform retrospective PCR ribotyping for C. diﬃcile RT001, RT017, RT027, RT078, and RT106. These strains were chosen as a test set representing global, long-term circulating and clinically relevant epidemic strains. The primary study goal was to develop a proof-of-principle procedure for sequence-based C. diﬃcile strain typing with retrospective compatibility to established PCR RTs. 2.2. DBGWAS-Mediated Discovery New RT-Speciﬁc Markers A total number of 160 C. diﬃcile genome assemblies (training set) of 14 diﬀerent RTs including hyper-virulent RT001, RT017, RT027, RT078, and RT106 were used for the discovery of unique RT genomic markers (Table 2). This small training set allowed for the development of discriminatory markers to characterize the ﬁve major RTs among the 14 diﬀerent RTs. Genomes were collected from the National Center for Biotechnology and Information (NCBI; www.ncbi.nlm.nih.gov), Creighton University, and the Enterobase databases (https://enterobase.warwick.ac.uk). Metadata of these genomes are summarized in Supplementary Table S2. Table 2. C. diﬃcile ribotypes included in the training dataset along with the number of genomes and their source of availability. Table 2. C. diﬃcile ribotypes included in the training dataset along with the number of genomes and their source of availability. C. diﬃcile Ribotype Number of Genomes Source RT001 24 Enterobase, NCBI, Creighton University RT002 2 NCBI, Creighton University RT003 19 NCBI, Creighton University RT005 19 NCBI, Creighton University RT010 3 NCBI, Creighton University RT014 11 NCBI, Creighton University RT015 2 NCBI, Creighton University RT017 15 NCBI, Creighton University RT023 3 NCBI, Creighton University RT027 15 NCBI, Creighton University RT046 4 NCBI, Creighton University RT078 15 NCBI, Creighton University RT106 22 Enterobase, NCBI, Creighton University RT126 6 NCBI, Creighton University TOTAL 160 To identify associations between variant genetic loci and PCR RT, a hypothesis-free DBGWAS method was used. DBGWAS deﬁnes genetic variants linked to phenotypic traits via single nucleotide polymorphism (SNP), insertions, deletions, and consequences of recombination [41,42]. We used an open source tool (https://gitlab.com/leoisl/dbgwas) [40]. The tool is able to cover variants in coding as well as non-coding regions of bacterial genomes. DBGWAS was performed keeping the tool parameters in the default setting for diﬀerent C. diﬃcile ribotypes (RT001, RT017, RT027, RT078, and RT106) and their RT-speciﬁc genetic variants observed in the training set. Each C. diﬃcile RT was considered independently in this study. DBGWAS identiﬁed short signature sequences called (overlapping) k-mers, yielding a compact summary of all variations across a set of genomes [40]. Overlapping k-mers are called unitigs and were selected on the basis of their speciﬁc and unique presence in a particular RT. Q-values deﬁne test sensitivity and speciﬁcity and are Benjamini–Hochberg-transformed p-values for controlling the false-positive results in case of multiple testing [40,43]. 2.1. In Silico PCR-Based Ribotyping We performed in silico PCR using canonical ribotyping PCR primers. Based on the reference sequences 16S-USA and 23S-USA (Table 1), in silico PCR was performed using the subsequence search tool in BioNumerics v7.6 software (Applied Maths NV, Sint Martens-Latem, Belgium). Besides 7 3 of 14 Diagnostics 2020, 10, 1078 genomes obtained from Creighton University, 23 C. diﬃcile genomes of ﬁve selected RTs were downloaded from NCBI to verify in silico ampliﬁcation of the ISR region (Supplementary Table S1). Table 1. Primer pair used for in silico PCR-based ribotyping of Clostridioides diﬃcile. Primer Gene Target GenBank Accession No. Sequence (5’–3’) Tm (◦C) Reference 16S-USA (Forward) 16S rRNA gene FN545816 (12293)GTGCGGCTGGATCACCTCCT (12312) 71.0 Xiao et al., 2012 (46) 23S-USA (Reverse) 23S rRNA gene FN545816 (12621)CCCTGCACCCTTAATAACTTGACC (12598) 67.1 Table 1. Primer pair used for in silico PCR-based ribotyping of Clostridioides diﬃcile. 2.3. Validation of Markers Validation of novel unitig markers was performed by means of BLAST searches against the test set of genomes (Table 3). A wide range of 2425 genomes covering 132 diﬀerent C. diﬃcile RTs was downloaded [44] and processed using a Linux shell script. These sequences represented PCR ribotyped strains from diﬀerent countries and clinical and environmental specimens for which phylogenetic analyses were already performed by Frentrup et al. [44] (Supplementary Table S3). A database of this test set was created to perform local command line BLAST searches against the set of signiﬁcant unitigs identiﬁed above. The speciﬁcity of all the unitigs was tested using strict parametric ﬁlters of 100% coverage and identity. 3. C. diﬃcile ribotypes downloaded from the Enterobase database as a test dataset and the num omes included in each ribotype. 2.2. DBGWAS-Mediated Discovery New RT-Speciﬁc Markers R scripting was used to deal with large matrices deﬁning the presence (1) or absence (0) of extracted unitigs in the training set of C. diﬃcile genomes. Diagnostics 2020, 10, 1078 4 of 14 2.3. Validation of Markers Ribotype Count Ribotype Count Ribotype Count Ribotype Count RT001 206 RT046 3 RT127 1 RT375 1 RT002 53 RT049 9 RT129 1 RT404 15 RT003 11 RT050 4 RT137 1 RT413 13 RT005 14 RT051 1 RT138 1 RT446 2 RT006 1 RT053 5 RT149 1 RT449 2 RT009 2 RT054 2 RT150 1 RT451 1 RT010 7 RT056 5 RT153 1 RT453 1 RT011 3 RT058 1 RT156 1 RT454 1 RT012 45 RT060 1 RT157 1 RT456 1 RT013 1 RT062 2 RT158 1 RT470 1 RT014 113 RT063 1 RT176 13 RT500 21 RT015 36 RT066 3 RT193 1 RT534 1 RT017 272 RT067 1 RT194 1 RT547 1 RT018 55 RT069 1 RT212 1 RT559 1 RT019 1 RT070 4 RT220 4 RT563 1 RT020 44 RT072 1 RT225 1 RT569 1 RT022 1 RT073 2 RT226 1 RT581 1 RT023 16 RT075 1 RT236 3 RT585 1 RT024 1 RT076 2 RT238 1 RT586 1 RT026 6 RT077 1 RT239 2 RT591 1 RT027 652 RT078 492 RT241 5 RT598 8 RT029 3 RT081 2 RT244 9 RT614 1 RT031 2 RT083 1 RT251 1 RT620 2 RT032 1 RT084 2 RT262 1 RT629 1 RT033 5 RT087 8 RT284 1 RT651 1 RT035 2 RT090 1 RT289 1 RT666 1 RT036 1 RT094 1 RT290 1 RT668 1 RT037 1 RT102 1 RT305 1 RT678 1 RT039 5 RT103 2 RT316 1 RT708 1 RT042 2 RT106 55 RT321 1 RT719 1 RT043 2 RT117 2 RT328 2 RT720 1 RT044 2 RT125 1 RT336 1 RT721 1 RT045 2 RT126 79 RT356 8 RT722 1 Table 3. C. diﬃcile ribotypes downloaded from the Enterobase database as a test dataset and the number of genomes included in each ribotype. Table 3. C. diﬃcile ribotypes downloaded from the Enterobase database as a test dataset and the number of genomes included in each ribotype. 2.4. Statistically Reliable Ribotype Prediction tistically Reliable Ribotype Prediction 3.1. In Silico PCR 3.1. In Silico PCR The visualiz The visualization of ampliﬁed DNA sequences from the intergenic region between 16S and 23S ribosomal genes is the current Gold Standard for C. diﬃcile typing [25,47]. In our study, in silico PCR for 30 randomly selected, well-characterized C. diﬃcile genome sequences was essentially unsuccessful (Figure 1 and Supplementary Table S1). Genome sequences included generated insuﬃcient numbers of diﬀerently sized fragments. The fragment sizes that were calculated were veriﬁed with the online tool available at http://insilico.ehu.es/PCR [10]. On the other hand, more recently sequenced C. diﬃcile genomes were showing only one or even none of the expected ampliﬁed fragments (Supplementary Table S1). There is a substantial possibility that the PCR ribotyping fragments observed upon laboratory experimentation for these strains may not derive from ISR variants but rather from random ampliﬁcation. Thus, in silico PCR failed to generate reliable RTs which prompted us to explore the feasibility of DBGWAS-based typing. Of note, we presume here that NGS-based methods are very likely to be more reliable than any of the many other molecular typing methods. The visualization of amplified DNA sequences from the intergenic region between 16S and 23S ribosomal genes is the current Gold Standard for C. difficile typing [25,47]. In our study, in silico PCR for 30 randomly selected, well-characterized C. difficile genome sequences was essentially unsuccessful (Figure 1 and Supplementary Table S1). Genome sequences included generated insufficient numbers of differently sized fragments. The fragment sizes that were calculated were verified with the online tool available at http://insilico.ehu.es/PCR [10]. On the other hand, more recently sequenced C. difficile genomes were showing only one or even none of the expected amplified fragments (Supplementary Table S1). There is a substantial possibility that the PCR ribotyping fragments observed upon laboratory experimentation for these strains may not derive from ISR variants but rather from random amplification. Thus, in silico PCR failed to generate reliable RTs which prompted us to explore the feasibility of DBGWAS-based typing. Of note, we presume here that NGS-based methods are very likely to be more reliable than any of the many other molecular typing methods. Figure 1. In silico ribotyping of different C. difficile genome sequences using the ISR 16S and 23S USA primer pair. The five panels represent the results obtained for examples of five different ribotypes. 2.4. Statistically Reliable Ribotype Prediction To evaluate the potential typing signiﬁcance of the unitigs as compared to the classical ribotyping of C. diﬃcile strains, sensitivity and speciﬁcity (selectivity) were computed for all the unitigs [45]. The eﬃciency of GWAS can be measured by assessing the false discovery rate (FDR) [46]. To increase the potential typing signiﬁcance of our new method, combination statistics were performed. Sensitivity 5 of 14 Diagnostics 2020, 10, 1078 and speciﬁcity were also computed for certain combinations of two or more selected unitig sequences. The parameters deﬁned were, next to the FDR, TP (true-positives, correctly predicting positive values, e.g., number of true RT017 predicted as RT017), FP (false-positives, missed negative values, e.g., number of non-RT017 genomes still predicted as RT017), FN (false-negatives, missed positive values), and TN (true-negatives, correctly rejected values). Diagnostics 2020, 10, x FOR PEER REVIEW 5 of 14 3. Results and Discussion 3. Results and Discussion 3.1. In Silico PCR 3.1. In Silico PCR The isuali 2.5. Functional Annotation of Unitigs 2.5. Functional Annotation of Unitigs Selected unitigs were annota Selected unitigs were annotated using BLASTn alignment. Well-characterized C. diﬃcile genomes were used as a reference to locate these new markers. Speciﬁc annotation for each marker was ﬁltered out using minimum E-value, 100% identity, 100% coverage, and 0 gap score. Selected unitigs were annotated using BLASTn alignment. Well characterized C. difficile genomes were used as a reference to locate these new markers. Specific annotation for each marker was filtered out using minimum E-value, 100% identity, 100% coverage, and 0 gap score. 3 R lt d Di i 3.1. In Silico PCR 3.1. In Silico PCR The visualiz Bar graphs show the number of theoretical PCR bands (vertical axis, number of bands labeled on each bar) in the ribosomal region of respective genome sequences (horizontal axis), whereas the genomes without any fragments depict the complete absence of primer binding sites in those genomes. Note that the expected outcome would be an identical number of fragments for each of the strains belonging to a single ribotype. We indicated this number as the first marker of reproducibility; it has to be stated that besides this variation of numbers of fragments, the size of the fragment was also determined as a variable as well. Figure 1. In silico ribotyping of diﬀerent C. diﬃcile genome sequences using the ISR 16S and 23S USA primer pair. The ﬁve panels represent the results obtained for examples of ﬁve diﬀerent ribotypes. Bar graphs show the number of theoretical PCR bands (vertical axis, number of bands labeled on each bar) in the ribosomal region of respective genome sequences (horizontal axis), whereas the genomes without any fragments depict the complete absence of primer binding sites in those genomes. Note that the expected outcome would be an identical number of fragments for each of the strains belonging to a single ribotype. We indicated this number as the ﬁrst marker of reproducibility; it has to be stated that besides this variation of numbers of fragments, the size of the fragment was also determined as a variable as well. Figure 1 In silico ribotyping of different C difficile genome sequences using the ISR 16S and 23S USA Figure 1. In silico ribotyping of diﬀerent C. diﬃcile genome sequences using the ISR 16S and 23S USA Figure 1. In silico ribotyping of different C. difficile genome sequences using the ISR 16S and 23S USA primer pair. The five panels represent the results obtained for examples of five different ribotypes. Bar graphs show the number of theoretical PCR bands (vertical axis, number of bands labeled on each bar) in the ribosomal region of respective genome sequences (horizontal axis), whereas the genomes without any fragments depict the complete absence of primer binding sites in those genomes. Note that the expected outcome would be an identical number of fragments for each of the strains belonging to a single ribotype. 3.1. In Silico PCR 3.1. In Silico PCR The visualiz We indicated this number as the first marker of reproducibility; it has to be stated that besides this variation of numbers of fragments, the size of the fragment was also determined as a variable as well. Figure 1. In silico ribotyping of diﬀerent C. diﬃcile genome sequences using the ISR 16S and 23S USA primer pair. The ﬁve panels represent the results obtained for examples of ﬁve diﬀerent ribotypes. Bar graphs show the number of theoretical PCR bands (vertical axis, number of bands labeled on each bar) in the ribosomal region of respective genome sequences (horizontal axis), whereas the genomes without any fragments depict the complete absence of primer binding sites in those genomes. Note that the expected outcome would be an identical number of fragments for each of the strains belonging to a single ribotype. We indicated this number as the ﬁrst marker of reproducibility; it has to be stated that besides this variation of numbers of fragments, the size of the fragment was also determined as a variable as well. Diagnostics 2020, 10, 1078 6 of 14 3.2. New Genotyping Markers A total number of 47 RT-speciﬁc unitigs (13 for RT017, 12 for RT078, 9 for RT106, 7 for RT027, and 6 for RT001) were identiﬁed. The unitigs shared an average length of 56 base pairs (Table 4). DBGWAS generated compacted De Bruijn graphs (cDBG) containing the speciﬁc unitigs as nodes deﬁning a genotypic association between a particular RT and the C. diﬃcile genomes included (Figure 2). Unitigs that were speciﬁc for a particular RT were color-coded according to their association to the RT (red for positive association, blue for negative association) and minimum q-values were provided by subgraphs. Q-values for selected unitigs ranged from 0 to 7.40 × 10−5. Signiﬁcantly associated unitigs were extracted as FASTA formatted sequences (Supplementary Table S4). Table 4. Number of unique markers identiﬁed for each C. diﬃcile ribotype, their average length, and annotation. Ribotype Number of Markers Average Length (Base Pairs) Annotation (Number of Unitigs) RT001 06 59 1. Intergenic (4) 2. tRNA uridine-5-carboxymethylaminomethyl synthesis enzyme MnmG (1) 3. rRNA-23S ribosomal RNA (1 excluded from the list) 4. Unknown (1) RT017 13 69 1. Intergenic (3) 2. Membrane spanning protein (1) 3. Ribosome small subunit-dependent GTPase A (1) 4. Hypothetical protein (1) 5. EAL domain-containing protein (2) 6. Glutamate 2,3-aminomutase (1) 7. MurR/RpiR family transcriptional regulator (1) 8. GGDEF domain-containing protein (1) 9. Glyoxalase-like domain protein (1) 10. Radical SAM protein (1) RT027 07 53 1. Collagen-like exosporium glycoprotein BclA2 (1) 2. Intergenic (5) 3. Unknown (1) RT078 12 42 1. IS200/IS605 family element transposase accessory protein TnpB (1) 2. Spore surface glycoprotein BclB (4) 3. Collagen-like exosporium glycoprotein (BclA2) (3) 4. Unknown (partial with ABC transporter permease) (1) 5. Intergenic (1) 6. Site-speciﬁc integrase (1) 7. S8 family peptidase (1) RT106 09 55 1. Intergenic (3) 2. ABC transporter permease (2) 3. Hypothetical Protein (1) 4. 3-Hydroxybutyryl-CoA dehydrogenase (1) 5. Potassium transporter (2) Table 4. Number of unique markers identiﬁed for each C. diﬃcile ribotype, their average length, and annotation. 4. Number of unique markers identiﬁed for each C. diﬃcile ribotype, their average length, i 7 of 14 7 of 14 Diagnostics 2020, 10, 1078 Figure 2. Compacted De Bruijn graphs (cDBG) generated by De Bruijn graph-based Genome Wide Association Studies (DBGWAS) for RT001 genome sequences. 3.2. New Genotyping Markers Additionally, the q-value linked to the first node is very significantly below 0.05 and hence, the estimated effect is high (represented by the red color of the node). 3.2. New Genotyping Markers The figure illustrates the significance of the nodes (representing the selective sequences called unitigs), which are denoted by their estimated effect ranging from high (28.304; red) to low (4.00; blue). Allele frequency is represented by the size of the node. The table explains that from the two selected significant nodes in terms of their association with ribotype, the node on the top right (n180654) is specific to RT001 (called Pheno 1 in the table) and completely absent in the other ribotypes in the training set (Pheno 0). Additionally, the q-value linked to the first node is very significantly below 0.05 and hence, the estimated effect is high (represented by the red color of the node). 3.3. Validation of Markers Figure 2. Compacted De Bruijn graphs (cDBG) generated by De Bruijn graph-based Genome Wide Association Studies (DBGWAS) for RT001 genome sequences. The ﬁgure illustrates the signiﬁcance of the nodes (representing the selective sequences called unitigs), which are denoted by their estimated eﬀect ranging from high (28.304; red) to low (4.00; blue). Allele frequency is represented by the size of the node. The table explains that from the two selected signiﬁcant nodes in terms of their association with ribotype, the node on the top right (n180654) is speciﬁc to RT001 (called Pheno 1 in the table) and completely absent in the other ribotypes in the training set (Pheno 0). Additionally, the q-value linked to the ﬁrst node is very signiﬁcantly below 0.05 and hence, the estimated eﬀect is high (represented by the red color of the node). nostics 2020, 10, x FOR PEER REVIEW 7 o Figure 2. Compacted De Bruijn graphs (cDBG) generated by De Bruijn graph-based Genome Wide Association Studies (DBGWAS) for RT001 genome sequences. The figure illustrates the significance of the nodes (representing the selective sequences called unitigs), which are denoted by their estimated effect ranging from high (28.304; red) to low (4.00; blue). Allele frequency is represented by the size of the node. The table explains that from the two selected significant nodes in terms of their association with ribotype, the node on the top right (n180654) is specific to RT001 (called Pheno 1 in the table) and completely absent in the other ribotypes in the training set (Pheno 0). Additionally, the q-value linked to the first node is very significantly below 0.05 and hence, the estimated effect is high (represented by the red color of the node). 3.2. New Genotyping Markers nostics 2020, 10, x FOR PEER REVIEW 7 Figure 2. Compacted De Bruijn graphs (cDBG) generated by De Bruijn graph-based Genome Wide Association Studies (DBGWAS) for RT001 genome sequences. The figure illustrates the significance of the nodes (representing the selective sequences called unitigs), which are denoted by their estimated effect ranging from high (28.304; red) to low (4.00; blue). Allele frequency is represented by the size of the node. The table explains that from the two selected significant nodes in terms of their association with ribotype, the node on the top right (n180654) is specific to RT001 (called Pheno 1 in the table) and completely absent in the other ribotypes in the training set (Pheno 0). Additionally, the q-value linked to the first node is very significantly below 0.05 and hence, the estimated effect is high (represented by the red color of the node). 3.3. Validation of Markers Figure 2. Compacted De Bruijn graphs (cDBG) generated by De Bruijn graph-based Genome Wide Association Studies (DBGWAS) for RT001 genome sequences. The ﬁgure illustrates the signiﬁcance of the nodes (representing the selective sequences called unitigs), which are denoted by their estimated eﬀect ranging from high (28.304; red) to low (4.00; blue). Allele frequency is represented by the size of the node. The table explains that from the two selected signiﬁcant nodes in terms of their association with ribotype, the node on the top right (n180654) is speciﬁc to RT001 (called Pheno 1 in the table) and completely absent in the other ribotypes in the training set (Pheno 0). Additionally, the q-value linked to the ﬁrst node is very signiﬁcantly below 0.05 and hence, the estimated eﬀect is high (represented by the red color of the node). Figure 2. Compacted De Bruijn graphs (cDBG) generated by De Bruijn graph-based Genome Wide Association Studies (DBGWAS) for RT001 genome sequences. The figure illustrates the significance of the nodes (representing the selective sequences called unitigs), which are denoted by their estimated effect ranging from high (28.304; red) to low (4.00; blue). Allele frequency is represented by the size of the node. The table explains that from the two selected significant nodes in terms of their association with ribotype, the node on the top right (n180654) is specific to RT001 (called Pheno 1 in the table) and completely absent in the other ribotypes in the training set (Pheno 0). Unitigs showin demonstrated the ef 3.3. Validation of Markers 3.3. Validation of Markers y q p y yp g g individual unitig-based characterization of C. difficile strains was not absolute, it allowed RT determination with approximate sensitivity and specificity of between 0.90 and 1.0 (Figure 3). FDR for all the unitigs for RT017 was the lowest (0.06) followed by RT027 (0.08), RT078 (0.23), RT001 (0.30), and RT106 (0.46) (Figure 3). Overall effectivity of identified Unitigs Unitigs showing 100% identity in all genomes belonging to a single RT in the validation set demonstrated the eﬃciency of these unique patterns to carry out in silico ribotyping. Although the individual unitig-based characterization of C. diﬃcile strains was not absolute, it allowed RT determination with approximate sensitivity and speciﬁcity of between 0.90 and 1.0 (Figure 3). FDR for all the unitigs for RT017 was the lowest (0.06) followed by RT027 (0.08), RT078 (0.23), RT001 (0.30), and RT106 (0.46) (Figure 3). Unitigs showing 100% identity in all genomes belonging to a single RT in the validation set demonstrated the efficiency of these unique patterns to carry out in silico ribotyping. Although the individual unitig-based characterization of C. difficile strains was not absolute, it allowed RT determination with approximate sensitivity and specificity of between 0.90 and 1.0 (Figure 3). FDR for all the unitigs for RT017 was the lowest (0.06) followed by RT027 (0.08), RT078 (0.23), RT001 (0.30), and RT106 (0.46) (Figure 3). 0 0.2 0.4 0.6 0.8 1 1.2 RT001 RT017 RT027 RT078 RT106 Sigificance of Unitigs C. difficile Ribotypes Sensitivity Specificity FDR Figure 3. Statistical comparison of genome typing efficiency of discovered unique patterns for selected C. difficile ribotypes in terms of specificity, sensitivity, and false discovery rate (FDR). Figure 3. Statistical comparison of genome typing eﬃciency of discovered unique patterns for selected C. diﬃcile ribotypes in terms of speciﬁcity, sensitivity, and false discovery rate (FDR). 8 1 Figure 3. Statistical comparison of genome typing efficiency of discovered unique patterns for selected C. difficile ribotypes in terms of specificity, sensitivity, and false discovery rate (FDR). Figure 3. Statistical comparison of genome typing eﬃciency of discovered unique patterns for selected C. diﬃcile ribotypes in terms of speciﬁcity, sensitivity, and false discovery rate (FDR). Diagnostics 2020, 10, 1078 Diagnostics 2020, 10, 8 of 14 f 15 Some of the unitigs were shared by closely related RTs. Unitigs showin demonstrated the ef 3.3. Validation of Markers 3.3. Validation of Markers Unitigs for RT001 were able to identify the genetically closely related RT087, RT241, and RT012, which altogether form a clonal complex (CC) 141 [44]. One of the markers identiﬁed for RT017 showed no false-positives or false negatives. Other markers for RT017 initially generated a small number of false-positives, but 100% true-positives in the validation dataset. Markers for RT078 identiﬁed 78 out of 79 isolates of RT126 and all of the RT413 strains from the test dataset, likely due to the close genetic relatedness of these RTs (CC 1) [44,48,49]. Unitig sequences for RT106 were also able to identify C. diﬃcile RT500 along with RT106 from the test set. Phylogenetic grouping of C. diﬃcile genomes [44] showed that C. diﬃcile core genome multi locus sequence typing (cgMLST) of RT106 and RT500 (CC 22) generated completely indistinguishable groupings. Considering closely related strains as true-positives based on their respective RTs, the FDRs for each unitig subset were found to be smaller, underscoring the biological consistency of the results. Adding genomes of RT413, RT126, and RT500 to the training set resulted in a decreased FDR rate. The continuously increasing number of publicly available C. diﬃcile genome sequences will provide substantial opportunities for improvement of our new characterization technique. Figure 3. Statistical comparison of genome typing efficiency of discovered unique patterns for selected C. difficile ribotypes in terms of specificity, sensitivity, and false discovery rate (FDR). Some of the unitigs were shared by closely related RTs. Unitigs for RT001 were able to identify the genetically closely related RT087, RT241, and RT012, which altogether form a clonal complex (CC) 141 [44]. One of the markers identified for RT017 showed no false-positives or false negatives. Other markers for RT017 initially generated a small number of false-positives, but 100% true-positives in the validation dataset. Markers for RT078 identified 78 out of 79 isolates of RT126 and all of the RT413 strains from the test dataset, likely due to the close genetic relatedness of these RTs (CC 1) [44,48,49]. Unitig sequences for RT106 were also able to identify C. difficile RT500 along with RT106 from the test set. Phylogenetic grouping of C. difficile genomes [44] showed that C. difficile core genome multi locus sequence typing (cgMLST) of RT106 and RT500 (CC 22) generated completely indistinguishable groupings. 3.4. Marker Combination Study 3.4. Marker Combination Stu For the ribotypes RT027, RT078, RT106, and RT001, every possible combination of RT-speciﬁc unitigs was created and tested for statistical signiﬁcance. A combination of two unitigs was shown to increase sensitivity and speciﬁcity up to 1 and to reduce the FDR to 0.05 (Figure 4A–D). Each combination was deﬁned on the basis of logical operators “AND/OR”. The AND operator symbolizes that both the markers in a combination need to be present with 100% identity, whereas the OR operator means that any one of the two markers in a combination need to be present at one time, again with 100% sequence identity. There is no combination required in the case of RT017. Conclusively, as clearly exempliﬁed in Figure 4A–D, in certain cases, the combination of markers improves RT testing by suppression of the false discovery rate. Marker’s SEQ ID numbers and their sequences are given in Supplementary Table S4. y For the ribotypes RT027, RT078, RT106, and RT001, every possible combination of RT-specific unitigs was created and tested for statistical significance. A combination of two unitigs was shown to increase sensitivity and specificity up to 1 and to reduce the FDR to 0.05 (Figure 4A–D). Each combination was defined on the basis of logical operators “AND/OR”. The AND operator symbolizes that both the markers in a combination need to be present with 100% identity, whereas the OR operator means that any one of the two markers in a combination need to be present at one time, again with 100% sequence identity. There is no combination required in the case of RT017. Conclusively, as clearly exemplified in Figure 4A–D, in certain cases, the combination of markers improves RT testing by suppression of the false discovery rate. Marker’s SEQ ID numbers and their sequences are given in Supplementary Table S4. Figure 4. A–D: Statistical reliability in terms of sensitivity, specificity, and false discovery rate (FDR) for the combination of two selected markers using OR operator for the identification of C. difficile RT027 (Panel A) and RT078 (Panel B). Panels C and D display similar analyses but then using the AND operator for identification of RT106 and RT001, respectively. Figure 4. (A–D) Statistical reliability in terms of sensitivity, speciﬁcity, and false discovery rate (FDR) for the combination of two selected markers using OR operator for the identiﬁcation of C. diﬃcile RT027 (Panel A) and RT078 (Panel B). Unitigs showin demonstrated the ef 3.3. Validation of Markers 3.3. Validation of Markers Considering closely related strains as true-positives based on their respective RTs, the FDRs for each unitig subset were found to be smaller, underscoring the biological consistency of the results. Adding genomes of RT413, RT126, and RT500 to the training set resulted in a decreased FDR rate. The continuously increasing number of publicly available C. difficile genome sequences will provide substantial opportunities for improvement of our new characterization technique 3.4. Marker Combination Study 3.4. Marker Combination Stu Panels C and D display similar analyses but then using the AND operator for identiﬁcation of RT106 and RT001, respectively. Figure 4. A–D: Statistical reliability in terms of sensitivity, specificity, and false discovery rate (FDR) for the combination of two selected markers using OR operator for the identification of C. difficile RT027 (Panel A) and RT078 (Panel B). Panels C and D display similar analyses but then using the AND operator for identification of RT106 and RT001, respectively. Figure 4. (A–D) Statistical reliability in terms of sensitivity, speciﬁcity, and false discovery rate (FDR) for the combination of two selected markers using OR operator for the identiﬁcation of C. diﬃcile RT027 (Panel A) and RT078 (Panel B). Panels C and D display similar analyses but then using the AND operator for identiﬁcation of RT106 and RT001, respectively. 3.5. Functional Annotation of Markers Functional characterization of the regions from which our unitigs originated demonstrated that 34% of the unitigs were localized in intergenic regions (ﬁve for RT027, four for RT001, three for of RT017 and RT106 each, and one for RT078 (Figures 5–9). Six percent of all markers were left unannotated in 9 of 14 Diagnostics 2020, 10, 1078 Diagnostics 2020, 1 3 5 F ti l RT001, RT027, and RT078 (one marker for each) (Table 4). Only RT001 was identiﬁed with a unitig marker residing within the rRNA-23S ribosomal gene showing at least some correspondence with ribotyping (Figure 5). This marker did not show suﬃcient diagnostic power and was thus not selected in the ﬁnal set of markers. All other markers were observed to be scattered throughout the C. diﬃcile genome. In RT078, one of these markers was identiﬁed in a mobile genetic element (Figure 8). Mostly, genes and intergenic regions, apart from the conserved ribosomal ISR, were observed to play a potential role in the unitig-mediated C. diﬃcile typing. Functional characterization of the regions from which our unitigs originated demonstrated that 34% of the unitigs were localized in intergenic regions (five for RT027, four for RT001, three for of RT017 and RT106 each, and one for RT078 (Figures 5–9). Six percent of all markers were left unannotated in RT001, RT027, and RT078 (one marker for each) (Table 4). Only RT001 was identified with a unitig marker residing within the rRNA-23S ribosomal gene showing at least some correspondence with ribotyping (Figure 5). This marker did not show sufficient diagnostic power and was thus not selected in the final set of markers. All other markers were observed to be scattered throughout the C. difficile genome. In RT078, one of these markers was identified in a mobile genetic element (Figure 8). Mostly, genes and intergenic regions, apart from the conserved ribosomal ISR, were observed to play a potential role in the unitig-mediated C. difficile typing. g ﬃ yp g were observed to play a potential role in the unitig-mediated C. difficile typing. Figure 5. Functional annotation and location of DBGWAS markers on the reference genome of C. difficile RT001. Both central rings represent the genome annotation (reverse inside, forward outside), while the outer and inner rings represent the signature sequences (unitigs) (reverse inside, forward outside). Figure 5. Functional annotation and location of DBGWAS markers on the reference genome of C. diﬃcile RT001. 3.5. Functional Annotation of Markers Both central rings represent the genome annotation (reverse inside, forward outside), while the outer and inner rings represent the signature sequences (unitigs) (reverse inside, forward outside). Diagnostics 2020, 10, x FOR PEER REVIEW 10 of 15 Figure 6. Functional annotation and location of DBGWAS markers on the reference genome of C. difficile RT017. Figure 6. Functional annotation and location of DBGWAS markers on the reference genome of C. diﬃcile RT017. Figure 5. Functional annotation and location of DBGWAS markers on the reference genome of C. difficile RT001. Both central rings represent the genome annotation (reverse inside, forward outside), while the outer and inner rings represent the signature sequences (unitigs) (reverse inside, forward outside). Figure 5. Functional annotation and location of DBGWAS markers on the reference genome of C. diﬃcile RT001. Both central rings represent the genome annotation (reverse inside, forward outside), while the outer and inner rings represent the signature sequences (unitigs) (reverse inside, forward outside). Diagnostics 2020, 10, x FOR PEER REVIEW 10 of 15 Figure 5. Functional annotation and location of DBGWAS markers on the reference genome of C. difficile RT001. Both central rings represent the genome annotation (reverse inside, forward outside), while the outer and inner rings represent the signature sequences (unitigs) (reverse inside, forward outside). Figure 5. Functional annotation and location of DBGWAS markers on the reference genome of C. diﬃcile RT001. Both central rings represent the genome annotation (reverse inside, forward outside), while the outer and inner rings represent the signature sequences (unitigs) (reverse inside, forward outside). Diagnostics 2020, 10, x FOR PEER REVIEW 10 of 15 Figure 6. Functional annotation and location of DBGWAS markers on the reference genome of C. difficile RT017. Figure 6. Functional annotation and location of DBGWAS markers on the reference genome of C. diﬃcile RT017. Figure 6. Functional annotation and location of DBGWAS markers on the reference genome of C. difficile RT017. Figure 6. Functional annotation and location of DBGWAS markers on the reference genome of C. diﬃcile RT017. 10 of 14 10 of 14 Diagnostics 2020, 10, 1078 Figure 6. Func g Figure 6. Functional annotation and location of DBGWAS markers on the reference genome of C. difficile RT017. Figure 7. Functional annotation and location of DBGWAS markers on the reference genome of C. difficile RT027. Figure 7. Functional annotation and location of DBGWAS markers on the reference genome of C. diﬃcile RT027. 4. Conclusions Strain typing has a proven value in monitoring the persistence and spread of bacterial pathogens in human populations. For C. diﬃcile, PCR ribotyping is the current ﬁrst choice but may be challenged now that genome sequencing is an option. No single-step test or algorithm is available so far for correlating C. diﬃcile RTs with WGS data. This implies that there may be an issue with the correlation between WGS-based epidemiological analysis and PCR ribotyping for C. diﬃcile. Here, we show that DBGWAS identiﬁed unique genomic markers that would suit that speciﬁc purpose. A combination of two unitigs led to 100% sensitive and speciﬁc discrimination between ﬁve important RTs. We believe that this approach is highly promising, providing a clear opportunity to deﬁne backward compatibility between classical RTs and WGS data. Supplementary Materials: The following are available online at http://www.mdpi.com/2075-4418/10/12/1078/s1. Table S1 describes a collection of genome sequences that were used for the in silico search of ribotypes based on ampliﬁcation of tentative ISRs. Table S2 contains a training set of C. diﬃcile genomes used for the initial DBGWAS. Table S3 contains the C. diﬃcile genomes used for DBGWAS validation. Table S4 shows a review of all unitigs that are statistically signiﬁcantly associated with speciﬁc ribotypes. Author Contributions: Conceptualization, A.v.B., M.G., L.H., M.J., K.D.B.and R.V.G.; Formal analysis, M.G.; Investigation, M.G., A.v.B., L.H., M.J. and R.V.G.; Methodology, M.G. and K.D.B.; Software, M.J.; Supervision, A.v.B.; Writing—original draft, M.G.; Writing— review & editing, A.v.B., L.H., H.P., M.J., K.D.B.and R.G. All authors have read and agreed to the published version of the manuscript. Author Contributions: Conceptualization, A.v.B., M.G., L.H., M.J., K.D.B.and R.V.G.; Formal analysis, M.G.; Investigation, M.G., A.v.B., L.H., M.J. and R.V.G.; Methodology, M.G. and K.D.B.; Software, M.J.; Supervision, A.v.B.; Writing—original draft, M.G.; Writing— review & editing, A.v.B., L.H., H.P., M.J., K.D.B.and R.G. All authors have read and agreed to the published version of the manuscript. Funding: This research was supported and funded by bioMérieux, France; Creighton University School of Medicine, NE, USA; and the European Union’s Horizon 2020 research and innovation program entitled as Viral and Bacterial Adhesin Network Training (ViBrANT) under Marie Skłodowska-Curie Grant Agreement No. 765042. Acknowledgments: During this study, M.G., L.H., H.P., M.J., K.D.B. and A.v.B. were employees of bioMérieux, a company designing, developing, and marketing tests in the domain of infectious diseases. 3.5. Functional Annotation of Markers Diagnostics 2020, 10, x FOR PEER REVIEW 11 of 15 Figure 8. Functional annotation and location of DBGWAS markers on the reference genome of C. difficile RT078. Figure 8. Functional annotation and location of DBGWAS markers on the reference genome of C. diﬃcile RT078. Figure 7. Functional annotation and location of DBGWAS markers on the reference genome of C. difficile RT027. Figure 7. Functional annotation and location of DBGWAS markers on the reference genome of C. diﬃcile RT027. Diagnostics 2020, 10, x FOR PEER REVIEW 11 of 15 Figure 7. Functional annotation and location of DBGWAS markers on the reference genome of C. difficile RT027. Figure 7. Functional annotation and location of DBGWAS markers on the reference genome of C. diﬃcile RT027. Diagnostics 2020, 10, x FOR PEER REVIEW 11 of 15 Figure 8. Functional annotation and location of DBGWAS markers on the reference genome of C. difficile RT078. Figure 8. Functional annotation and location of DBGWAS markers on the reference genome of C. diﬃcile RT078. Figure 8. Functional annotation and location of DBGWAS markers on the reference genome of C. difficile RT078. Figure 8. Functional annotation and location of DBGWAS markers on the reference genome of C. diﬃcile RT078. 11 of 14 Diagnostics 2020, 10, 1078 Figure 8. Fun Figure 9. Functional annotation and location of DBGWAS markers on the reference genome of C. difficile RT106. Figure 9. Functional annotation and location of DBGWAS markers on the reference genome of C. diﬃcile RT106. Figure 9. Functional annotation and location of DBGWAS markers on the reference genome of C. difficile RT106. Figure 9. Functional annotation and location of DBGWAS markers on the reference genome of C. diﬃcile RT106. References 1. Walk, S.T.; Micic, D.; Jain, R.; Lo, E.S.; Trivedi, I.; Liu, E.W.; Almassalha, L.M.; Ewing, S.A.; Ring, C.; Galecki, A.T.; et al. Clostridium diﬃcile Ribotype Does Not Predict Severe Infection. Clin. Infect. Dis. 2012, 55, 1661–1668. [CrossRef] [PubMed] 1. Walk, S.T.; Micic, D.; Jain, R.; Lo, E.S.; Trivedi, I.; Liu, E.W.; Almassalha, L.M.; Ewing, S.A.; Ring, C.; Galecki, A.T.; et al. Clostridium diﬃcile Ribotype Does Not Predict Severe Infection. Clin. Infect. Dis. 2012, 55, 1661–1668. [CrossRef] [PubMed] 2. 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[CrossRef] Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional aﬃliations. © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
https://openalex.org/W3006052785	https://europepmc.org/articles/pmc7040399?pdf=render	English	null	Identification of a Modified <i>HOXB9</i> mRNA in Breast Cancer	Journal of oncology	2,020	cc-by	7,173	Department of Surgery, Keio University School of Medicine, Shinanomachi 35 Shinjuku-ku, Tokyo 160-0016, Correspondence should be addressed to Tetsu Hayashida; tetsu@keio.jp Received 3 August 2019; Revised 29 December 2019; Accepted 7 January 2020; Published 13 February 2020 Copyright © 2020 Ayako Nakashoji et al. 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. First identiﬁed as a developmental gene, HOXB9 is also known to be involved in tumor biological processes, and its aberrant expression correlates with poor prognosis of various cancers. In this study, we isolated a homeodomain-less, novel HOXB9 variant (HOXB9v) from human breast cancer cell line-derived mRNA. We conﬁrmed that the novel variant was produced from variationless HOXB9 genomic DNA. RT-PCR of mRNA isolated from clinical samples and reanalysis of publicly available RNA- seq data proved that the new transcript is frequently expressed in human breast cancer. Exogenous HOXB9v expression sig- niﬁcantly enhanced the proliferation of breast cancer cells, and gene ontology analysis indicated that apoptotic signaling was suppressed in these cells. Considering that HOXB9v lacks key domains of homeobox proteins, its behavior could be completely diﬀerent from that of the previously described variationless HOXB9. Because none of the previous studies on HOXB9 have considered the presence of HOXB9v, further research analyzing the two transcripts individually is warranted to re-evaluate the true role of HOXB9 in cancer. promotes metastasis by activating the WNT signaling pathway [5, 6]. In breast cancer, the gene induces the ex- pression of proangiogenic factors, increasing the cell mo- tility and supporting epithelial-mesenchymal transition (EMT) [7, 8]. HOXB9 also promotes the growth of colon cancer by activating IL6 signaling, inducing the secretion of angiogenic factors and increasing proliferation of tumor cells [9]. Similar observations are found in ovarian cancer and hepatocellular carcinoma [10, 11]. Tus, HOXB9 acti- vates the WNT signaling pathway and enhances the ac- quisition of capabilities critical to the transformation of normal cells to cancer, including EMTand the growth of new vasculature within the tumor microenvironment. Hindawi Journal of Oncology Volume 2020, Article ID 6065736, 10 pages https://doi.org/10.1155/2020/6065736 Hindawi Journal of Oncology Volume 2020, Article ID 6065736, 10 pages https://doi.org/10.1155/2020/6065736 Hindawi Journal of Oncology Volume 2020, Article ID 6065736, 10 pages https://doi.org/10.1155/2020/6065736 1. Introduction Homeobox (HOX) genes were initially characterized as developmental genes, which code for transcription factors that play critical roles in embryogenesis. Evolutionarily, they are highly conserved and share a high degree of homology, especially within the same paralog groups. All 39 mam- malian HOX genes consist of two exons and a single intron. Te homeobox domain, encoded in the second exon [1], includes the DNA binding site. Te diverse and speciﬁc transcriptional activities of the HOX proteins often depend on key cofactors including PBX, MEIS, and PREP, which interact with hexapeptide motifs of HOX proteins [2]. HOX genes play key roles in both solid and hemato- logical malignancies, including cancers of the colon, breast, prostate, lung, brain, thyroid, ovary, bladder, kidney, skin, and blood [3, 4]. HOXB9, the ninth paralog in the HOX-B cluster, is associated with the growth and progression of multiple cancers. In lung adenocarcinoma, HOXB9 HOXB9 is also directly associated with cancer-induced patient mortality. Te duration of disease-free and overall survival of patients with HOXB9-positive breast cancer is signiﬁcantly shorter compared with patients with HOXB9- negative breast cancer [12]. Increased HOXB9 expression signiﬁcantly correlates with decreased overall survival for 2 Journal of Oncology patients with colorectal cancer [9]. Patients of laryngeal squamous cell carcinoma, hepatocellular carcinoma, glioma, and endometrial cancer also present poor outcomes or tumor progression, resulting from aberrant HOXB9 ex- pression [13–16]. Minilys homogenizer (Bertin Instruments, Bretonneux, France) using 2.4 mm metal beads prior to mRNA extrac- tion. Total RNA was converted to cDNA using the High- Capacity RNA-to-cDNA Kit (Termo Fischer Scientiﬁc). p [ ] Te recent elucidation of the critical and diverse roles HOXB9 plays in various cancers have led us to explore the mechanism of this gene’s role in cancer progression. In the present study, we identiﬁed and characterized a HOXB9 variant (HOXB9v) of mRNA from human breast cancer cell lines. Te sequence of HOXB9v largely diﬀers from the previously known HOXB9 normal transcript (HOXB9n), and we found it lacks some important domains of HOX genes. Based on these ﬁndings, we inferred its role and function were diﬀerent from HOXB9n. Tis study aimed to conﬁrm the presence of HOXB9v in clinical breast cancer specimens and investigate the role of HOXB9v in breast cancer progression. 2.4. HOXB9 Cloning. 1. Introduction HOXB9 transcripts and genomic DNA were ampliﬁed by PCR and subsequently cloned into the pME-HA vector (Lucigen, Middleton, WI, USA) using the Expresso CMV Cloning & Expression system (Lucigen). Te primers used were as follows: sense 5′ GAAGGAGA- TACCACCATGTCCATTTCTGGGACGCTTAGC 3′ and antisense 5′ GGGCACGTCATACGGATACTCTTTGCCC TGCTCCTTATT 3′. After transformation into Competent Quick DH5α cells (Toyobo, Osaka, Japan) and culturing in kanamycin-con- taining LB plates, at least 4 colonies were selected for each sample. 2. Materials and Methods 2.5. Sequence Analysis. Sequences were analyzed using the BigDye Terminator V3.1 Cycle Sequencing kit (Termo Fisher Scientiﬁc) and Applied Biosystems 3500 Genetic analyzer (Termo Fisher Scientiﬁc). Te GENETYX-MAC Ver.19 software (GENETYX, Osaka, Japan) was used for homology alignment. 2.1.CellCulture. We cultured eight human breast cancer cell lines (MCF7, MDA-MB-231, MDA-MB-468, Hs578T, HCC38, BT-474, BT-549, and SKBR3), a human colon cancer cell line (WiDR), and a mouse breast cancer cell line (4T1) in DMEM supplemented with 10% FBS (Termo Fisher Scientiﬁc, Waltham, MA, USA) with the addition of antibiotic and antimycotic agent (antibiotic-antimycotic mixed stock solution, Nacalai Tesque, Inc., Kyoto, Japan). T47D cells were grown in RPMI1640 medium with 10% FBS and antibiotic and antimycotic agents. MCF10A cells were grown in DMEM/F12 (1 :1) medium with 5% horse serum (Termo Fischer Scientiﬁc), 20 ng/mL EGF (PeproTech, Rocky Hill, NJ, USA), 0.5 mg/mL hydrocortisone, 100 ng/ mL cholera toxin, 10 µg/mL insulin (all Sigma-Aldrich, St. Louis, MO, USA), and antibiotic and antimycotic agents. Cells were maintained at 37°C in a humidiﬁed 5% CO2 incubator. 2.6. RT-PCR Analysis. Te expressions of HOXB9n and HOXB9v were detected by RT-PCR using the following primers: sense, 5′ TGTCCATTTCTGGGACGCTT 3′; an- tisense, 5′ CTACGGTCCCTGGTGAGGTA 3′. Te genomic DNA of HOXB9 was detected by PCR using the following primers: sense, 5′ CGAGAGAGCTGCAAGTCGAT 3′; antisense, 5′ CTGCCGTCCGTCTACCAC 3′. Te primers for genomic DNA were designed against exon 1 and the intron region of the HOXB9 gene to ensure that the pair will speciﬁcally amplify only genomic DNA and not cDNA derived from mRNA. Te conditions applied for ampliﬁ- cation were as follows: 94°C for 1 minute, followed by 35 cycles at 95°C for 5 seconds, 55°C for 5 seconds, and 72°C for 5 seconds, and run on the Life ECO thermal cycler (Hangzhou Bioer Technology, Hangzhou, China) using the SapphireAmp Fast PCR Master Mix (Takara Bio, Shiga, Japan) or KOD-Plus-Neo (Toyobo). 2.2. Patients and Samples. Clinical specimens of human breast cancer (n 14) were collected from patients with primary operable breast cancer who underwent total or partial mastectomy between July and November 2018 in Keio University Hospital (Tokyo, Japan). Patient-matched healthy breast epithelium samples (n 6) were collected from healthy breast tissue from patients who underwent total mastectomy. 3. Results 3.1.IdentiﬁcationofaNovelTranscriptHOXB9v,WhichLacks Important Domains of HOX Gene. We isolated total RNA from human breast cancer cell lines (MCF7, T47D, MDA- MB-231, MDA-MB-468, HCC38, BT-474, BT-549, Hs578T, and SKBR3), a normal human mammary gland cell line (MCF10A), and a human colon cancer cell line (WiDR). We cloned and sequenced the HOXB9 gene from the nucleic acids derived from these cell lines. We isolated a novel variant of the HOXB9 transcript from the MCF7, T47D, MDA-MB-231, MDA-MB-468, Hs578T, HCC38, and MCF10A cell lines. Te sequence homology of the new transcript is shown in Figures 1(a) and S1. 2.9. Transient Overexpression of HOXB9n and HOXB9v in MDA-MB-468 Cells. MDA-MB-468 cells were transfected with aforementioned HiBiT-tagged HOXB9n or HOXB9v vector using the jetPRIME (Polyplus-transfection, Illkirch, France) as per the manufacturer’s instructions. 2.9. Transient Overexpression of HOXB9n and HOXB9v in MDA-MB-468 Cells. MDA-MB-468 cells were transfected with aforementioned HiBiT-tagged HOXB9n or HOXB9v vector using the jetPRIME (Polyplus-transfection, Illkirch, France) as per the manufacturer’s instructions. 2.10. Quantitative Real-Time PCR. Quantitative real-time PCR was run on ViiA7 (Termo Fisher Scientiﬁc) using Fast SYBR Green Master Mix (Termo Fisher Scientiﬁc). Pre- incubation was performed for 20 seconds at 95°C and ampliﬁcation for 41 cycles (1 second of denaturation at 95°C and 20 seconds of annealing and extension at 60°C), followed by melt-curve analysis. GAPDH served as an internal con- trol, and QuantStudio Real-Time PCR Software v1.2 (Termo Fisher Scientiﬁc) was used for quantiﬁcation. Te relative standard curve method was used for linear regres- sion analysis of unknown samples, and data are presented as fold change between samples. Te primers used were as follows: HOXB9: sense, 5′ CGGTGGCTGTCGTGAAATT 3′; antisense, 5′ CGAGACAATCACCCCCAAAG 3′; GAP DH: sense, 5′ATCATCCCTGCCTCTACTGG 3′; antisense, 5′ TTTCTAGACGGCAGGTCAGGT 3′. We will refer to the new truncated transcript as HOXB9v, to distinguish from the full-length HOXB9 transcript (HOXB9n). A 100-base deletion in exon 1 in the new transcript leads to a frameshift and the formation of a stop codon (TAG), which truncates the protein coding at AA167. Figure 1(b) is a schematic diagram of HOXB9 transcripts showing the exons and the deleted lesion. Te encoded protein will therefore possibly lack the homeobox domain, DNA binding domain, and the hexapeptide motif, a major player in cofactor interactions (Figure 1(c)). Te HOXB9v sequence has been submitted to GenBank under Accession No. LC466645. 3. Results We next performed PCR analyses to verify the presence of the HOXB9v transcript and identify genomic DNA variations of the HOXB9 gene in human breast cancer cell lines. Te primer target region included the deletion site of HOXB9v and the amplicon for HOXB9n was 643 bp and 543 bp for HOXB9v. We detected both HOXB9n and HOXB9v transcripts in breast cancer cell lines (Figure 2(a)). Te PCR products were sequenced and were conﬁrmed that each band corresponded to the exact sequence of the HOXB9n or HOXB9v (Figure 2(b)). By PCR of breast cancer cell lines’ genomic DNA, no bands indicative of genomic DNA variations in HOXB9 were detected. Te amplicon of variation-less HOXB9 genomic DNA was 569 bp (Figure 2(c)). Sequencing of the PCR products conﬁrmed that they had no variations or deletions in genomic DNA (Figure S2). Tese ﬁndings show that the two transcripts (HOXB9n and HOXB9v) were produced from variation-less HOXB9 genomic DNA. 2.11. Cell Proliferation Assay. Cell proliferation in three- dimensional (3D) culture was measured using the 24-well Bio-Assembler kit and NanoShuttle-PL (Greiner Bio-One, Kremsm¨unster, Austria). Further, 20,000 cells/well were incubated for 48 hours before taking photomicrographs. 2.11. Cell Proliferation Assay. Cell proliferation in three- dimensional (3D) culture was measured using the 24-well Bio-Assembler kit and NanoShuttle-PL (Greiner Bio-One, Kremsm¨unster, Austria). Further, 20,000 cells/well were incubated for 48 hours before taking photomicrographs. 2.11. Cell Proliferation Assay. Cell proliferation in three- dimensional (3D) culture was measured using the 24-well Bio-Assembler kit and NanoShuttle-PL (Greiner Bio-One, Kremsm¨unster, Austria). Further, 20,000 cells/well were incubated for 48 hours before taking photomicrographs. Cell proliferation in ﬂat culture was measured using Cell Count Reagent SF (Nacalai Tesque). Brieﬂy, 5000 cells/well in 96-well microtiter plates (Sumilon, Sumitomo Bakelite, Tokyo Japan) were incubated for 5 days or after 24 hour continuous exposure to either 30 nM HXR9 or 30 nM CXR9 for 4 days. Te absorbance in the wells was measured on days 1, 3, and 5 using a Sunrise Rainbow-RC (TECAN, M¨annedorf, Switzerland) microplate spectrophotometer at 450 nm, using 600 nm as reference. Te HXR9 (WYPWMKKHHRRRRRRRRR-) and control CXR9 (WYPAKKHHRRRRRRRRR) peptides were synthesized by Euroﬁns Genomics K. K. (Tokyo, Japan). g p g p Cell proliferation in ﬂat culture was measured using Cell Count Reagent SF (Nacalai Tesque). 2. Materials and Methods Ethics approval for the present study was provided by the Ethics Committee at the Keio University School of Medicine (approval number: 20180090), and the study was performed in accordance with the provisions of the Declaration of Helsinki (as revised in Fortaleza, Brazil, October 2013). All included patients gave informed consent. 2.2. Patients and Samples. Clinical specimens of human breast cancer (n 14) were collected from patients with primary operable breast cancer who underwent total or partial mastectomy between July and November 2018 in Keio University Hospital (Tokyo, Japan). Patient-matched healthy breast epithelium samples (n 6) were collected from healthy breast tissue from patients who underwent total mastectomy. Ethics approval for the present study was provided by the Ethics Committee at the Keio University School of Medicine (approval number: 20180090), and the study was performed in accordance with the provisions of the Declaration of Helsinki (as revised in Fortaleza, Brazil, October 2013). All included patients gave informed consent. 2.7. Public Data Reanalysis. Te RNA sequence data set (GSE119937) in FASTQ format was downloaded via SRA (SRP161704) using the SRA Toolkit (version 2.3.4-2). RNA- seq reads were aligned by STAR (version 2.6.1b) against the hg38 reference genome. All reads mapped on the HOXB9 gene were visually conﬁrmed by taking snapshots in IGV (version 2.4.15). 2.3. mRNA and Genomic DNA Extraction from Cell Lines and Clinical Specimens. Total RNA and genomic DNA were extracted using the RNeasy Mini Kit and QIAmp DNA Mini Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. Clinical specimens were homogenized using a 2.8. Establishment of Stable MCF7 Cell Lines Overexpressing HOXB9n or HOXB9v. Te HOXB9n and HOXB9v se- quences were ampliﬁed by PCR and subsequently cloned into the pBiT3.1-N [CMV/HiBiT/Blast] expression vector (Promega, Tokyo, Japan) at the Xhol and BamHI sites using 3 Journal of Oncology results were analyzed by TAC 4.0 software (Termo Fisher Scientiﬁc). Genes having a false discovery rate (FDR) under 0.05 and upregulated in HOXB9v samples were considered as upregulated diﬀerentially expressed genes (up-DEGs). DAVID Bioinformatics Resources 6.8 [17] was used for gene ontology and pathway analysis of DEGs. R ver. 3.5.0 software was used to draw the heatmap of 37 genes included in GO: 0043069 (negative regulation of programmed cell death). Ligation high Ver.2 (Toyobo). Te primers used for am- pliﬁcation were as follows: sense, 3′ ATACCTCGAGG TCCATTTCTGG 5′; antisense, 3′ CACGTCATACGGAT CCTCTTTG 5′. 2. Materials and Methods MCF7 cells were transfected with the HiBiT-tagged HOXB9n or HOXB9v vector using the Viafect Transfection reagent (Promega) and selected with 10 µg/mL of blasticidin for at least 4 weeks. Te expression of HiBiT- tagged HOXB9n and HOXB9v proteins was detected using the Nano-Glo HiBiT Blotting System (Promega) as per the manufacturer’s instructions. 3. Results HOXB9 gDNA 445 TACATCCAGCCCCAGGGCGTCCCGCCGGCCGAGAGCAGGTACCTCCGCACCTGGCTGGAGCCGGCGCCGCGCGGCGAAGCGGCCCCGGGGCAGGGCCAGGCGGCGGTGAAGGCGGAGCCGCTGCTGGGCG 574 HOXB9n mRNA 241 TACATCCAGCCCCAGGGCGTCCCGCCGGCCGAGAGCAGGTACCTCCGCACCTGGCTGGAGCCGGCGCCGCGCGGCGAAGCGGCCCCGGGGCAGGGCCAGGCGGCGGTGAAGGCGGAGCCGCTGCTGGGCG 370 HOXB9v mRNA 241 TACATCCTGCCCC–GGGG – CCGCTGCTGGGCG 270 (a) 205 1 ATG 721 722 955 2701 TAA Exon I Exon II 205 1 ATG 721 722 804 2701 TAG 462 562 HOXB9n HOXB9v (b) 1 528 555 1 176 185 1 257 357 501 653 1 85fs 167∗ HOXB9n HOXB9v Frameshift Nucleotide AA Nucleotide AA Hexapeptide & linker N-terminal flexible region Homeodomain 251∗ 753 (c) (a) HOXB9n HOXB9n (c) Figure 1: Structure and sequence of HOXB9n and HOXB9v. (a) Sequences of genomic DNA HOXB9, mRNA HOXB9n, and mRNA HOXB9v (black and grey highlighting indicates homology between sequences). (b) Schematic diagram of HOXB9n (upper) and HOXB9v (lower) transcripts showing the exons, splicing regions, and the deleted region. In HOXB9v, a 100 bp deletion in exon 1 leads to a frameshift and a stop codon formation (TAA). (c) Protein structure of HOXB9n and HOXB9v. Transcription from the start codon (ATG) to the stop codon (TAA) results in the translation of a full-length HOXB9n protein (upper). Te 100 bp deletion in HOXB9v (shown in black) leads to a frameshift from AA85 (shown in grey) and truncation of protein coding by a stop codon (TAG) at AA167, which results in HOXB9v protein without hexapeptide and homeodomain.∗Stop codon. MCF7 T47D MDA- MB-231 Hs578T MDA- MB-468 SKBR3 MCF10A HOXB9n HOXB9v 600bp –– 500bp –– 700bp –– 600bp –– 500bp –– 700bp –– (a) (b) MCF7 T47D MDA- MB-231 Hs578T MDA- MB-468 MCF10A 500bp 400bp 600bp –– –– –– (c) Figure 2: Detection of mRNA and genomic DNA of HOXB9 in cell lines. (a) Both HOXB9n and HOXB9v transcripts were detected in breast cancer cell line mRNA (cDNA). (b) Sequencing conﬁrmation of PCR product of Figure 2(a); SKBR3 (HOXB9n, upper column) and MCF10A (HOXB9v, lower column). (c) No genomic variation was detected in genomic DNA of breast cancer cell line genomic DNA. HOXB9v transcripts are commonly found in human breast cancer specimens. MCF7 T47D MDA- MB-231 Hs578T MDA- MB-468 SKBR3 MCF10A HOXB9n HOXB9v 600bp –– 500bp –– 700bp –– 600bp –– 500bp –– 700bp –– (a) (b) MDA- MB-468 (a) (b) MDA- MB-231 MCF7 T47D MB-231 Hs578T MB-468 MCF10A 500bp 400bp 600bp –– –– –– (c) (c) Figure 2: Detection of mRNA and genomic DNA of HOXB9 in cell lines. (a) Both HOXB9n and HOXB9v transcripts were detected in breast cancer cell line mRNA (cDNA). 3. Results Brieﬂy, 5000 cells/well in 96-well microtiter plates (Sumilon, Sumitomo Bakelite, Tokyo Japan) were incubated for 5 days or after 24 hour continuous exposure to either 30 nM HXR9 or 30 nM CXR9 for 4 days. Te absorbance in the wells was measured on days 1, 3, and 5 using a Sunrise Rainbow-RC (TECAN, M¨annedorf, Switzerland) microplate spectrophotometer at 450 nm, using 600 nm as reference. Te HXR9 (WYPWMKKHHRRRRRRRRR-) and control CXR9 (WYPAKKHHRRRRRRRRR) peptides were synthesized by Euroﬁns Genomics K. K. (Tokyo, Japan). To determine the presence of HOXB9v transcripts in human breast cancer samples, we performed PCR analysis. HOXB9v was commonly detected from clinical breast cancer samples (Figure 3(a)), regardless of their hormone receptor and HER2 status. However, HOXB9v was not detected in normal mammary gland samples (Figure 3(b)). 2.12. Microarray and Diﬀerential Expression Analyses. Total RNA was isolated using the RNeasy Mini kit (Qiagen). Microarray was performed using the human Clariom S assay (Termo Fisher Scientiﬁc) by the GeneChip Scanner 3000 7G system (Aﬀymetrix, Santa Clara, CA, USA), and the 4 Journal of Oncology HOXB9 gDNA 445 TACATCCAGCCCCAGGGCGTCCCGCCGGCCGAGAGCAGGTACCTCCGCACCTGGCTGGAGCCGGCGCCGCGCGGCGAAGCGGCCCCGGGGCAGGGCCAGGCGGCGGTGAAGGCGGAGCCGCTGCTGGGCG 574 HOXB9n mRNA 241 TACATCCAGCCCCAGGGCGTCCCGCCGGCCGAGAGCAGGTACCTCCGCACCTGGCTGGAGCCGGCGCCGCGCGGCGAAGCGGCCCCGGGGCAGGGCCAGGCGGCGGTGAAGGCGGAGCCGCTGCTGGGCG 370 HOXB9v mRNA 241 TACATCCTGCCCC–GGGG – CCGCTGCTGGGCG 270 (a) 205 1 ATG 721 722 955 2701 TAA Exon I Exon II 205 1 ATG 721 722 804 2701 TAG 462 562 HOXB9n HOXB9v (b) 1 528 555 1 176 185 1 257 357 501 653 1 85fs 167∗ HOXB9n HOXB9v Frameshift Nucleotide AA Nucleotide AA Hexapeptide & linker N-terminal flexible region Homeodomain 251∗ 753 (c) Figure 1: Structure and sequence of HOXB9n and HOXB9v. (a) Sequences of genomic DNA HOXB9, mRNA HOXB9n, and mRNA HOXB9v (black and grey highlighting indicates homology between sequences). (b) Schematic diagram of HOXB9n (upper) and HOXB9v (lower) transcripts showing the exons, splicing regions, and the deleted region. In HOXB9v, a 100 bp deletion in exon 1 leads to a frameshift and a stop codon formation (TAA). (c) Protein structure of HOXB9n and HOXB9v. Transcription from the start codon (ATG) to the stop codon (TAA) results in the translation of a full-length HOXB9n protein (upper). Te 100 bp deletion in HOXB9v (shown in black) leads to a frameshift from AA85 (shown in grey) and truncation of protein coding by a stop codon (TAG) at AA167, which results in HOXB9v protein without hexapeptide and homeodomain.∗Stop codon. 3. Results (b) Sequencing conﬁrmation of PCR product of Figure 2(a); SKBR3 (HOXB9n, upper column) and MCF10A (HOXB9v, lower column). (c) No genomic variation was detected in genomic DNA of breast cancer cell line genomic DNA. HOXB9v transcripts are commonly found in human breast cancer specimens. 5 Journal of Oncology Journal of Oncology +/+ +/– +/– +/– +/– –/– +/– –/– +/– +/– –/– +/– +/– –/– HR/HER2 Sample# 1 2 3 4 5 6 7 8 9 10 11 12 13 14 (a) 1 2 5 11 12 14 HOXB9n HOXB9v Sample# (b) Figure 3: Detection of HOXB9v in clinical samples. (a) HOXB9n and HOXB9v were detected in breast cancer clinical samples. (b) HOXB9v was not detected in normal mammary gland samples. Hormone receptor status and HER2 status of each cancer sample are shown beneath each sample number. We assigned the same sample number if cancer and normal gland samples were acquired from the same patient. (a) (a) 11 (a) 11 12 (b) (b) Figure 3: Detection of HOXB9v in clinical samples. (a) HOXB9n and HOXB9v were detected in breast cancer clinical samples. (b) HOXB9v was not detected in normal mammary gland samples. Hormone receptor status and HER2 status of each cancer sample are shown beneath each sample number. We assigned the same sample number if cancer and normal gland samples were acquired from the same patient. SRR7826861 SRR7826774 SRR7826783 Exon II Exon I 5′ 3′ HOXB9 Intron ATG ATG TAG HOXB9n HOXB9v TAA T3 N1 IDC (primary) ER – PgR – HER2–, ki67 60% T1cN0 IDC (primary) ER + PgR – HER2–, ki67 5% T1aN1 IDC (primary) ER + PgR + HER2–, ki67 0% Figure 4: NGS data indicates the presence of HOXB9v in breast cancer. Showing RNA sequence breast cancer sample data (SRR782677) mapped on HOXB9 exon 1. Te blue band on the bottom shows the coding region (the thick part) and the 5′ UTR (the thin part) of exon 1. Tere is a region where data are sparsely mapped (indicated with the red arrow), which matches the deletion region in HOXBv. HOXB9v overexpressing MCF7 and MDA-MB-468 cells proliferated faster compared with HOXB9n overexpressing cells. Figure 4: NGS data indicates the presence of HOXB9v in breast cancer. Showing RNA sequence breast cancer sample data (SRR782677) mapped on HOXB9 exon 1. 3. Results We also 6 Journal of Oncology 1 10 100 HOXB9v HOXB9n HOXB9 mRNA expression relative to control log10 (a) 35kDa 17kDa HOXB9n HOXB9v 25kDa (b) HOXB9v HOXB9n 0 200 400 600 800 1000 1200 1400 1600 1800 2000 ∗p = 1.7247E – 06 1601.0 1834.8 (μm) ∗ (c) 0 0.2 0.4 0.6 0.8 1 1.2 1.4 Day1 Day3 Day5 ∗p = 0.0134 HOXB9n HOXB9v ∗ (d) HOXB9v HOXB9n 1 10 100 1000 10000 100000 HOXB9 mRNA expression relative to control log10 (e) Day1 Day3 Day5 0 0.1 0.2 0.3 0.4 0.5 ∗p = 4.40E – 07 HOXB9n HOXB9v ∗ (f) re 5: (a–d) Conﬁrmation and proliferation of stable MCF7 cell lines that overexpress HOXB9n or HOXB9v. Levels of HOXB9n and XB9v (a) mRNA and (b) protein in stable cell lines. Proliferation of the HOXB9n and HOXB9v stable cell lines using a (c) 3D cell culture el and a (d) 2D cell culture model. (e-f) Conﬁrmation and proliferation of MDA-MB-468 cells with transient overexpression of XB9n or HOXB9v. (e) Levels of HOXB9n and HOXB9v mRNA expression. (f) Proliferation of HOXB9n and HOXB9v overexpressing by 2D cell culture model. 1 10 100 HOXB9v HOXB9n HOXB9 mRNA expression relative to control log10 ( ) 35kDa 17kDa HOX HOXB9v 25kDa (b) HOXB9v HOXB9n 0 200 400 600 800 1000 1200 1400 1600 1800 2000 ∗p = 1.7247E – 06 1601.0 1834.8 (μm) ∗ (c) (a) (b) (c) Day3 Day5 ∗p = 0.0134 B9n B9v ∗ (d) HOXB9v HOXB9n 1 10 100 1000 10000 100000 HOXB9 mRNA expression relative to control log10 (e) Day1 Day3 Day5 0 0.1 0.2 0.3 0.4 0.5 ∗p = 4.40E – 07 HOXB9n HOXB9v ∗ (f) (a) (b) (c) (b) HOXB9v HOXB9n 1 10 100 1000 10000 100000 HOXB9 mRNA expression relative to control log10 (e) 0 0.2 0.4 0.6 0.8 1 1.2 1.4 Day1 Day3 Day5 ∗p = 0.0134 HOXB9n HOXB9v ∗ (d) Day1 Day3 Day5 0 0.1 0.2 0.3 0.4 0.5 ∗p = 4.40E – 07 HOXB9n HOXB9v ∗ (f) HOXB9 mRNA expression (d) (f) (e) Figure 5: (a–d) Conﬁrmation and proliferation of stable MCF7 cell lines that overexpress HOXB9n or HOXB9v. Levels of HOXB9n and HOXB9v (a) mRNA and (b) protein in stable cell lines. Proliferation of the HOXB9n and HOXB9v stable cell lines using a (c) 3D cell culture model and a (d) 2D cell culture model. 3. Results Te blue band on the bottom shows the coding region (the thick part) and the 5′ UTR (the thin part) of exon 1. Tere is a region where data are sparsely mapped (indicated with the red arrow), which matches the deletion region in HOXBv. HOXB9v overexpressing MCF7 and MDA-MB-468 cells proliferated faster compared with HOXB9n overexpressing cells. transiently overexpressed HOXB9n or HOXB9v in MDA- MB-468 cells, and the cell proliferation assay showed that HOXB9v overexpression increased MDA-MB468 cell growth (Figures 5(e) and 5(f)). Tus, to further conﬁrm the presence of HOXB9v in human breast cancer samples, we reanalyzed a publicly available breast cancer RNA sequence data set (GSE119937) [18] and mapped the reads onto the HOXB9 gene sequence. We identiﬁed a region in exon 1 where the number of mapped reads was low in numerous samples; this region matched the deletion region of HOXB9v (Figure 4). 3.2. HXR9 and CXR9 Treatment. HXR9, an 18-amino acid peptide, competently inhibits the hexapeptide motif of HOX proteins and prevents HOX-PBX binding [19]. HOXB9n and HOXB9v expressing MCF7 cells were treated with HXR9 or a control peptide, CXR9 (Figures 6(a) and 6(b)). Although HOXB9v lacks the hexapeptide motif, which is known to interact with PBX proteins [20], HXR9 signiﬁcantly inhibited proliferation of both cell lines. 3.2. HXR9 and CXR9 Treatment. HXR9, an 18-amino acid peptide, competently inhibits the hexapeptide motif of HOX proteins and prevents HOX-PBX binding [19]. HOXB9n and HOXB9v expressing MCF7 cells were treated with HXR9 or a control peptide, CXR9 (Figures 6(a) and 6(b)). Although HOXB9v lacks the hexapeptide motif, which is known to interact with PBX proteins [20], HXR9 signiﬁcantly inhibited proliferation of both cell lines. To determine the role of HOXB9v in breast cancer, we established stable MCF7 cell lines overexpressing HOXB9n or HOXB9v. Gene and protein expressions of HOXB9n and HOXB9v were veriﬁed in both cell lines (Figures 5(a) and 5(b)). Cell proliferation assays in both 3D culture and ﬂat culture showed that HOXB9v overexpression increased MCF7 cell growth (Figures 5(c) and 5(d)). 3. Results (e-f) Conﬁrmation and proliferation of MDA-MB-468 cells with transient overexpression of HOXB9n or HOXB9v. (e) Levels of HOXB9n and HOXB9v mRNA expression. (f) Proliferation of HOXB9n and HOXB9v overexpressing cells by 2D cell culture model. 3.3. Microarray and Gene Ontology Analysis. To explore the reason behind the faster proliferation of HOXB9v over- expressing cells, we performed microarray and gene on- tology analyses using HOXB9n and HOXB9v-expressing MCF7 cells. We chose 1056 genes as DEGs, and gene ontology analysis showed that up-DEGs between HOXB9n and HOXB9v expressing cells presented sig- niﬁcant diﬀerences in pathways relevant to apoptosis suppression (GO:0060548∼negative regulation of cell death, GO:0043069∼negative regulation of programmed cell death, and GO:0043066∼negative regulation of apo- ptotic process) and steroid hormone response (GO:004 8545∼response to steroid hormone, GO:0032870∼cellular response to hormone stimulus, and GO:0071383∼cellular response to steroid hormone stimulus) (Table 1). Te genomic expression heatmap comparing 37 genes in- volved in the apoptotic process (GO:0043069∼negative regulation of programmed cell death) shows that apo- ptosis is highly suppressed in HOXB9v expressing cells (Figure 7). 3.3. Microarray and Gene Ontology Analysis. To explore the reason behind the faster proliferation of HOXB9v over- expressing cells, we performed microarray and gene on- tology analyses using HOXB9n and HOXB9v-expressing MCF7 cells. We chose 1056 genes as DEGs, and gene ontology analysis showed that up-DEGs between HOXB9n and HOXB9v expressing cells presented sig- niﬁcant diﬀerences in pathways relevant to apoptosis suppression (GO:0060548∼negative regulation of cell death, GO:0043069∼negative regulation of programmed cell death, and GO:0043066∼negative regulation of apo- ptotic process) and steroid hormone response (GO:004 8545∼response to steroid hormone, GO:0032870∼cellular response to hormone stimulus, and GO:0071383∼cellular response to steroid hormone stimulus) (Table 1). Te 4. Discussion In the present study, we identiﬁed a novel modiﬁed transcript of HOXB9, in which a deletion in exon 1 causes a frameshift, formation of a stop codon, and truncation of protein coding. Tis leads to a defect in the homeodomain and hexapeptide regions, which are both crucial for HOX gene function. We conﬁrmed that HOXB9v is widely present in human breast cancer cell lines and clinical breast cancer samples, however not 7 Journal of Oncology 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 Day1 Day3 Day5 ∗p = 0.00015 HOXB9n HXR9 HOXB9n CXR9 ∗ (a) 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 Day1 Day3 Day5 ∗p = 0.00675 HOXB9v HXR9 HOXB9v CXR9 ∗ (b) Figure 6: (a) HOXB9n-MCF7 cells and (b) HOXB9v-MCF7 cells were treated with HXR9 or with a control peptide, CXR9. HXR9 signiﬁcantly inhibited the proliferation of both cell lines. 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 Day1 Day3 Day5 ∗p = 0.00015 HOXB9n HXR9 HOXB9n CXR9 ∗ (a) 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 Day1 Day3 Day5 ∗p = 0.00675 HOXB9v HXR9 HOXB9v CXR9 ∗ (b) 1 (a) Figure 6: (a) HOXB9n-MCF7 cells and (b) HOXB9v-MCF7 cells were treated with HXR9 or with a control peptide, CXR9. HXR9 signiﬁcantly inhibited the proliferation of both cell lines. Table 1: Gene ontology analysis by diﬀerential gene expression. Apoptosis was suppressed in HOXB9v-MCF7 cells compared with HOXB9n MCF7 cells Table 1: Gene ontology analysis by diﬀerential gene expression. Apoptosis was suppressed in HOXB9v-MCF7 cells compared with HOXB9n-MCF7 cells. Term P value Bonferroni Negative regulation of cell death 7.36E−07 0.002961 Response to steroid hormone 2.24E−06 0.008994 Cellular response to zinc ion 2.66E−06 0.01065 Negative regulation of programmed cell death 3.30E−06 0.013228 Cellular response to hormone stimulus 6.93E−06 0.027551 Negative regulation of apoptotic process 7.26E−06 0.028853 Cellular response to steroid hormone stimulus 1.15E−05 0.045436 Negative regulation of transcription, DNA-template 1.17E−05 0.045904 Table 1: Gene ontology analysis by diﬀerential gene expression. Apoptosis was suppressed in HOXB9v-M HOXB9n-MCF7 cells. diﬀerential gene expression. Apoptosis was suppressed in HOXB9v-MCF7 cells compared with in normal gland samples. Detection of HOXB9v in MCF10A may be attributed to the fact that this cell line is not karyo- typically normal, while it maintains major characteristics of normal breast epithelium [21]. We further conﬁrmed that HOXBv and the previously known HOXB9n were both pro- duced from variationless HOXB9 genomic DNA. 4. Discussion Genomic heatmap compares expression of 37 gene involved in suppression of apoptotic process (GO:0043069), with red and green color intensities indicating high and low e ti l P d ll d th i d i HOXB9 MCF7 ll 8 Journal of Journal of Oncology IER3 CAV1 IL6ST EFNA1 TGFB3 BNIP3 PRDX5 SYCP2 PDCD4 SHH RGL2 CREB3L1 BCL6 CEACAM5 DDAH2 THBS1 MUC1 AR SOCS2 MET ANXA1 NR4A1 BAD NTSR1 POR FOXP1 ASCL1 DHRS2 CORO1A UCP2 AAMDC HIPK2 BNIP3L THOC6 TFAP2A TNFAIP3 BMP7 Sample 2 1 0 –1 –2 Scaled Journal of Oncology IER3 CAV1 IL6ST EFNA1 TGFB3 BNIP3 PRDX5 SYCP2 PDCD4 SHH RGL2 CREB3L1 BCL6 CEACAM5 DDAH2 THBS1 MUC1 AR SOCS2 MET ANXA1 NR4A1 BAD NTSR1 POR FOXP1 ASCL1 DHRS2 CORO1A UCP2 AAMDC HIPK2 BNIP3L THOC6 TFAP2A TNFAIP3 BMP7 Sample 2 1 0 –1 –2 Scaled Journal of Oncology Figure 7: Microarray data heatmap of HOXB9n-MCF7 and HOXB9v-MCF7. Genomic heatmap compares expression of 37 genes which are involved in suppression of apoptotic process (GO:0043069), with red and green color intensities indicating high and low expressions, respectively. Programmed cell death is suppressed in HOXB9v-MCF7 cells. Figure 7: Microarray data heatmap of HOXB9n-MCF7 and HOXB9v-MCF7. Genomic heatmap compares expression of 37 genes which are involved in suppression of apoptotic process (GO:0043069), with red and green color intensities indicating high and low expressions, respectively. Programmed cell death is suppressed in HOXB9v-MCF7 cells. cancer. HOXB9n and HOXB9v should be assessed separately. have been reported to regulate apoptosis in cancer [29, 30], and HOX-regulated apoptosis is a general mechanism used during development to maintain metameric patterns [31, 32]. However, the role of HOXB9 in apoptosis is yet to be investigated. In the growth assay, MCF7 and MDA-MB-468 cells expressing HOXB9v at high levels presented more rapid proliferation than did those expressing HOXB9v. It is meaningful that similar results were observed in two cell lines: one with hardly any expression of HOXB9n and HOXB9v (MCF7) and the other with high expression of both (MDA-MB-468). Fostered proliferation by HOXB9v may be attributed to the suppressed apoptosis observed in our microarray studies. Gene ontology analysis of HOXB9v-expressing cells compared to HOXB9n- expressing cells indicated signiﬁcant upregulation of pathways related to apoptosis suppression, further underscoring its role in this regard. Several HOX genes p p y g Te binding selectivity of HOX proteins is inﬂuenced by cofactors, including members of the PBX, MEIS, and PREP families [2]. 4. Discussion Two distinct mRNA products from a single variationless genomic DNA may indicate that HOXB9v is a novel splice variant of HOXB9. It may be assumed that HOXB9v has a typical splice site of GU as a 5′ donor site and AG as a 3′ acceptor site [22]; however, the single-nucleotide transitions at 248 and 258 and the single- nucleotide deletion at 254 must be taken into account. Further investigation and discussions are needed to decide whether HOXB9v is a splice variant or an mRNA modiﬁed by a diﬀerent mechanism. Nonetheless, the modiﬁed transcript is certainly not a result of variations in genomic DNA. Additionally, HOXB9v results in a diﬀerent stop codon site from that of HOXB9n and may become a target of nonsense-mediated mRNA decay (NMD), an mRNA surveillance mechanism that eliminates premature translation-termination codons. How- ever, NMD is known to be initiated if an exon-junction complex is present more than 50–55 bases downstream of the stop codon [23]. HOXB9v forms its exon-junction complex upstream from its stop codon; therefore, we infer it escapes from NMD. Nevertheless, the production of splice variants of HOX genes lacking the homeodomain is likely a common phenomenon. Murine Hoxb9, which shares sequence similarity with human HOXB9, is reported to generate a splice variant without a homeodomain. Other HOX genes, including human HOXA1, HOXB6, HOXA9, HOXA10, murine Hoxb6 and Meis1, and Xenopus XlHbox2, have also been reported to generate splice variants lacking homeodomains [24–28]. Interestingly, HOXA9T, a homeodomain-less isoform of HOXA9, which is structurally similar to HOXB9v, has been demonstrated to act as an oncogene in leukemia without directly binding to DNA [24]. HOXB9 has been reported to promote tumorigenesis in various types of cancers; however, no previous research on HOXB9 has shown a mRNA variant of HOXB9. Considering that the structure of the HOXB9v protein diﬀers from that of the previously studied HOXB9n protein and lacks important domains such as homeodomain and hexapeptide motif, its function is likely to diﬀer from that of HOXB9n. Further study is required to re-evaluate the role of HOXB9 in 8 IER3 CAV1 IL6ST EFNA1 TGFB3 BNIP3 PRDX5 SYCP2 PDCD4 SHH RGL2 CREB3L1 BCL6 CEACAM5 DDAH2 THBS1 MUC1 AR SOCS2 MET ANXA1 NR4A1 BAD NTSR1 POR FOXP1 ASCL1 DHRS2 CORO1A UCP2 AAMDC HIPK2 BNIP3L THOC6 TFAP2A TNFAIP3 BMP7 Sample 2 1 0 –1 –2 Sample HOXB9n HOXB9v Scaled Figure 7: Microarray data heatmap of HOXB9n-MCF7 and HOXB9v-MCF7. 4. Discussion Additionally, HOX-PBX interactions involve a short HOX protein motif, the hexapeptide, located upstream of the homeodomain [33]. HXR9 is an 18-amino acid peptide and suppresses tumor proliferation by inhibiting HOX-PBX binding [19]. In our proliferation assay, HOXB9v expressing cell lines were sensitive to the HOX-PBX in- hibitor HXR9, even though HOXB9v lacks the hexapeptide. Tis may be because HOXB9v, in consort with HOXB9n or 9 Journal of Oncology other HOX proteins, indirectly promotes HOX-PBX binding. [6] O.-S. Kwon, E. Oh, J.-R. Park et al., “GalNAc-T14 promotes metastasis through Wnt dependent HOXB9 expression in lung adenocarcinoma,” Oncotarget, vol. 6, no. 39, pp. 41916–41928, 2015. 5. Conclusions [7] T. Hayashida, F. Takahashi, N. Chiba et al., “HOXB9, a gene overexpressed in breast cancer, promotes tumorigenicity and lung metastasis,” Proceedings of the National Academy of Sciences, vol. 107, no. 3, pp. 1100–1105, 2010. We report a modiﬁed HOXB9 mRNA variant that results in a homeodomain defect. We conﬁrmed its presence in human breast cancer cell lines and breast cancer clinical samples and also revealed that HOXB9v may promote breast cancer proliferation by suppression of apoptosis. 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Yamamura, “Analysis of the murine Hoxa-9 cDNA: an alternatively spliced transcript encodes a truncated protein lacking the homeodomain,” Gene, vol. 209, no. 1-2, pp. 77–85, 1998. [26] Y. S. Hong, S. Y. Kim, A. Bhattacharya, D. R. Pratt, W. K. Hong, and M. A. Tainsky, “Structure and function of the HOX A1 human homeobox gene cDNA,” Gene, vol. 159, no. 2, pp. 209–214, 1995. [27] W.-F. Shen, K. Detmer, T. A. Simonitch-Eason, C. Lawrence, and C. Largman, “Alternative splicing of the HOX 2.2 ho- meobox gene in human hematopoietic cells and murine embryonic and adult tissues,” Nucleic Acids Research, vol. 19, no. 3, pp. 539–545, 1991. pp [28] C. V. Wright, K. W. Cho, A. Fritz, E. M. B¨urglin, and E. M. De Robertis, “A Xenopus laevis gene encodes both homeobox-containing and homeobox-less transcripts,” Te EMBO Journal, vol. 6, no. 13, pp. 4083–4094, 1987. [29] N. Shah and S. Sukumar, “Te Hox genes and their roles in oncogenesis,” Nature Reviews Cancer, vol. 10, no. 5, pp. 361–371, 2010. 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https://openalex.org/W2313524073	https://zenodo.org/records/2236067/files/article.pdf	English	null	“Is God's Moral Government Out of Order?” the Burden of Habakkuk	The Baptist review and expositor	1,904	public-domain	4,607	The Burden of Habakkuk. The Burden of Habakkuk. 45 at The University of Iowa Libraries on June 2, 2015 rae.sagepub.com Downloaded from "IS GOD'S MORAL GOVERNMENT OUT OF ORDER?" THE BURDEN OF HABAKKUK. By REv. E. B. POLLARD, PH.D., D.D. Geor~etown, Ky., Formerly Professor in Columbian University. They are sweeping away with irresistible momentum all that stands in their path. Judah cannot hope to escape, unless all human signs fail, or unless God Himself should intervene. The ten tribes, the Northern Kingdom. a little over a century ago (B.C. 722) had been brought low by the Assyrians; Samaria had been razed to the ground, and the flower of the land transported to foreign parts, never again to return. Was Judah now to share the same fate, at the hand of the Babylonian! The in- ternal picture was not a whit less dark. The uncertain- ties without led to confusion within. Nor was King J ehoiakim the man to grapple .with such difficulties. Rather did his weakness and his wickedness tend only to aggravate the perplexity of the situation. For not only did he show a lack of sagacity, by reversing the policy of Josiah, and siding with Pharaoh-Neche, as against Nebuchadnezzar, but also unlike his father, "did that which was evil in the sight of the Lord." Burden- some taxation, oppression, lawlessness and greed ran riot in Judah, find there was no hand to stay. The judges were corrupt and the society utterly demoralized. It was at such a crisis that Habakkuk begins to re- giddo, brought the brief era of righteousness to a sud- den and unhappy close. The reign of Jehoahaz was nipped by Pharaoh-Neche in its very budding, and Jeho- iakim came to the throne, about the year 609 B.C. The political problem of the day for Judah's new king was, how to maintain his government in the midst of the threatening dangers from Babylonia on the one hand and Egypt on the other-the two powers, of the North and of the South, which for years had been contending against each other for supremacy. Poor Judah lay as a prize between the two powerful contestants; or rather was kicked hither and thither as a foot-ball by the ruth- less players. The Babylonians, or Chaldeans, under the sway of the great Nebuchadnezzar, are on the march. They are sweeping away with irresistible momentum all that stands in their path. Judah cannot hope to escape, unless all human signs fail, or unless God Himself should intervene. The ten tribes, the Northern Kingdom. a little over a century ago (B.C. "IS GOD'S MORAL GOVERNMENT OUT OF ORDER?" THE BURDEN OF HABAKKUK. By REv. E. B. POLLARD, PH.D., D.D. Geor~etown, Ky., Formerly Professor in Columbian University. T HE man with a message is the man with an exper- ience. The Old Testament prophets were pre- eminently men of hope. But often it was necessary for them to win their optimism by struggling through the gloom of doubt and fear into the light of an unconquer- able faih, Thus their messages, though born from above, were not foreign to their own souls, having pas- sed through the furnace of their own burning experience. So, the prophecy came forth not only with a warmth and intimacy as respects the prophet himself, but it also became a weight upon his own heart, until he should give his fellowmen the benefit of his own struggle. Hence the deliverances of the prophet, as respects the prophet himself, were an experience; as respects the di- vine will, a message; as respects the needs of men, a burden on the prophet's soul-pressing heavily, until the word of the Lord be told. The prophet Habakkuk had an experience. God made it a message. He discharged the burden. "The burden which Habakkuk the prophet did see!" The message came on this wise:- It had been but a few years since under the reforming hands of Josiah, the King, Hilkiah the priest and Hul- dah the prophetess, idolatry had outwardly at least been ground to powder, and many wholesome efforts at social and religious purification undertaken. The sad end of the vigorous young reformer however, failing as he did in the battle with Pharaoh-Necho on the plains of Me- The Baptist Review and Expositor. 46 giddo, brought the brief era of righteousness to a sud- den and unhappy close. The reign of Jehoahaz was nipped by Pharaoh-Neche in its very budding, and Jeho- iakim came to the throne, about the year 609 B.C. The political problem of the day for Judah's new king was, how to maintain his government in the midst of the threatening dangers from Babylonia on the one hand and Egypt on the other-the two powers, of the North and of the South, which for years had been contending against each other for supremacy. Poor Judah lay as a prize between the two powerful contestants; or rather was kicked hither and thither as a foot-ball by the ruth- less players. The Babylonians, or Chaldeans, under the sway of the great Nebuchadnezzar, are on the march. at The University of Iowa Libraries on June 2, 2015 rae.sagepub.com Downloaded from at The University of Iowa Libraries on June 2, 2015 rae.sagepub.com Downloaded from "IS GOD'S MORAL GOVERNMENT OUT OF ORDER?" THE BURDEN OF HABAKKUK. By REv. E. B. POLLARD, PH.D., D.D. Geor~etown, Ky., Formerly Professor in Columbian University. 722) had been brought low by the Assyrians; Samaria had been razed to the ground, and the flower of the land transported to foreign parts, never again to return. Was Judah now to share the same fate, at the hand of the Babylonian! The in- ternal picture was not a whit less dark. The uncertain- ties without led to confusion within. Nor was King J ehoiakim the man to grapple .with such difficulties. Rather did his weakness and his wickedness tend only to aggravate the perplexity of the situation. For not only did he show a lack of sagacity, by reversing the policy of Josiah, and siding with Pharaoh-Neche, as against Nebuchadnezzar, but also unlike his father, "did that which was evil in the sight of the Lord." Burden- some taxation, oppression, lawlessness and greed ran riot in Judah, find there was no hand to stay. The judges were corrupt and the society utterly demoralized. It was at such a crisis that Habakkuk begins to re- p y y It was at such a crisis that Habakkuk begins to re- flect sadly upon the horrible situation. Is not Jehovah The Burden of Habakkuk. 47 a God of justiceY Why should He permit such conditions to exist ~ Is Israel not His chosen one ~ Why should the late reforms go for nothing,and evil stalk boldly forth trampling the good and holy under its cruel heel-and there be none to deliver? Surely God has not lost His grip upon human affairs, nor His goodness toward His people, that wickedness is allowed to triumph and those that would do good are crushed into the dust. And what of the recent effort at reforming Israel ~ True Josiah's successors were not imbued with his spirit, and there was every indication of the inevitable failure of his reforms; but even at this instant, is not Judah bet- ter than Babylonia, and has the chosen people no stronger claim in Jehovah's favor than the Chaldeans ~ Is the worse to destroy the better; and will God stand calmly by and let the awful work of destruction go on? "IS GOD'S MORAL GOVERNMENT OUT OF ORDER?" THE BURDEN OF HABAKKUK. By REv. E. B. POLLARD, PH.D., D.D. Geor~etown, Ky., Formerly Professor in Columbian University. Can it be that God's moral government is out of order and that the wicked and the heathen are, after all, to inherit the earth t a God of justiceY Why should He permit such conditions to exist ~ Is Israel not His chosen one ~ Why should the late reforms go for nothing,and evil stalk boldly forth trampling the good and holy under its cruel heel-and there be none to deliver? Surely God has not lost His grip upon human affairs, nor His goodness toward His people, that wickedness is allowed to triumph and those that would do good are crushed into the dust. And what of the recent effort at reforming Israel ~ True Josiah's successors were not imbued with his spirit, and there was every indication of the inevitable failure of his reforms; but even at this instant, is not Judah bet- ter than Babylonia, and has the chosen people no stronger claim in Jehovah's favor than the Chaldeans ~ Is the worse to destroy the better; and will God stand calmly by and let the awful work of destruction go on? Can it be that God's moral government is out of order and that the wicked and the heathen are, after all, to inherit the earth t It is with such thoughts as these raging in his bosom that Habakkuk makes his appearance before us. Gen- erally we first see prophets of the Lord uttering their unmistakable message to the people. Habakkuk, how- ever, leads us through the very processes by which God gives him his 'burden' to the world. There is a tradi- tion that Habakkuk was a priest, as well as a prophet. Certainly he had the true priestly heart Indeed, in that portion of Habakkuk's work which he has left us in this book which bears his name, it is the priest that speaks more than the prophet. Here we find him 'Standing not so much before the people on behalf of God, but before God on behalf of His people. It is while he is struggling in priestly solicitude and intercession for his countrymen that God gives him a revelation which first quiets his own soul and then comes as a message of hope and sted- fastness to others. Habakkuk's struggle with his peo- ple '8 problem seemed to come to him almost as an inner The Baptist Review and Expositor. at The University of Iowa Libraries on June 2, 2015 rae.sagepub.com Downloaded from "IS GOD'S MORAL GOVERNMENT OUT OF ORDER?" THE BURDEN OF HABAKKUK. By REv. E. B. POLLARD, PH.D., D.D. Geor~etown, Ky., Formerly Professor in Columbian University. 48 tragedy. Scepticism and Pessimism stalk over the stage of his soul. 'I'he spirits of Atheism and Despair hover over the scene. Shall they in the next act, take complete possession of his soul l No. Habakkuk and God have won land the prophet gives the history of the struggle to victory in the boldly dramatic form in which it passed through his own life. Habakkuk: "0, Lord how long shall I cry (for the deliverance of my people) and thou will not hear7 I cry out of violence and thou wilt not save. Why dost thou show me iniquity and cause me to look upon perverseness, for spoiling and violence are before ine: and there is strife, and contention riseth up. Therefore the law is slacked, and judgment never goeth forth: for the wicked doth compass about the righteous, therefore judgment goeth forth preverted." (Chap.I :1-4.) Jehovah: "Behold ye among the nations and regard and wonder marvelously, for I work a work in your days which ye will not believe though it were told you. For 10, I am raising up the Chaldeans, that bitter and hasty nation, which march through the breadth of the earth to possess dwelling places not their own. They are ter- rible and dreadful, their judgment and their dignity pro- ceed from themselves. Their horses are swifter than leopards, and are more fierce than evening wolves; and their horsemen spread themselves, yea, their horsemen come from far, they fly as an eagle that hasteth to devour. 'I'hey come all of them for violence; their faces are set eagerly as the east wind; and they gather captives as the sand. Yea, he scoffeth at kings, and princes are a derision unto him: he derideth every strong-hold, he heapeth up dust and taketh it. Then shall he sweep by as a wind, and shall pass over and be guilty: even he whose might is his god." But how can Habakkuk '8 questioning mind be satis- fied with such a reply f He knows all too well about the cruel and oppressive Chaldean, Does it help matters 49 The Burden of Habakkuk. at The University of Iowa Libraries on June 2, 2015 rae.sagepub.com Downloaded from "IS GOD'S MORAL GOVERNMENT OUT OF ORDER?" THE BURDEN OF HABAKKUK. By REv. E. B. POLLARD, PH.D., D.D. Geor~etown, Ky., Formerly Professor in Columbian University. First, Habakkuk is told to remember that wickedness, even when it seems strongest, is weakness, it will surely be destroyed. Second, that in the midst of evil, the salva- tion of the just is in his steadfast faith. Should God's moral government be regarded as out of order because wickedness stalks proudly over the prostrate form of righteousness ~ No, for evil will 'yet meet its overthrow, and the righteous man, in his faithfulness, standeth sure! "Behold, his soul (the wicked) is puffed up, it is not upright in him, but the just shall live by his faith," v, 4. The proud and defiant Chaldean, type of all self-reliant wickedness in the world, drunken with lust and fury, large as hell, shall meet his moral waterloo, and all na- tions shall see his defeat and sing the five-fold song taunting and of woe over his fall : Woe to him that in- creaseth that which is not his ownI" " Woe to him that getteth an evil gain for his house, that he may set his nest on high." "Woe to him that buildeth a town with blood, and establisheth a city with iniquity." " Woe to him that giveth his neighbor drink and addeth thy venom thereto. " " Woe to him who saith to the wood, awake; to the dumb stone, arise!', No idle god can come to the rescue when Jehovah has decreed the utter overthrow of the wicked! He has sought for it from heaven. He has stood and waited, till it came. The answer from Jehovah is given in Chap. 2 :4-20. It is composed of two parts. First, Habakkuk is told to remember that wickedness, even when it seems strongest, is weakness, it will surely be destroyed. Second, that in the midst of evil, the salva- tion of the just is in his steadfast faith. Should God's moral government be regarded as out of order because wickedness stalks proudly over the prostrate form of righteousness ~ No, for evil will 'yet meet its overthrow, and the righteous man, in his faithfulness, standeth sure! "Behold, his soul (the wicked) is puffed up, it is not upright in him, but the just shall live by his faith," v, 4. "IS GOD'S MORAL GOVERNMENT OUT OF ORDER?" THE BURDEN OF HABAKKUK. By REv. E. B. POLLARD, PH.D., D.D. Geor~etown, Ky., Formerly Professor in Columbian University. to know that God is raising him up, and that the internal sins of which the prophet has complained will be avenged by so wicked and relentless a foe from withoutt There is surely little consolation in the thought that Judah for her wrong doing is to be punished and plundered by the still more wicked Chaldean, even though Jehovah shall 'regard the invader as guilty. And so Habakkuk dares again to answer. Habakkuk: "0 Lord, my God, my Holy One, we shall not die 1 0 Lord thou hast ordained him for judgment, and thou, 0 Rock, hath established correction. Thou that art of purer eyes than to behold evil and that canst not look upon perverseness, wherefore then lookest thou up- on them that deal treacherously, and boldest thy peace when the wicked swallow up the man that is more right- eous than he, and maketh them as the fishes of the sea, or creeping things that have no ruler over themT•••••• Shall he therefore empty his net) and not spare to slay the nations continuallyT" Anxious to know how Jehovah will answer the question why even the wicked should be punished by being given over to the plunderer of the more wicked, Habakkuk says to himself: "I will stand upon my watch, and set me upon my tower and will look forth to see what he will speak with me, and what I shall answer concerning my complaint." Ashe waited the Lord came in a vision which said: Jehovah: "Write the vision out and make it plain upon tables that he may run that readeth it., For the vision is yet for the appointed; and it hasteth toward the end and shall not lie. Though it tarry, wait for it, because it will surely come; it will not delay." God always gives vision to the honest seeker after light. The "more faith in honest doubt" than lives in unfelt creeds is always rewarded. "Lord I believe, help thou my unbelief." With this attitude of mind, Habakkuk receives the reply which is to quiet his soul, 50 The Baptist Review and Expositor. He has sought for it from heaven. He has stood and waited, till it came. The answer from Jehovah is given in Chap. 2 :4-20. It is composed of two parts. at The University of Iowa Libraries on June 2, 2015 rae.sagepub.com Downloaded from "IS GOD'S MORAL GOVERNMENT OUT OF ORDER?" THE BURDEN OF HABAKKUK. By REv. E. B. POLLARD, PH.D., D.D. Geor~etown, Ky., Formerly Professor in Columbian University. The proud and defiant Chaldean, type of all self-reliant wickedness in the world, drunken with lust and fury, large as hell, shall meet his moral waterloo, and all na- tions shall see his defeat and sing the five-fold song taunting and of woe over his fall : Woe to him that in- creaseth that which is not his ownI" " Woe to him that getteth an evil gain for his house, that he may set his nest on high." "Woe to him that buildeth a town with blood, and establisheth a city with iniquity." " Woe to him that giveth his neighbor drink and addeth thy venom thereto. " " Woe to him who saith to the wood, awake; to the dumb stone, arise!', No idle god can come to the rescue when Jehovah has decreed the utter overthrow of the wicked! The question at the first raised by Habakkuk, "How long?" is not answered, to be sure. But the prophet sees the point that God would teach him: ''I will surely over- throw the wicked, TRUST ME!" Habakkuk now bursts out in a wondrous confession and prayer. (Chapter III.) God has. spoken, and the prophet's soul is now calm. For he now remembers well that Jehovah's past deal- ing with the world has always given assurance both of his power and his goodness. In the light of history, the 51 The Burden of Habakkuk. good prophet is ashamed that he could ever have doubt- ed.. "0 Lord, I have heard the report of thee and am afraid. "0 Lord, I have heard the report of thee and am afraid. o Lord, revive thy work in the midst of the years; I th id t f th k it k o Lord, revive thy work in the midst of the In the midst of the years make it known; o Lord, revive thy work in the midst of t In the midst of the years make it known; In the midst of the years make it known; y In wrath, remember mercy." In wrath, remember mercy." The quieted prophet calls to mind how God led his people through the wilderness, appearing from Teman and Mount Paran; how the tents of Cushan in the land of Midian were overthrown for the sake of his people. Both in history (vv. 3-7) and in nature (vv .. at The University of Iowa Libraries on June 2, 2015 rae.sagepub.com Downloaded from "IS GOD'S MORAL GOVERNMENT OUT OF ORDER?" THE BURDEN OF HABAKKUK. By REv. E. B. POLLARD, PH.D., D.D. Geor~etown, Ky., Formerly Professor in Columbian University. 8-16) the Lord has shown His ability and His willingness to aid His faithful people against the foe. He cannot fail the people now! "Thy bow was made quite bare, Thine oaths to the tribes was a sure word. Thou didst cleave the earth with rivers, The mountains saw thee and were afraid. "Thy bow was made quite bare, Thine oaths to the tribes was a sure word. Thou didst cleave the earth with rivers, The mountains saw thee and were afraid. The mountains saw thee and were afraid. The mountains saw thee and were afraid. The sun and moon stood still in their habitation At the light of thine arrows as they went, At the shining of thy glittering spear. Thou didst march through the land in indignation, Thou didst thresh the nations in anger. Thou wentest forth for the salvation of thy people. For the salvation of thine anointed. Thou woundedst the head out of the house of the wicked The sun and moon stood still in their habitation At the light of thine arrows as they went, At the shining of thy glittering spear. Thou didst march through the land in indignation, Thou didst thresh the nations in anger. Thou wentest forth for the salvation of thy people. For the salvation of thine anointed. Thou woundedst the head out of the house of the wicked The sun and moon stood still in their habitation Thou woundedst the head out of the house of the wicked Laying bare the foundation even to the neck." Laying bare the foundation even to the neck." What God has done he will do. History confirms His promise to save the faithful and destroy the evil-doer. What God has done he will do. History confirms His promise to save the faithful and destroy the evil-doer. Habakkuk's calmness and his confidence are now com- plete. He has learned the lesson, for the want of which his soul was but lately shrouded in gloom. And this is the lesson: p y Habakkuk's calmness and his confidence are now com- plete. He has learned the lesson, for the want of which his soul was but lately shrouded in gloom. And this is the lesson: 52 The Baptist Review and Expositor. at The University of Iowa Libraries on June 2, 2015 rae.sagepub.com Downloaded from "IS GOD'S MORAL GOVERNMENT OUT OF ORDER?" THE BURDEN OF HABAKKUK. By REv. E. B. POLLARD, PH.D., D.D. Geor~etown, Ky., Formerly Professor in Columbian University. "That I should rest in the day of trouble, "That I should rest in the day of trouble, When it cometh upon the people to invade them in troops." p Hence he breaks out with the robust faith of one who can leave all with God: Hence he breaks out with the robust faith of one who can leave all with God: ,'Though the fig tree should not blossom ,'Though the fig tree should not blossom Neither shall fruit be in the vines ; The labor of the olive may fail, And the fields yield no meat, The flocks may be cut off from the fold, And there be no herd in the stalls, Yet will I rejoice in the Lord, I will joy in the God of my salvation. Jehovah, the Lord, is my strength, He maketh my feet like the hind's, And will make me to walk upon my high places." This remarkably beautiful prophecy, so full of moral earnestness and dramatic power, has carried in its own bosom its lessons. They need not be further drawn out. But in our own age, or indeed, in every age, we need to learn afresh the chief teachings in Habakkuk's message from heaven. The problems of sin, and of suffering. are the most momentus with which we have to deal. Why sin is permitted in the world, why the righteous suffer, and kindred questions are left unanswered, some lessons ring out clear and distinct. g First, God can be trusted. In all the perplexing moral problems that arise in the manifold experiences of men and nations, the Almighty is worthy of all confidence. His government is not hopelessly deranged even when it seems most so. His hand is on the helm. Many times He is compelled to say to those of his creatures who have lost confidence in moral order and would passion- ately fly into complaint and displeasure with God, just as Emerson says Nature would remark to some-"Why so hot my little man?" Or as Jehovah himself aforetime 53 The Burden of Habakkuk. said to His doubting people, "Be still, and know that I am God." Not only so, but the very life of the righteous lies in his faith in the Almighty. Confidence in God is his only hope, his stay, his salvation. "The just shall live by his faith, " 2:4. at The University of Iowa Libraries on June 2, 2015 rae.sagepub.com Downloaded from at The University of Iowa Libraries on June 2, 2015 rae.sagepub.com Downloaded from "IS GOD'S MORAL GOVERNMENT OUT OF ORDER?" THE BURDEN OF HABAKKUK. By REv. E. B. POLLARD, PH.D., D.D. Geor~etown, Ky., Formerly Professor in Columbian University. In contrast with the pride and arrogance of the Chaldean whose "soul is puffed up within him," and whose "dignity proceedeth from himself," the right- eous ceasing from all confidence in his own strength re- lies wholly upon God. Turning from human assistance he seeks the divine power to save him from calamity and sin. The past faithfulness of God has begotten faith and steadfastness in his own soul. He can therefore af- ford to "rest in the day of trouble," being assured that his- soul and God stand sure. But the voice of Jehovah brought for the prophet another message. It "is the lesson of the inherent weak- ness of sin. The Lord pointed out to Habakkuk the fact that, notwithstanding the apparent successes of the Chal- dean by which he had made his "portion fat and his meat plenteous," destruction awaited "him; that sin contains the very germs of its own dissolution, the pledge of its own undoing. The wicked man "hath sinned against his own soul," Chap. 2:10. "Woe! Woe! Woe!" is written upon every line of the sinful programme. God has set himself against wrong, even when he gives it reins. His very character is ample warrant that sin shall be overthrown; that when it is finished it will bring forth death. "For right is right, and God is God, "For right is right, and God is God, An right the day must win; To doubt would be disloyalty, To falter would be sin." God's moral government is found therefore not to be a chaos, simply because evil seems to be triumphant and shameless. God will at last overthrow sin and save those that trust in him. 54: The Baptist Review and Expositor. The Baptist Review and Expositor. The Baptist Review and Expositor. What confidence should this give to those who would battle for the right, and against the wrong. Sin shall be destroyed and "the just shall live by faith." It was this word spoken to Habakkuk that became the central and sustaining thought of Paul's message, a gospel,which had to be preached in the midst of bitterest persecution. It was this that became the keynote to Luther's life and the Reformation which he instituted. It still brings us to the center of the gospel for a lost world. "The just shall live by faith."
https://openalex.org/W2168015727	http://escholarship.mcgill.ca/downloads/bv73c521w	English	null	Long-Lasting Rescue of Age-Associated Deficits in Cognition and the CNS Cholinergic Phenotype by a Partial Agonist Peptidomimetic Ligand of TrkA	The journal of neuroscience/The Journal of neuroscience	2,004	cc-by	10,147	Received June 25, 2003; revised July 29, 2004; accepted Aug. 3, 2004. This work was supported by Canadian Institutes of Health Research Grants MT-13265 (H.U.S.) and MOP 62735 (A.C.C.)andNationalInstitutesofHealthGrantCA82642(K.B.).M.A.B.receivedafellowshipfromConsejoNacional de Investigaciones Cientı´ficas y Te´cnicas, Argentina. We thank Dr. H. Nedev and M. C. Zaccaro (McGill University, Montreal, Quebec, Canada) for aid with compound synthesis and characterization and K. Bell and E. Tsang for aid with image analysis and histology. A.C.C. and H.U.S. contributed equally to this work. Correspondence should be addressed to Dr. H. Uri Saragovi, Lady Davis Institute–Jewish General Hospital, 3755 Cote St. Catherine, F-223, Montreal, Quebec, H3T 1E2 Canada. E-mail: Uri@pharma.mcgill.ca. DOI:10.1523/JNEUROSCI.1508-04.2004 Copyright © 2004 Society for Neuroscience 0270-6474/04/248009-10$15.00/0 The Journal of Neuroscience, September 15, 2004 • 24(37):8009–8018 • 8009 The Journal of Neuroscience, September 15, 2004 • 24(37):8009–8018 • 8009 Behavioral/Systems/Cognitive Long-Lasting Rescue of Age-Associated Deficits in Cognition and the CNS Cholinergic Phenotype by a Partial Agonist Peptidomimetic Ligand of TrkA Bruno,1 Paul B. S. Clarke,1 Alicia Seltzer,6 Re´mi Quirion,1,2 Kevin Burgess,7 A. Claudio Cuello,1,3 i1 4 5 Martin A. Bruno,1 Paul B. S. Clarke,1 Alicia Seltzer,6 Re´mi Quirion,1,2 Kevin Burgess,7 A. Clau H. Uri Saragovi1,4,5* Martin A. Bruno,1 Paul B. S. Clarke,1 Alicia Seltzer,6 Re´mi Quirion,1,2 Kevin Burgess,7 A. Claudio Cuello,1,3* and H. Uri Saragovi1,4,5* Departments of 1Pharmacology and Therapeutics, 2Psychiatry/Douglas Hospital Research Center, 3Anatomy and Cell Biology, and 4Oncology/Cancer Center, and 5Lady Davis Research Institute, McGill University, Montre´al, Quebec, H3T 1E2 Canada, 6Facultad de Medicina, Universidad de Cuyo, Mendoza 5500, Argentina, and 7Department of Chemistry, Texas A&M University, College Station, Texas 77842 g Departments of 1Pharmacology and Therapeutics, 2Psychiatry/Douglas Hospital Research Center, 3Anatomy and Cell Biology, and 4Oncology/Cancer Center, and 5Lady Davis Research Institute, McGill University, Montre´al, Quebec, H3T 1E2 Canada, 6Facultad de Medicina, Universidad de Cuyo, Mendoza 5500, Argentina, and 7Department of Chemistry, Texas A&M University, College Station, Texas 77842 Previously, we developed a proteolytically stable small molecule peptidomimetic termed D3 as a selective ligand of the extracellular domain of the TrkA receptor for the NGF. Ex vivo D3 was defined as a selective, partial TrkA agonist. Here, the in vivo efficacy of D3 as a potential therapeutic for cholinergic neurons was tested in cognitively impaired aged rats, and we compared the consequence of partial TrkAactivation(D3)versusfullTrkA/p75activation(NGF).WeshowthatinvivoD3bindstoTrkAreceptorsandaffordsasignificantand long-lived phenotypic rescue of the cholinergic phenotype both in the cortex and in the nucleus basalis. The cholinergic rescue was selectiveandcorrelateswithasignificantimprovementofmemory/learningincognitivelyimpairedagedrats.Theeffectsofthesynthetic ligand D3 and the natural ligand NGF were comparable. Small, proteolytically stable ligands with selective agonistic activity at a growth factor receptor may have therapeutic potential for neurodegenerative disorders. Key words: peptidomimetic; age; cognition; memory; TrkA; NGF; agonist; cholinergic; CNS Key words: peptidomimetic; age; cognition; memory; TrkA; NGF; agonist; cholinergic; CNS (Barker, 1998; Rabizadeh et al., 1999; Zaccaro et al., 2001; Sara- govi and Zaccaro, 2002). These features make p75 biology com- plex and the behavior of p75 ligands unpredictable. Received June 25, 2003; revised July 29, 2004; accepted Aug. 3, 2004. D3 distribution in brain D3 and its analog D3-biotin (D3 conjugated with biotin) bind TrkA with similar properties (Maliartchouk et al., 2000), and D3-biotin can be fol- lowed via the biotin label (Maliartchouk et al., 2000). To assess whether D3 can diffuse when given intracerebroventricularly, male Fischer-344 aged rats (n 2) were anesthetized, placed in a stereotaxic apparatus, and given a single injection of D3-biotin. A relatively high dose of D3-biotin (10 g in 10 l) was used because our pilot studies (data not shown) demonstrated it yields a good signal in these diffusion studies. D3-biotin was injected into the lateral ventricle (anteroposterior, 0.8 mm; lateral, 1.3 mm; vertical, 3.5 mm) (Paxinos and Watson, 1986). Without perfu- sion (to avoid washout during perfusion–fixation), rats were killed after an intracerebroventricular injection at 0.25, 2, 4, 8, 12, and 24 hr. Fixed (4% paraformaldehyde; 15 min), free-floating sections (50 m) were cut using a freezing sledge microtome, followed by 30 min of blocking with 3% normal mouse serum (Chemicon, Temecula, CA). After rinsing, sec- tions were incubated at room temperature for 2 hr with a mouse IgG anti-biotin–Alexa-488 (1:300; Molecular Probes, Eugene, OR). Sections were rinsed three times with PBS and then mounted onto gelatin-coated slides using Vectashield mounting medium for fluorescence (Vector Laboratories, Burlingame, CA). The latency to locate the escape platform was used to segregate rats into aged cognitively unimpaired and aged cognitively impaired groups. Aged rats whose individual mean latencies to locate the platform were within 0.5 SDs of the latency of young rats were considered cognitively unimpaired (n 19). Aged rats whose individual average latencies to locate the platform were 2 SDs from those of young rats on days 2–5 of the acquisition phase were considered cognitively impaired (n 31), as described (Tombaugh et al., 2002; Rowe et al., 2003). Rats displaying an intermediate performance between unimpaired and impaired (i.e., rats with 2 SDs but 0.5 SDs) were eliminated from this study (n 70) because this “cognitively mildly impaired population” was expected to yield highly variable behavioral data. Testing phase. Throughout, all tests were always carried in the same room and the same Morris water maze was set up. D3 distribution in brain Aged cognitively impaired rats were treated with saline, NGF, or D3 (see below) during 2 weeks, allowed to recover from surgery for 1 week (week 3), and then they were retested in the Morris water maze (week 4) with the platform sub- merged in the same position as in the acquisition phase. Animals were tested in nine trials over 3 consecutive days (three trials per day with an intertrial time of 20 min) with the platform 2 cm below the water. The latency of each aged cognitively impaired group (vehicle, NGF, or D3) was also analyzed as the average time spent in each quadrant of the pool using the same software program (HVS Image). Cellular distribution and D3–TrkA localization studies Aged rats (n 4) received a single intracerebroventricular injection of D3-biotin (1 g in 10 l), and 24 hr later, their brains were perfused– fixed (24 hr because the biodistribution studies above indicated good penetration). Then, 35-m-thick sections containing the nucleus basalis and cerebral cortex regions were prepared, and double immunostaining was done to identify neurons, glia, or TrkA receptors. Neurons were identified with mouse anti-neural nuclei (NeuN) antibody (1:100; Chemicon), followed by goat anti-mouse coupled with rhodamine (1: 100; red; Jackson ImmunoResearch, West Grove, PA), and D3-biotin on neurons was identified with goat anti-biotin–FITC (green; Sigma, St. Louis, MO). Glia were identified using mouse anti-glial fibrillary acidic protein (GFAP; 1:100; Chemicon), followed by goat anti-mouse coupled with rhodamine (1:100; red; Jackson ImmunoResearch), and D3-biotin on glia was identified with goat anti-biotin–FITC (green; Sigma). TrkA receptors were identified using rabbit anti-TrkA antibody (1:500; Chemicon), followed by donkey anti-rabbit coupled with rhodamine red X (1:100; red; Jackson ImmunoResearch), and D3-biotin colocalizing with TrkA was identified with mouse IgG anti-biotin–Alexa-488 (1:300; green; Molecular Probes). In all cases, sections were preincubated and blocked with the corresponding normal serum secondary antibody. The colocalization of D3-biotin and neuronal, glial, or TrkA markers was examined with an LSM 510 laser scanning confocal microscope (Zeiss, Oberkochen, Germany). Memory test. At the 5th and 12th weeks after drug delivery, rats were placed in the water maze with no platform, for three trials of 60 sec with an intertrial interval of 20 min in a single day. The data are expressed as percentage of time spent in each quadrant SEM, the “target quadrant” being where the platform had been hidden before. Introduction • Age-Associated Deficits Rescued by a Mimetic TrkA Ligand 8010 • J. Neurosci., September 15, 2004 • 24(37):8009–8018 that bind TrkA and exclude p75. To this end, we developed a TrkA-selective NGF peptidomimetic termed D3. D3 induces TrkA tyrosine phosphorylation, stabilizes TrkA–TrkA ho- modimers, enhances ChAT production/activity, and induces neurotrophic activities in PC12 cells and primary neuronal cul- tures (Maliartchouk et al., 2000). D3 has partial agonistic activity but could be best described as a potentiator of NGF action. In addition, D3 is a small (mass, 580 Da), water-soluble and proteo- lytically stable molecule. In view of these drug-like features, we tested whether D3 has superior in vivo pharmacological proper- ties compared with the NGF polypeptide. water maze test (Fischer et al., 1987; Gage et al., 1988; Chen et al., 1995), to avoid the potential confounding effects of studying drug effects on a global and heterogeneous population of aged rats. Acquisition phase. Aged (n 120) and young (n 10) rats were screened for spatial memory deficits using the Morris water maze as described previously (Issa et al., 1990; Aubert et al., 1995). The animals were required to find a submerged platform in a 1.4 m diameter pool of white, nontoxic colored water using only distal and spatial clues available in the testing room. Throughout, all tests were always carried in the same room and set up. The center of the escape platform (15 cm diameter) was located 45 cm from the pool wall, in the northeast quadrant. Animals were tested in 15 trials over 5 consecutive days (three trials per day with an intertrial time of 20 min) with the platform 2 cm below the water. At the end of the testing periods, all animals were given three trials in which the platform was raised 2 cm above the water to exclude visual deficits as the cause of poor performance, as described (Rowe et al., 2003). The swim speeds and distances were recorded using a video tracking system (HVS Image, Buckingham, UK) to exclude motor deficits as the cause of poor performance, as described (Rowe et al., 2003). For excluding thig- motaxis and amotivational status as the cause of poor performance (Gra- ziano et al., 2003; Rowe et al., 2003), concentric zones of the pool defined by the software program (HVS Image) were used to average swim pat- terns during days 2–5 of the acquisition phase. Introduction Neurotrophins are growth factors that regulate the development and maintenance of the peripheral and the central nervous sys- tems (Lewin and Barde, 1996; Saragovi and Burgess, 1999). Neu- rotrophins and their receptors are targets for therapeutic inter- vention in neurodegeneration (Eide et al., 1993; Hefti, 1994; Thoenen, 1995; Saragovi and Burgess, 1999; Saragovi and Ge- hring, 2000). Mature cholinergic basal forebrain (CBF) neurons, which in- nervate the cerebral cortex and hippocampus, play a key role in cognition and memory (Lad et al., 2003). These neurons express NGF receptors (Backman et al., 1997) and are dependent for their health and cholinergic phenotype on NGF receptor activation (Debeir et al., 1999; Sofroniew et al., 2001). In aging and cognitive disorders such as Alzheimer’s disease (AD), CBF neurons un- dergo atrophy and eventually degenerate (Turrini et al., 2001; Casu et al., 2002). This process correlates with a progressive re- duction of TrkA density before cellular death (Mufson et al., 1997, 2000; Sendera et al., 2000). Indeed, TrkA (but not of p75) depletion is a reliable marker of progressively deteriorating cog- nitive ability (Counts et al., 2004). Thus, exogenous NGF has been used as an experimental therapeutic agent in aging and stroke animal models (Garofalo et al., 1992; Backman et al., 1997; Frick et al., 1997) and in human AD patients (Gage et al., 1988). Exogenous NGF reversed the age-dependent changes in CBF neurons and improved performance in spatial memory tasks. The effect of NGF was long-lived after discontinuation of deliv- ery. However, NGF therapy for AD patients failed because of CNS delivery problems and pleiotropic and adverse side effects (Bari- naga, 1994; Verrall, 1994; Jonhagen, 2000). Moreover, it is hard to interpret the outcome of NGF–p75 interactions in vivo in the aged brain. The neurotrophin nerve growth factor (NGF) acts by binding to receptors TrkA and p75. NGF–TrkA binding (approximate Kd, 10 11 M) activates the intrinsic tyrosine kinase of the receptor, causing tyrosine phosphorylation of TrkA and associated signal- ing partners, and activates “traditional” neurotrophic biological signals. These signals promote cell survival or differentiation (Kaplan and Miller, 2000). The p75 receptor is a member of the tumor necrosis factor receptor superfamily. Depending on the cellular environment and the type of ligand, p75 can transduce pro-survival, pro-apoptotic, or pro-differentiation signals We reasoned that it would be advantageous to apply ligands Bruno et al. Materials and Methods Animals Aged (24 months old) and young (6 months old) male Fisher-344 rats were used. Efforts were made to minimize the number of animals used and their suffering. All procedures were approved beforehand by the Animal Care Committee of McGill University and followed the guide- lines of the Canadian Institutes of Health Research. D3 distribution in brain This experiment is reflective of memory because there is no new learning. Surgical procedures and CNS administration Cognitively impaired rats were anesthetized and placed in a stereotaxic apparatus (David Kopf Instruments, Tujunga, CA). They were im- planted with cannulas (Alzet) into the lateral ventricle, according to the following coordinates from bregma: anteroposterior, 0.8 mm; lateral, 1.3 mm; vertical, 3.5 mm (Paxinos and Watson, 1986). The cannulas were connected to sterile coiled polyethylene tubing filled with an air–oil spacer (Vahlsing et al., 1989; Varon et al., 1989) at the pump end. Rats receiving control vehicle (saline group; n 8) had pumps filled with phosphate-buffered artificial CSF (150 mM NaCl, 1.8 mM CaCl2, 1.2 mM MgSO4, 2 mM K2HPO4, 10 mM glucose, and 0.001% rat serum, pH 7.4). Rats receiving positive control (NGF group; n 7) had 1.4 g/d of 2.5 S NGF (Cedarlane Laboratories, Ontario, Canada) diluted in saline vehicle (a total of 20 g NGF). This concentration of NGF was established previously to be neuroprotective in dose–response studies in vivo (Garo- falo and Cuello, 1995). Rats receiving test peptidomimetic (D3 group; Behavioral task We resorted to segregating animals in spatially cognitively impaired and unimpaired groups applying well accepted protocols using the Morris J. Neurosci., September 15, 2004 • 24(37):8009–8018 • 8011 Bruno et al. • Age-Associated Deficits Rescued by a Mimetic TrkA Ligand Figure1. PeptidomimeticD3efficientlypenetratestheCNSparenchyma.Imagesofthelateralventricleoffresh,unfixedtissue afterD3-biotininjectionattimepoints:15min(A);2hr(B);4hr(C);8hr(D);12hr(E);24hr(F).At2hrafterinjection,D3-biotin started to penetrate the CNS parenchyma and gave a maximal signal at the 8 and 12 hr. At 24 hr, no D3-biotin is apparent in the choroid plexus or ventricular surface. Scale bar, 200 m. lateral, 4.7–5.7 mm; ventral, 3.0–4.2 mm) and the size and density of the VAChT-IR neurons at the nucleus basalis (lateral, 2.3–3.1 mm; an- teroposterior, 1.2 to 1.8 mm; ventral, 6.3– 7.3 mm) per 1000 m 2 were measured using a BH-2 Olympus microscope connected to an image analysis system (MCID Elite; Imaging Research, St. Catharines, Ontario, Canada) as described (Debeir et al., 1999). Briefly, five im- ages per area, from both hemispheres, were taken from five different slices per rat (50 im- ages per rats for each region basalis and cortex). For lamina V of the parietal cortex and the nu- cleus basalis, five animals were used in each group: young adult, aged unimpaired, aged im- paired, aged impaired plus saline, and aged im- paired plus D3. For the NGF group, 4 animals were analyzed. D3 distribution in brain Because our previous studies have shown that sham surgeries (no infusion of saline control) on mature or in aged rats have no impact on behavior or basal forebrain cho- linergic markers (Garofalo and Cuello, 1994, 1995; Garofalo, 1995), we opted to forgo these additional surgical controls. For quantification of lamina V of the parietal cortex, a total area of 80,000 m 2 were analyzed per rat. For the nu- cleus basalis, a total of 50,000 m 2 were ana- lyzed per rat. Results are expressed per 1000 m 2. For analyses of cell size, a total of 10,000 VAChT-IR cells per rat were studied. Figure1. PeptidomimeticD3efficientlypenetratestheCNSparenchyma.Imagesofthelateralventricleoffresh,unfixedtissue afterD3-biotininjectionattimepoints:15min(A);2hr(B);4hr(C);8hr(D);12hr(E);24hr(F).At2hrafterinjection,D3-biotin started to penetrate the CNS parenchyma and gave a maximal signal at the 8 and 12 hr. At 24 hr, no D3-biotin is apparent in the choroid plexus or ventricular surface. Scale bar, 200 m. n 7) had 2.8 g/d D3 diluted in saline vehicle (a total of 40 g of D3). This concentration of D3 was chosen based on its potency and affinity relative to NGF. The tubing was connected from its oil spacer end to Alzet 2002 (14 d life span) osmotic minipumps filled with dye (0.1% methylene blue), which were pretested to confirm their delivery rates. After the end of thedrugdelivery,thepumpplacedsubcutaneouslyintheneck/shoulderarea and the tubing were removed from anesthetized rats, and the total infusion volumewasdeterminedforeachanimaltoconfirmthedosageadministered. Animalswereallowedtorestfor1week,andthetestingphaseofthememory task was performed as described above. Preparation of D3 and NGF D3 and D3-biotin were prepared as described (Maliartchouk et al., 2000). The material was analyzed as homogeneous single peak by analytical HPLC, by matrix-assisted desorption ionization mass spectrometry, and by 1H NMR (300 MHz; DMSO-d6) (Pattarawarapan and Burgess, 2000; Pattarawarapan et al., 2002), and the expected structure was confirmed. D3 was prepared in saline buffer at 3.5 mg/ml. 2.5 S NGF (Cedarlane Laboratories, Ontario, Canada) was prepared in saline buffer. Phenotypic analyses Statistical analyses were performed using commercial software (Systat 10.0; SPSS Inc., Chicago, IL). Data were subjected to univariate and multivariate ANOVA; for repeated measures, the Huyhn-Feldt p value is reported. Between-group comparisons were made using Tukey’s test. All probability values were two-tailed; a level of 5% was considered signifi- cant. Data are reported as the mean SEM. yp y After completion of the behavioral studies, rats were anesthetized and perfused transcardially with a mixture of 4% paraformaldehyde, 0.5% glutaraldehyde, and 15% saturated picric acid in 0.1 M phosphate buffer (PB), pH 7.4, for 30 min, followed by the same fixative without glutaral- dehyde for an additional 30 min. The tissue was subsequently postfixed for 4 hr in the latter fixative mixture and left at 4°C with a solution of 30% sucrose in 0.1 M PBS. Then, 35-m-thick sections between coordinates from bregma (0.5 mm and 3.0 mm, which comprise the parietal region of the cerebral cortex and the nucleus basalis area) (Paxinos and Watson, 1986) were cut using a sledge microtome (Sliding Microtome, SM2000 R; Leica Microsystems, Quebec, Canada) equipped with a freezing stage. After rinsing in PBS, the free-floating sections were incubated for 1 hr in a solution containing 1% bovine serum and 10% normal goat serum in PB. Sections were then incubated overnight at 4°C with an anti- vesicular acetylcholine transporter (VAChT) antibody (1:10,000; a gift from Dr. R. Edwards, University of California–San Francisco, San Francisco, CA) or anti-synaptophysin antibody (1:200) (Gilmor et al., 1996) as described. After washing, the tissue was incubated in biotinylated goat anti-rabbit antibody (1:800; 2 hr at room temperature; Vector Laboratories). After several washes, the tissue was incubated in 0.6% diaminobenzidine (Sigma) in Tween 20 in PBS (15 min at room temperature). Subse- quently, H2O2 was added to the diaminobenzidine solution. After wash- ing, the sections were mounted on gelatin-coated glass slides and were dehydrated and coverslipped with Entellan (Merck, Darmstadt, Ger- many). Omission of the primary antibody served as a negative control. Results Th D The D3 peptidomimetic TrkA ligand distributes throughout the CNS parenchyma and colocalizes with the TrkA receptor D3-biotin was given intracerebroventricularly to assess whether small peptidomimetics penetrate the CNS parenchyma effi- ciently. After an intracerebroventricular injection of D3-biotin (10 g), rats were killed without perfusion at times 0.25, 2, 4, 8, 12, and 24 hr and stained with mouse anti-biotin–Alexa-488 (Fig. 1). At 2 hr (Fig. 1B) and 4 hr (Fig. 1C) after injection, D3-biotin started to penetrate the CNS parenchyma. A maximal signal is seen at the 8 hr (Fig. 1D) and 12 hr (Fig. 1E) time points. At 24 hr (Fig. 1F), no signal is apparent in the choroid plexus or ventric- ular surface. Hence, D3-biotin is efficient at penetrating brain parenchyma. The D3 peptidomimetic TrkA ligand distributes throughout the CNS parenchyma and colocalizes with the TrkA receptor More detailed colocalization studies were performed using D3-biotin and subjecting the rats to a perfusion–fixation step to remove unbound D3-biotin 24 hr after injection (Fig. 2). Most of the D3-biotin was washed off by perfusion, and it is presumed that those D3-biotin molecules were not bound to a receptor. Specific analyses of target areas where TrkA is expressed (e.g., Image analysis g y The density of VAChT-immunoreactive (IR) or synaptophysin-IR pre- synaptic boutons in lamina V of the parietal cortex (bregma coordinates: 8012 • J. Neurosci., September 15, 2004 • 24(37):8009–8018 Bruno et al. • Age-Associated Deficits Rescued by a Mimetic TrkA Ligand Figure2. NeuronalbindingbyD3andcolocalizationwithTrkAreceptors.ConfocalmicroscopyofparietalcortexlaminaV(A,B) ornucleusbasalis(C)24hrafterinjectionofD3-biotin.Brainswereperfused,andsectionswereimmunostained.A,Colocalization ofD3-biotinandNeuN.B,LackofcolocalizationofD3-biotinandGFAP.C,ColocalizationofD3-biotinandTrkA.Scalebar,20m. cerebral cortex and nucleus basalis) re- vealed bound D3-biotin remaining after perfusion and could not be washed off, suggesting that it is tightly bound to a re- ceptor. These D3-biotin molecules accu- mulated at the cell surface of lamina V pa- rietal cortical neurons, the nuclei of which were identified with the NeuN marker (Fig. 2A). Additional analysis using glial markers demonstrated no colocalization of D3-biotin signal with GFAP in parietal cortex lamina V (Fig. 2B). Finally, studies in the nucleus basalis demonstrated colo- calization of the D3-biotin signal with TrkA immunoreactivity (Fig. 2C). To- gether, these data indicate that D3 binds to cell surface TrkA expressed in neurons. Preselection of aged, cognitively impaired rats An aged rat model of cognitive impair- ment was used to test in vivo the TrkA par- tial agonist D3 (Fig. 3A). From of a pool of 120 aged rats (24-month-old males), the Morris water maze allowed preselection of 31 rats that were severely cognitively im- paired (Fig. 3B). In the Morris water maze rats, use visual clues to learn and recall the position of a platform hidden underwater. Visual impairment was ruled out as the cause of poor performance by testing with the platform exposed (Fig. 3B) (day 6). Figure2. NeuronalbindingbyD3andcolocalizationwithTrkAreceptors.ConfocalmicroscopyofparietalcortexlaminaV(A,B) ornucleusbasalis(C)24hrafterinjectionofD3-biotin.Brainswereperfused,andsectionswereimmunostained.A,Colocalization ofD3-biotinandNeuN.B,LackofcolocalizationofD3-biotinandGFAP.C,ColocalizationofD3-biotinandTrkA.Scalebar,20m. p p g y All data were analyzed for swim speed to rule out motor deficits. Thirty-one cognitively impaired aged rats were thus selected. In the visual platform task (Fig. 3B) (day 6), latencies to reach the platform were low in all three groups. However, Tukey’s test revealed significant between-group differ- ences ( p 0.05 to p 0.0001) in which young animals tended to reach the platform soonest and the aged impaired last. This dif- ference may be attributable to swim speed because this measure differed between the three groups of animals (F 37.1; df 2, 57; p 0.0001). This group difference did not differ across days (day group interaction: F 1.1; df 8, 228; p 0.3), and the young animals swam fastest (22.3 0.5 cm/sec). cortex, and the density and size of cholinergic neurons in the nucleus basalis in rats not exposed to drugs (see Figs. 6, 7). The peptidomimetic TrkA ligand, D3, affords rescue of age-associated cognitive deficits in vivo Twenty-four cognitively impaired aged rats were randomly allo- cated to three treatments. One group received vehicle (saline; n 8), another received NGF-positive control (n 8), and another received D3 (n 8). Over 14 d, a total of 20 g of NGF was administered, a dose previously shown to be neuroprotective (Garofalo and Cuello, 1995). Over 14 d, a total of 40 g of D3 was administered, a dose chosen from previous pilot studies with aged rats (data not shown) reflecting the lower ex vivo TrkA bind- ing affinity and efficacy of D3 compared with NGF. On week 4 after initiation of drug delivery, rats were retested for learning and memory in the Morris water maze, as shown in the experi- mental flowchart of Figure 4. All rats in the saline and the D3 groups survived surgery and testing with no signs of side effects. In the NGF group, there were two rats with tremors and weight loss (a known side effect of applying NGF therapeutically). All the Morris water maze testing data from these two rats in the NGF group were completely discarded. Also, there were two deaths each in the NGF and the D3 groups attributable to natural causes. Thus, the n values were n 8 (vehicle), n 6 (D3), and n 4 (NGF). Importantly, the swim speed of the two aged groups (unim- paired and impaired) were indistinguishable (18.7 0.1 and 18.7 0.2, respectively). Therefore, swim speed did not account for poor performance in the aged rats. Additionally, poor perfor- mance by cognitively impaired aged rats was not attributable to thigmotaxis, which is a tendency of the animal to swim in contact with the pool wall (Graziano et al., 2003). All rats (young, aged unimpaired, aged impaired) swam in each zone (Fig. 3C) for a similar relative amount of time (Fig. 3D). Therefore, thigmotaxis did not account for poor performance in the aged impaired rats. Importantly, the swim speed of the two aged groups (unim- paired and impaired) were indistinguishable (18.7 0.1 and 18.7 0.2, respectively). Therefore, swim speed did not account for poor performance in the aged rats. Additionally, poor perfor- mance by cognitively impaired aged rats was not attributable to thigmotaxis, which is a tendency of the animal to swim in contact with the pool wall (Graziano et al., 2003). D3 rescues the cholinergic neuronal phenotype in vivo ll h d d f b h l k ll d All the aged rats used for behavioral testing were killed 12 weeks after drug delivery, their brains were perfused and fixed, and multiple serial cryosections were prepared. As controls, an addi- tional cohort not subjected to drug treatment or surgery (five young rats, five aged unimpaired rats, and five aged impaired rats) were studied as well. Sections were immunostained with antibodies to VAChT. This marker was chosen because cortical changes in VAChT accompany age-dependent cognitive impair- ments in mammals (Bartus et al., 1982; Hasselmo, 1999; Turrini et al., 2001; Casu et al., 2002). The density of VAChT-IR presyn- aptic boutons in the lamina V of the parietal cortex and the size of soma in the nucleus basalis were studied quantitatively. Concom- itant analyses of synaptophysin immunostaining and density were also done to test for cholinergic specificity. Figure3. Preselectionofcognitivelyimpairedagedrats.A,TrkApartialagonistD3peptido- mimetic.B,SegregationofcohortsofagedimpairedratsintheMorriswatermaze(seeMaterials andMethodsfordetails).Latencyscoresweresubjectedtoatwo-wayANOVAwithtwofactors: days (within-subject) and group (between-subject). This confirmed that the three groups did indeed differ. The main effects of day and group, and their interaction, were all highly signifi- cant(F99.0;df2,228;p0.0001;F18.1;df4,228;p0.0001;F10.3;df8,228;p 0.0001,respectively).Inthevisualplatformtask(day6),allthreegroupsreachedtheplatform rapidly.C,Graphicrepresentationofthepooldividedintothreeconcentriczones(ZoneA,20cm diameter; Zone B, 45 cm width; Zone C, 15 cm from the outer wall). The location of the escape platform is indicated in the northeast, with its center 45 cm from the outer wall. D, The young, agedunimpaired,andagedimpairedgroupsspendsimilarrelativeamountsoftimeinthethree zones averaged for days 2–5 of the acquisition phase. In particular, there was no group differ- enceintheproportionoftimespentintheouterZoneC,whichisameasureofthigmotaxis(F 0.99; df 2.57; p 0.3). In untreated rats, computer-assisted image analysis of the number of VAChT-IR boutons in lamina V of the parietal cortex demonstrated a reduction in cognitively impaired aged rats com- pared with young rats ( p 0.001) and between cognitively im- paired aged rats and unimpaired aged rats ( p 0.01). There were no statistical differences between the young rats and unimpaired aged rats (Fig. 6A). In cognitively impaired aged rats, treatment with D3 or NGF significantly ( p 0.001) increased the density of cortical cholin- ergic (i.e., VAChT-IR) presynaptic boutons to a similar extent, 40% (Fig. 6A). It is noteworthy that the phenotypic changes in forebrain VAChT expression brought about by D3 and NGF treatments were long-lived and lasted up to 12 weeks after drug treatment. mean swim speeds (centimeters per second) for the vehicle, D3, and NGF groups were, respectively, 18.2 0.2, 18.2 0.2, and 18.1 0.2 (ANOVA main effects and interaction; p 0.7). The peptidomimetic TrkA ligand, D3, affords rescue of age-associated cognitive deficits in vivo All rats (young, aged unimpaired, aged impaired) swam in each zone (Fig. 3C) for a similar relative amount of time (Fig. 3D). Therefore, thigmotaxis did not account for poor performance in the aged impaired rats. Thus, 31 aged cognitively impaired rats were defined and se- lected. Of these 31, two died of natural causes. Of the remaining 29 aged cognitively impaired rats, 24 were used for drug testing, followed by behavioral analyses (see Figs. 4, 5) and studies of the cholinergic phenotype (see Figs. 6, 7). The remaining five aged cognitively impaired rats (together with five aged unimpaired rats and five young adult rats) were used as controls for analyses of the density of presynaptic cholinergic boutons in the parietal Thus, 31 aged cognitively impaired rats were defined and se- lected. Of these 31, two died of natural causes. Of the remaining 29 aged cognitively impaired rats, 24 were used for drug testing, followed by behavioral analyses (see Figs. 4, 5) and studies of the cholinergic phenotype (see Figs. 6, 7). The remaining five aged cognitively impaired rats (together with five aged unimpaired rats and five young adult rats) were used as controls for analyses of the density of presynaptic cholinergic boutons in the parietal Rats that received D3 or NGF had significantly improved per- formance compared with the saline group from trial 2 onward of the testing phase (Fig. 4A). However, NGF and D3 were not significantly different from each other. Swim speeds of aged im- paired animals receiving treatments were not a factor because they were closely similar between groups and across trials. The Bruno et al. • Age-Associated Deficits Rescued by a Mimetic TrkA Ligand J. Neurosci., September 15, 2004 • 24(37):8009–8018 • 8013 Figure3. Preselectionofcognitivelyimpairedagedrats.A,TrkApartialagonistD3peptido- mimetic.B,SegregationofcohortsofagedimpairedratsintheMorriswatermaze(seeMaterials andMethodsfordetails).Latencyscoresweresubjectedtoatwo-wayANOVAwithtwofactors: days (within-subject) and group (between-subject). This confirmed that the three groups did indeed differ. The main effects of day and group, and their interaction, were all highly signifi- cant(F99.0;df2,228;p0.0001;F18.1;df4,228;p0.0001;F10.3;df8,228;p 0.0001,respectively).Inthevisualplatformtask(day6),allthreegroupsreachedtheplatform rapidly.C,Graphicrepresentationofthepooldividedintothreeconcentriczones(ZoneA,20cm diameter; Zone B, 45 cm width; Zone C, 15 cm from the outer wall). The location of the escape platform is indicated in the northeast, with its center 45 cm from the outer wall. D, The young, agedunimpaired,andagedimpairedgroupsspendsimilarrelativeamountsoftimeinthethree zones averaged for days 2–5 of the acquisition phase. In particular, there was no group differ- enceintheproportionoftimespentintheouterZoneC,whichisameasureofthigmotaxis(F 0.99; df 2.57; p 0.3). (where the platform had been placed) does not reflect new learn- ing but only memory. D3 rescues the cholinergic neuronal phenotype in vivo ll h d d f b h l k ll d p The data in Figure 4A were further evaluated by quadrant analyses, specifically comparing trial 1 and trial 7 on week 4 post- drug delivery. The data are presented as the percentage of time the animals spend in the trained quadrant (where the platform is located) versus the other three quadrants (Fig. 4B). The analyses for the vehicle, NGF, and D3 groups are shown for trial 1 (Fig. 4C) and trial 7 (Fig. 4D). As expected, in trial 1, there were no signif- icant differences among the vehicle, NGF, or D3 groups (Fig. 4C), but in trial 7, the NGF and the D3 groups had a significant im- provement at targeting the trained quadrant compared with ve- hicle (Fig. 4D). Tukey’s test showed that this measure differed significantly between groups on trial 7 but not on trial 1 (ANOVA; p 0.0001 and p 0.09, respectively). In trial 7, comparisons for all three groups were significant ( p 0.05 to p 0.0001). These results are suggestive of improved learning and/or memory by the NGF and D3 groups. Control immunostaining of all cortical presynaptic sites with synaptophysin antibodies in adjacent serial sections demon- strated no variation in that marker (Fig. 6B), except a slight dif- ference between the young and aged impaired groups ( p 0.05). This control indicates that D3 is a fairly selective agent for NGF- sensitive cholinergic neurons. The microscopic appearance of images of VAChT cortical presynaptic sites is shown to illustrate the VAChT changes in each of the test groups (Fig. 6C). The size of VAChT-immunostained cell somata in the nucleus basalis was determined by computer-assisted image analysis. In untreated rats, mean somal size was significantly ( p 0.01) re- duced in cognitively impaired aged rats compared with young rats (Fig. 7A). There was a small but significant difference be- tween unimpaired aged rats compared with impaired aged rats ( p 0.05), but not between young rats and unimpaired aged rats. The peptidomimetic TrkA ligand, D3, affords rescue of age-associated cognitive deficits in vivo Representative swim paths from individual rats at 12 weeks after drug delivery are shown in Figure 5A. The NGF and the D3 groups spent a significantly higher proportion of time in the tar- get quadrant at week 5 (Fig. 5B) and week 12 (Fig. 5C) after drug delivery. In contrast, saline-treated rats spent very similar amounts of time in each quadrant (Fig. 5B,C). These data indi- cate that the behavioral improvement afforded by D3 and NGF is very long-lived and attributable, at least in part, to improved memory. Targeting TrkA in cognitive disorders In the normal adult human CNS, the cortical and nucleus basalis neurons that comprise the cholinergic system participate in higher brain functions such as learning and memory. These neu- rons express the TrkA receptor (Mufson et al., 1997) and are responsive to NGF. In neurodegenerative processes such as mild cognitive impairment (MCI), loss of TrkA density correlates with neuronal atrophy and precedes neuronal death and severe cogni- tive impairment in AD (Counts et al., 2004). Indeed, in MCI–AD progression, and in Down’s syndrome, loss of TrkA correlates with cognitive decline (Mufson et al., 2000, 2002). Likewise, in basal forebrain neurons of the aged rat, there is reduced expres- sion of NGF receptors, which is reversed by delivery of NGF (Backman et al., 1997). Although published evidence linking a compromised TrkA– NGF system and progressive cognitive impairment is substantial, consistent for many models (MCI, AD, aging, and Down’s syn- drome), and NGF reverts cognitive impairment, the relationship seems to be casual rather than causal. Additional studies will be needed to address causality. presented as the percentage of time spent in the trained quadrant (where the platform is located) versus the other three quadrants. C, Analyses for the vehicle, NGF, and D3 groups for trial1.D,Analysesforthevehicle,NGF,andD3groupsfortrial7.Statisticalcomparisonsreferto Tukey’s test. Discussion We demonstrated the biological actions and usefulness of a pep- tidomimetic small molecule TrkA agonist named D3 (Maliart- chouk et al., 2000) in an in vivo model of cognitive impairment. D3 affords a significant, long-lasting, and selective enhancement of the CNS cholinergic phenotype in cognitively impaired aged rats, accompanied with a significant, long-lasting improvement in the cognitive ability of aged rats thus treated. In these investi- gations, D3 and NGF afforded comparable results at the doses tested. D3 affords a long-lasting rescue of age-associated memory deficits in vivo In cognitively impaired aged rats, treatment with either D3 or NGF increased significantly ( p 0.001) the somal size of nucleus basalis neurons by 30% (Fig. 7A). Because the size of D3- or NGF-treated rats was not significantly different from each other or from young rats, the data would suggest that drug treatment reversed shrinkage. As was the case with cholinergic terminals, To discriminate between learning and memory, the rats were tested in the same pool for a fixed time of 1 min, but without the hidden platform, thus offering no escape and no new learning. Because there is no successful resolution of the task, because there is no platform to find, the time spent in the target quadrant 8014 • J. Neurosci., September 15, 2004 • 24(37):8009–8018 Bruno et al. • Age-Associated Deficits Rescued by a Mimetic TrkA Ligand Figure4. D3andNGFimproveperformanceincognitivelyimpairedagedrats.A,Cognitively impaired rats were treated as indicated and then were retested as described in Materials and 8014 • J. Neurosci., September 15, 2004 • 24(37):8009–8018 the phenotypic changes in cell somata brought about by D3 and NGF were long-lived because they lasted up to 12 weeks after drug treatment. There were no significant changes observed in the density of nucleus basalis cholinergic cell bodies (Fig. 7B), thus reinforcing the notion that atrophy (rather than cholinergic forebrain cell loss) is a feature in age-related cholinergic decline. Selective improvement to cognition and the cholinergic system We demonstrate that the age-dependent cognitive impairment is accompanied by marked cholinergic synaptic loss, reduction in the size of cholinergic cell soma (but not in cell numbers), and by reduced expression of VAChT. These behavioral and phenotypic deficits are reverted by NGF and D3. The pharmacological effects are long lasting and were observed for 3 months after the initia- tion of drug treatment. It is gratifying that our data using exoge- nous NGF as control is consistent with previous reports (Fischer et al., 1987; Chen et al., 1995). The in vivo cholinergic selectivity of D3 was evidenced by a specific enhancement of VAChT-IR boutons, but an absence of an effect on the overall presynaptic population (e.g., synaptophysin-IR boutons), and was anticipated because D3 is a TrkA ligand. Figure4. D3andNGFimproveperformanceincognitivelyimpairedagedrats.A,Cognitively impaired rats were treated as indicated and then were retested, as described in Materials and Methods,intheMorriswatermazeonweek4afterinitiationofdrugdelivery.ANOVAoflaten- cies to find the platform revealed significant main effects of group (F 44.8; df 2, 15; p 0.0001)andtrial(F18.6;df6,90;p0.0001).ThelatenciesofD3-andNGF-treatedrats were similar in all trials, whereas both groups diverged significantly from the vehicle-treated controlgroup(grouplineartrendoftrial:F4.62;df2,15;p0.05).Drugeffectswere evident,forexample,fordatapooledacrossthelastfourtrials(Tukey’stest:D3orNGFvsvehicle, p 0.0001). B, Schematic graph of the pool showing the quadrants and the position of the submerged escape platform. Data for Figure 4A were evaluated by quadrant analyses and One interesting finding corollary to our study of D3 provides additional confirmation that structural changes (reduced density of VAChT-IR presynaptic boutons in the neocortex and smaller Bruno et al. • Age-Associated Deficits Rescued by a Mimetic TrkA Ligand J. Neurosci., September 15, 2004 • 24(37):8009–8018 • 8015 Figure5. D3andNGFimprovememoryincognitivelyimpairedagedrats.A,Typicalswimpathforindividualratsrecordedover a 1 min period in week 12. Target quadrant is where the escape platform had been located formerly (northeast quadrant). The summaryofquadrantanalysisforthevehicleandD3groupsandNGFgroupatweek5(B)andweek12(C)afterdrugtreatment. Statistical comparisons refer to Tukey’s test. the cytoplasma or inside neurite exten- sions. There are five possible explanations for these differences. First, there may be lower relative signal intensity for D3 versus anti-TrkA serum because the latter has multiple reactivities. Second, the TrkA protein present in neu- rite terminals (seen with anti-TrkA serum after fixation/permeabilization) may be mostly intracellular and not cell surfaced, and hence it is not accessible to the D3- biotin injected in vivo. Third, relative re- ceptor density in the neurites may be lower than in the cell somata. Fourth, in- tracellular retrogradely transported D3 may be processed (i.e., the biotin label is cleaved off) and becomes undetectable. Selective improvement to cognition and the cholinergic system Fifth, perhaps the D3–TrkA complex does not internalize efficiently. In vitro studies of neuronal cultures documented that ligand-activated TrkA neurotrophic signals do not require internalization of the NGF–TrkA complex (Neet and Campenot, 2001); thus, our in vivo studies would be compatible with those results. rindividualratsrecordedover rly (northeast quadrant). The k12(C)afterdrugtreatment. First, there may be lower relative signal intensity for D3 versus anti-TrkA serum because the latter has multiple reactivities. Second, the TrkA protein present in neu- rite terminals (seen with anti-TrkA serum after fixation/permeabilization) may be mostly intracellular and not cell surfaced, and hence it is not accessible to the D3- biotin injected in vivo. Third, relative re- ceptor density in the neurites may be lower than in the cell somata. Fourth, in- tracellular retrogradely transported D3 may be processed (i.e., the biotin label is cleaved off) and becomes undetectable. Fifth, perhaps the D3–TrkA complex does ently. In vitro studies of neuronal cultures and-activated TrkA neurotrophic signals do ation of the NGF–TrkA complex (Neet and us, our in vivo studies would be compatible Figure5. D3andNGFimprovememoryincognitivelyimpairedagedrats.A,Typicalswimpathforindividualratsrecordedover a 1 min period in week 12. Target quadrant is where the escape platform had been located formerly (northeast quadrant). The summaryofquadrantanalysisforthevehicleandD3groupsandNGFgroupatweek5(B)andweek12(C)afterdrugtreatment. Statistical comparisons refer to Tukey’s test. size of somata in the nucleus basalis) may be directly implicated in aged-associated cognitive decline because such changes occur only in the cognitively impaired aged rats but not in the unim- paired aged rats. Our results suggest that it may be possible to delay the chronic and progressive cholinergic neurodegeneration in aging or in pathological states with the use of neurotrophin receptor ago- nists. If selective agonists of TrkA were to be applied before ex- tensive CNS damage (e.g., in MCI), it is conceivable that selective protection of cholinergic forebrain neurons and cognitive im- provements could be achieved. Additionally, single or concomi- tant use of pharmacological modulators of p75 action or poten- tiators of NGF activity may also prove to be useful (Maliartchouk and Saragovi, 1997; Saragovi and Burgess, 1999; Saragovi and Maliartchouk, 1999). Pharmacological advantages Two properties of D3 are desirable in a drug candidate: proteo- lytic stability and no incidence of side effects compared with NGF. The ex vivo proteolytic stability of D3 has been shown previously (Maliartchouk et al., 2000). D3 is extremely stable to pepsin, chymotrypsin, papain, trypsin, and other peptidases. Pre- liminary pharmacokinetic studies on D3 (data not shown) showed a serum half-life of 4.5 hr and the appearance of two metabolites. In contrast, the serum half-life of NGF is in the order of minutes (Verrall, 1994). To our knowledge, D3 is the first reported small molecule ligand capable of inducing selective survival of the cholinergic neurons. Other small molecules with drug-like properties, such as GM1 or the immunophilins, have been nonselective (Saragovi and Burgess, 1999; Saragovi and Gehring, 2000). The effective dose of NGF (nearly the lowest effective dose iden- tifiedinpublishedreports)(GarofaloandCuello,1995)causednote- worthy weight loss and tremors as side effects in two of eight (25%) of the tested rats. In contrast, the effective dose of D3 did not appear to cause these side effects or any obvious signs of toxicity, but we have not performed objective pathological studies. Note that all the data with NGF toxemic rats were discarded. Had that data been considered, the neuroprotective and behavioral effect of D3 would have been significantly better than NGF. Efficacy and selectivity Selectivity, rather than potency or affinity, is the most relevant feature that impacts on drug efficacy in vivo (Mattie et al., 1989; Troke et al., 1990; Brook, 1991; Vella and Floridia, 1998; Wald- man, 2002). For example, despite its high TrkA binding affinity (Kd, 10–100 pM), NGF has a narrow therapeutic window partly because of poor selectivity (e.g., it binds p75 receptors), partly because of pleiotropic activation, and partly because of a short half-life. In contrast, the TrkA binding affinity of D3 is 5 M. However, D3 does not bind another target, and it does not acti- vate other Trk receptors (Maliartchouk et al., 2000). Therefore, D3 may afford a large therapeutic window. Although D3 is a partial agonist of TrkA, its intrinsic activity is relatively low and it is best revealed when acting as a synergistic potentiator of NGF (Maliartchouk et al., 2000). This property may be advantageous when testing in vivo because, in some neu- rogenerative disorders, NGF protein is increased (Fahnestock et al., 1996; Hock et al., 2000a,b); but this NGF protein is either less active or has different activity (Fahnestock et al., 2004a,b). For that reason, a selective small molecule NGF potentiator such as D3 may be extremely effective. D3 can be detected associated with TrkA receptors for a rela- tively long time (as shown in Fig. 2C). Improved penetration and stability for D3 (compared with NGF) may result in better access to target tissue from the site of administration. Also, D3 binds at the IgC2 ectodomain of TrkA, which is defined as a functional “receptor hot spot.” In contrast, NGF seems to bind at least two hot spots on TrkA (Zaccaro et al., 2001; Saragovi and Zaccaro, 2002). A potentially attractive feature is that small molecules could target receptors at single, defined, activation, or regulatory hot spots (e.g., only the Trk IgC2 ectodomain) while excluding coreceptors (e.g., p75 receptors). Thus, binding to a single receptor site could afford partial recep- tor activation and higher selectivity. Targeting TrkA in vivo Anti-TrkA antiserum revealed TrkA protein in four spatial loca- tions: at the surface of the cell soma and the neuritic terminals and intracellularly at the cytoplasma and the neurites. In contrast, we noted that D3 seems to localize preferentially at TrkA recep- tors present on the cell surface of the somata. There is very little D3 on the cell surface of neuritic terminals or inside cells either at Bruno et al. • Age-Associated Deficits Rescued by a Mimetic TrkA Ligand Summary Disregulation of the neurotrophins or their receptors is releva to human neuropathologies, including chronic or acute neur degeneration, pain, and cancer (Eide et al., 1993; Hefti, 199 Saragovi and Burgess, 1999; Saragovi and Gehring, 2000). Ne rotrophin clinical trials have been disappointing because of vivo instability, side effects produced by the activation of sign that were not intentionally targeted, and drawbacks inherent proteins when used as drugs. For these reasons, small molecu peptidomimetics that have better pharmacokinetic propert may be useful therapeutics. From a drug development perspe tive, it may be desirable to have small molecules that uncoup trophic and neuritogenic signals (Saragovi et al., 1998; Zhang al., 2000) because they can be selective therapeutics in conditio in which a single activity is required. For example, it may desirable to support existing neurons from death but witho inducing potentially maladapted de novo connections To th Figure6. SelectiveeffectofD3onthecholinergicphenotypeofcorticalneurons.A,Number (boutons/1000 m 2) SEM of cholinergic presynaptic boutons (VAChT immunoreactive). B, Totalpresynapticboutons(synaptophysinimmunoreactive)inlaminaVoftheparietalcortexof agedFischer-344rats.InAandB,atotalareaof80,000m 2wasanalyzedperrat(n4or5). Aged impaired versus aged impaired plus vehicle were not different from each other but pre- sentedasignificantlydecreasednumberofcholinergicpresynapticboutonsversusyoung.Res- toration of the size in the cholinergic presynaptic boutons in aged impaired plus NGF and aged impairedplusD3whencomparedwithagedimpairedandagedimpairedplusvehicleisshown Figure 7. Selective effect of D3 on the size and cholinergic phenotype of nucleus ba neurons.A,Cellsizeofthesoma(m 2)SEMofVAChT-IRcholinergicneuronsinthenuc basalis of Fischer-344 rats. For cell size analyses, a total of 10,000 VachT-positive cells pe (n 4 or 5) were studied. Aged impaired and aged impaired plus vehicle were not diffe fromeachotherbutdisplayedsignificantlylowercellsizethanyoung.Restorationofthecell ofcholinergicneuronsafterreceivingeitherD3orNGFwhencomparedwithagedimpaired aged impaired plus vehicle. B, Density of VAChT-IR neurons, representative of neuronal n bers in the nucleus basalis. Statistical comparisons refer to Tukey’s test. For VAChT densit totalof50,000VachT-positivecellsperrat(n4or5)werestudied,anddataareexpressed 1000 m 2. 8016 • J. Neurosci., September 15, 2004 • 24(37):8009–8018 Bruno et al. • Age-Associated Deficits Rescued by a Mimetic TrkA Lig 8016 • J. Neurosci., September 15, 2004 • 24(37):8009–8018 Figure 7. Targeting TrkA in vivo Selective effect of D3 on the size and cholinergic phenotype of nucleus basalis neurons.A,Cellsizeofthesoma(m 2)SEMofVAChT-IRcholinergicneuronsinthenucleus basalis of Fischer-344 rats. For cell size analyses, a total of 10,000 VachT-positive cells per rat (n 4 or 5) were studied. Aged impaired and aged impaired plus vehicle were not different fromeachotherbutdisplayedsignificantlylowercellsizethanyoung.Restorationofthecellsize ofcholinergicneuronsafterreceivingeitherD3orNGFwhencomparedwithagedimpairedand aged impaired plus vehicle. B, Density of VAChT-IR neurons, representative of neuronal num- bers in the nucleus basalis. Statistical comparisons refer to Tukey’s test. For VAChT density, a totalof50,000VachT-positivecellsperrat(n4or5)werestudied,anddataareexpressedper 1000 m 2. g y g Figure6. SelectiveeffectofD3onthecholinergicphenotypeofcorticalneurons.A,Number (boutons/1000 m 2) SEM of cholinergic presynaptic boutons (VAChT immunoreactive). B, Totalpresynapticboutons(synaptophysinimmunoreactive)inlaminaVoftheparietalcortexof agedFischer-344rats.InAandB,atotalareaof80,000m 2wasanalyzedperrat(n4or5). Aged impaired versus aged impaired plus vehicle were not different from each other but pre- sentedasignificantlydecreasednumberofcholinergicpresynapticboutonsversusyoung.Res- toration of the size in the cholinergic presynaptic boutons in aged impaired plus NGF and aged impairedplusD3whencomparedwithagedimpairedandagedimpairedplusvehicleisshown. For synaptophysin staining, there were no significant differences between any groups. C, Rep- resentativepicturesoflaminaVoftheparietalcorteximmunostainedwithanti-VAChTantibod- Figure6. SelectiveeffectofD3onthecholinergicphenotypeofcorticalneurons.A,Number (boutons/1000 m 2) SEM of cholinergic presynaptic boutons (VAChT immunoreactive). B, Totalpresynapticboutons(synaptophysinimmunoreactive)inlaminaVoftheparietalcortexof agedFischer-344rats.InAandB,atotalareaof80,000m 2wasanalyzedperrat(n4or5). Aged impaired versus aged impaired plus vehicle were not different from each other but pre- sentedasignificantlydecreasednumberofcholinergicpresynapticboutonsversusyoung.Res- toration of the size in the cholinergic presynaptic boutons in aged impaired plus NGF and aged impairedplusD3whencomparedwithagedimpairedandagedimpairedplusvehicleisshown. For synaptophysin staining, there were no significant differences between any groups. C, Rep- resentativepicturesoflaminaVoftheparietalcorteximmunostainedwithanti-VAChTantibod- ies. Statistical comparisons refer to Tukey’s test. Figure 7. Selective effect of D3 on the size and cholinergic phenotype of nucleus basalis neurons.A,Cellsizeofthesoma(m 2)SEMofVAChT-IRcholinergicneuronsinthenucleus basalis of Fischer-344 rats. For cell size analyses, a total of 10,000 VachT-positive cells per rat (n 4 or 5) were studied. Aged impaired and aged impaired plus vehicle were not different fromeachotherbutdisplayedsignificantlylowercellsizethanyoung.Restorationofthecellsize ofcholinergicneuronsafterreceivingeitherD3orNGFwhencomparedwithagedimpairedand aged impaired plus vehicle. B, Density of VAChT-IR neurons, representative of neuronal num- bers in the nucleus basalis. Statistical comparisons refer to Tukey’s test. For VAChT density, a totalof50,000VachT-positivecellsperrat(n4or5)werestudied,anddataareexpressedper 1000 m 2. 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Neurosci., September 15, 2004 • 24(37):8009–8018 • 8017 Bruno et al. • Age-Associated Deficits Rescued by a Mimetic TrkA Ligand Future studies will be directed at establishing optimal drug concentration and efficacy, blood brain barrier penetration, and maximal tolerated doses for these compounds. Graziano A, Petrosini L, Bartoletti A (2003) Automatic recognition of ex- plorative strategies in the Morris water maze. J Neurosci Methods 130:33–44. Hasselmo ME (1999) Neuromodulation: acetylcholine and memory con- solidation. Trends Cogn Sci 3:351–359. Summary Disregulation of the neurotrophins or their receptors is relevant to human neuropathologies, including chronic or acute neuro- degeneration, pain, and cancer (Eide et al., 1993; Hefti, 1994; Saragovi and Burgess, 1999; Saragovi and Gehring, 2000). Neu- rotrophin clinical trials have been disappointing because of in vivo instability, side effects produced by the activation of signals that were not intentionally targeted, and drawbacks inherent to proteins when used as drugs. For these reasons, small molecule peptidomimetics that have better pharmacokinetic properties may be useful therapeutics. From a drug development perspec- tive, it may be desirable to have small molecules that uncouple trophic and neuritogenic signals (Saragovi et al., 1998; Zhang et al., 2000) because they can be selective therapeutics in conditions in which a single activity is required. For example, it may be desirable to support existing neurons from death but without inducing potentially maladapted de novo connections. To that end, we have developed a family of analogs of D3 with single activity (Saragovi and Burgess, 2001) (M. C. Zaccaro, H. B. Lee, M. Pattarawarapan, Z. Xia, A. Caron, K. Burgess, and H. U. Sara- govi, unpublished observations). Figure6. SelectiveeffectofD3onthecholinergicphenotypeofcorticalneurons.A,Number (boutons/1000 m 2) SEM of cholinergic presynaptic boutons (VAChT immunoreactive). B, Totalpresynapticboutons(synaptophysinimmunoreactive)inlaminaVoftheparietalcortexof agedFischer-344rats.InAandB,atotalareaof80,000m 2wasanalyzedperrat(n4or5). Aged impaired versus aged impaired plus vehicle were not different from each other but pre- sentedasignificantlydecreasednumberofcholinergicpresynapticboutonsversusyoung.Res- toration of the size in the cholinergic presynaptic boutons in aged impaired plus NGF and aged impairedplusD3whencomparedwithagedimpairedandagedimpairedplusvehicleisshown. For synaptophysin staining, there were no significant differences between any groups. C, Rep- resentativepicturesoflaminaVoftheparietalcorteximmunostainedwithanti-VAChTantibod- ies. Statistical comparisons refer to Tukey’s test. Figure6. SelectiveeffectofD3onthecholinergicphenotypeofcorticalneurons.A,Number (boutons/1000 m 2) SEM of cholinergic presynaptic boutons (VAChT immunoreactive). B, Totalpresynapticboutons(synaptophysinimmunoreactive)inlaminaVoftheparietalcortexof agedFischer-344rats.InAandB,atotalareaof80,000m 2wasanalyzedperrat(n4or5). Aged impaired versus aged impaired plus vehicle were not different from each other but pre- sentedasignificantlydecreasednumberofcholinergicpresynapticboutonsversusyoung.Res- toration of the size in the cholinergic presynaptic boutons in aged impaired plus NGF and aged impairedplusD3whencomparedwithagedimpairedandagedimpairedplusvehicleisshown. For synaptophysin staining, there were no significant differences between any groups. C, Rep- resentativepicturesoflaminaVoftheparietalcorteximmunostainedwithanti-VAChTantibod- ies. Statistical comparisons refer to Tukey’s test. Figure6. SelectiveeffectofD3onthecholinergicphenotypeofcorticalneurons.A,Number (boutons/1000 m 2) SEM of cholinergic presynaptic boutons (VAChT immunoreactive). B, Totalpresynapticboutons(synaptophysinimmunoreactive)inlaminaVoftheparietalcortexof agedFischer-344rats.InAandB,atotalareaof80,000m 2wasanalyzedperrat(n4or5). Aged impaired versus aged impaired plus vehicle were not different from each other but pre- sentedasignificantlydecreasednumberofcholinergicpresynapticboutonsversusyoung.Res- toration of the size in the cholinergic presynaptic boutons in aged impaired plus NGF and aged impairedplusD3whencomparedwithagedimpairedandagedimpairedplusvehicleisshown. For synaptophysin staining, there were no significant differences between any groups. C, Rep- resentativepicturesoflaminaVoftheparietalcorteximmunostainedwithanti-VAChTantibod- ies. Statistical comparisons refer to Tukey’s test. 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https://openalex.org/W2048101653	https://europepmc.org/articles/pmc4004039?pdf=render	English	null	Clinical Features and Outcome of Sporadic Colorectal Carcinoma in Young Patients: A Cross-Sectional Analysis from a Developing Country	ISRN Oncology	2,014	cc-by	5,844	Hindawi Publishing Corporation ISRN Oncology Volume 2014, Article ID 461570, 8 pages http://dx.doi.org/10.1155/2014/461570 Hindawi Publishing Corporation ISRN Oncology Volume 2014, Article ID 461570, 8 pages http://dx.doi.org/10.1155/2014/461570 Hindawi Publishing Corporation ISRN Oncology Volume 2014, Article ID 461570, 8 pages http://dx.doi.org/10.1155/2014/461570 Muhammad Nauman Zahir,1 Eisha Mahpara Azhar,2 Sobia Rafiq,2 Kulsoom Ghias,2 and Munira Shabbir-Moosajee1 1 Department of Oncology, Aga Khan University Hospital, Stadium Road, P.O. Box 3500, Karachi 74800, Pakistan 2 Department of Biological and Biomedical Sciences, Aga Khan University Hospital, Stadium Road, P.O. Box 3500, Karachi 74800, Pakistan Correspondence should be addressed to Muhammad Nauman Zahir; nauman.zahir@gmail.com Received 7 February 2014; Accepted 12 March 2014; Published 1 April 2014 Academic Editors: W. Habano and S. Holdenrieder Academic Editors: W. Habano and S. Holdenrieder Copyright © 2014 Muhammad Nauman Zahir et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background. Early onset colorectal carcinoma (CRC) is rare and has been hypothesized to be a biologically and clinically distinct entity personifying aggressive disease and worse survival. Methods. Data for 131 patients was collected by retrospective chart review. Cox proportional hazard model was used to compute prevalence ratios and 95% confidence intervals. Results. Early onset sporadic CRC accounted for 32% of all CRC treated in the specified time period. The mean age was 33.3 ± 7.9 years and the male to female ratio was 2 : 1. Colon and rectal cancers accounted for 55% and 45% of patients, respectively. 96% of rectal carcinoma patients received appropriate therapy as opposed to 65% of colon cancers. On multivariable analysis, appropriate reception of therapy (PR 4.99; 95% CI, 1.21–20.6) and signet ring morphology (PR 2.40; 95% CI, 1.33–4.32) were significantly associated with rectal cancers as opposed to colon cancer. Kaplan-Meier analysis revealed a trend towards inferior survival for rectal carcinoma 2 years after diagnosis. Conclusion.A high prevalence of early onset CRC was noted in the study. A trend towards inferior survival was seen in patients with rectal cancer. This finding raises the possibility of rectal carcinoma being an aggressive subset of young CRC. 1. Introduction Age is considered a major risk factor for colon cancer. Diagnosis of CRC is a rare occurrence in patients less than 40 years. Thereafter, the incidence increases sharply with every decade of life. In developed countries, the risk of colon cancer is 1 in 10 males after the 8th decade of life [3]. The life time risk of sporadic CRC is 5% and it accounts for 90% of the cases diagnosed in patients above 50 years [3]. Colorectal carcinoma (CRC) is one of the most common cancers diagnosed worldwide. It has been documented that CRC is the second most common malignancy in females and the third most common amongst males [1]. Epidemiological data suggests a higher incidence of sporadic CRC in devel- oped countries, with Australia and New Zealand having an age adjusted incidence of 45.7 per 100,000 as compared to Southeast Asia which has a reported incidence of 15.9 per 100,000 [1]. Interestingly, a rapid increase in CRC incidence rate in economically transitioning countries has been recently reported in the literature [2] and has been attributed to a change in the dietary habits and physical activity patterns superimposed on genetic predisposition. Early onset CRC accounts for around 7% of the total CRC population in the West [4] but the problem has been reported to be of a much greater magnitude in several Asian and African countries [5–12]. Similarly, half of the incident cases of CRC in the Pakistani population have been estimated to be in young patients [13]. From this, coupled with the fact that greater than 80% of the Pakistani population is younger than 40 years [14], the “at-risk” population for early onset 2 ISRN Oncology ISRN Oncology data. A patient was labeled “lost to follow-up” if all attempts to establish contact with him/her failed. CRC in countries like Pakistan is much higher than the rest of the world. data. A patient was labeled “lost to follow-up” if all attempts to establish contact with him/her failed. Studies from the past have shown that in low incidence countries like Pakistan, CRC tends to be located in the distal colon and rectum while it is more likely to be located proxi- mally in countries where incidence is higher [15]. Further- more, dominance of left sided tumors in the young popu- lation has also been demonstrated in other studies [4]. 2. Methods p The mean age of the study population was 33.3 ± 7.9 years. Male to female ratio was 2 : 1. No significant genetic pre- disposing factors (such as familial adenomatous polyposis, Lynch syndrome) were suspected in included patients. Only 8 patients (6.1%) had a positive family history of colon cancer but were assumed to have sporadic CRC as they did not fulfill the Amsterdam criterion for hereditary nonpolyposis colorectal carcinoma (HNPCC) or the clinical criteria for familial adenomatous polyposis (FAP). The most common clinical presentations were abdominal pain (87%, 𝑛= 114), weight loss (66%, 𝑛= 86), and bleeding per rectum (52%, 𝑛= 68). Only 29 patients (22%) presented with intestinal obstruc- tion. 2.1. Patients. We retrospectively collected data of patients less than or equal to 45 years of age with newly diagnosed CRC, presenting to the Oncology Department, Aga Khan Uni- versity Hospital (AKUH), Karachi, between January 1, 2004, and December 31, 2011. Patients were identified using the dis- charge coding system. Patients with suspected familial syn- dromes (based upon clinical history and colonoscopic find- ings) incompletely documented histopathological reports and with biopsy reports from other centers were excluded from analysis.h The data was collected through chart review using a pre- designed and coded questionnaire which had been approved by the institutional Ethical Review Committee. Details of the demographics, symptomatology, risk factors, family history, histopathological features, and outcome were collected for all patients. Histopathology and surgical records were reviewed for tumor location, tumor grade, dissected lymph nodes, involved lymph nodes, and presence of lymphovascular inva- sion and perineural invasion. The American Joint Committee on Cancer Staging System (AJCC 7th edition) was used to stage the disease. 55% of the cohort (𝑛= 72) had colon cancer while the rest had presented with a diagnosis of rectal carcinoma (𝑛= 59). 38 patients (53%) with colon cancer had right sided disease. The colon and rectal cancer groups were similar in age of presentation and sex. However, bleeding per rectum was much more common in rectal carcinomas (85% versus 25%), whereas patients with colon cancer were more likely to present with an abdominal lump (17% versus 7%) and intestinal obstruction (29% versus 14%) and were more likely to have preexisting polyps (13% versus 2%) (Table 1). Follow-up records, data pertaining to therapies, and dates of last contact were also recorded. 1. Introduction This finding is consistent with our clinical experience where we see a large proportion of rectal cancers in our young CRC popu- lation. The exact proportion of this subgroup of patients pre- senting with distally located disease has however never been reported from our region. 2.2. Statistical Analysis. Data pertaining to 131 patients, who were found eligible for the study, were analyzed. Statistical Package for Social Sciences (SPSS) version 19 was used to perform data analysis which was reported as mean ± standard deviation for continuous variables and proportions and percentages for categorical data. Continuous variables not following the normal distribution were reported as medians along with interquartile ranges. Analysis was performed on the entire cohort followed by a subgroup analysis according to anatomical location of the tumor (colon versus rectal). Kaplan-Meier curves were generated for median overall survival (OS) for both colon and rectal carcinomas which were compared using the log-rank test. It has been hypothesized that early onset colorectal cancer is a biologically and clinically distinct disease where younger individuals have more aggressive disease and worse survival [16, 17]. A recent population based study from Pakistan also showed that younger patients presented with disease which was more likely to be poorly differentiated and at a more advanced stage at diagnosis [13]. Although there have been a few studies regarding local experience in treatment and man- agement of colon cancer in the past, data pertaining specif- ically to early onset colorectal cancer is lacking. Given the increased incidence of early onset CRC in Pakistan and the greater “at-risk” population, this study was undertaken to report the clinical and pathological characteristics along with the outcome of this unique subgroup of patients through data from a tertiary care oncology center in Karachi, Pakistan. Univariate and multivariable analysis was performed using the Cox proportional hazard model which was used to compute prevalence ratios and 95% confidence intervals. 𝑃 value less than 0.05 was considered significant. 3. Results 3.1. Patients. A total of 581 patients presented with newly diagnosed CRC to our centre during the study period. Of these 186 (32%) were younger than 45 years and 131 were found to be eligible for analysis. Most of the patients were diagnosed via a colonoscopy (83%), while 97 patients (74%) had advanced disease, defined as stage III/IV, at initial presentation. 2. Methods Patients who had not followed up for greater than 6 months were contacted on the telephone numbers obtained from the hospital database. Any additional information regarding outcome accrued through this telephonic contact was incorporated into the recorded 3.2. Histopathological Data. On histopathology, preexisting polyps were identified in only 10 patients (7.6%). 73 patients (56%) had moderately differentiated carcinoma and further 50 patients (38%) had poorly differentiated carcinoma. Signet ISRN Oncology 3 Table 1: Baseline characteristics of the study population. Table 1: Baseline characteristics of the study population. Table 1: Baseline characteristics of the study population. Variables All colorectal carcinoma (𝑛= 131) Colon cancer (𝑛= 72) Rectal cancer (𝑛= 59) Age, mean (SD) 33.3 (7.9) 33.74 (8.0) 32.80 (8.0) 16–25 years (%) 24 (18) 12 (17) 12 (20) 26–35 years (%) 44 (34) 24 (33) 20 (34) 36–45 years (%) 63 (48) 36 (50) 27 (46) Gender, male (%) 87 (66.4) 50 (69.4) 37 (62.7) Bleeding P/R (%) 68 (51.9) 18 (25) 50 (84.7) Abdominal lump (%) 16 (12.2) 12 (16.7) 4 (6.8) Intestinal obstruction (%) 29 (22.1) 21 (29.2) 8 (13.6) Stage at diagnosis (%) Stages 1 and 2 34 (26) 22 (31) 12 (20) Stages 3 and 4 97 (74) 50 (69) 47 (80) Grade of tumor (%) Grade 1 8 (6.1) 4 (5.6) 4 (6.8) Grade 2 73 (55.7) 47 (65.3) 26 (44.1) Grade 3 50 (38.2) 21 (29.2) 29 (49.2) Presence of signet ring cell morphology (%) 28 (21) 6 (8) 22 (37) Presence of polyps (%) 10 (7.6) 9 (12.5) 1 (1.7) carcinoma cohort (49% versus 41%). Of the patients who maintained regular follow-up, a higher proportion of patients were found to be in remission in the colon cancer group (46% versus 39%). Conversely, a higher proportion of patients with rectal carcinoma had died at the time of analysis (58% versus 51%) (Figure 2).h ring morphology was identified in 28 patients (21%), per- ineural invasion in 21 (22%), and lymphovascular invasion in 25 (26%) of surgical specimens. 76% of patients undergoing curative surgery had undergone appropriate lymph node dissection (>12 nodes).t When right sided colon cancers were compared to left sided colon cancers, no statistically significant differences were found in histopathological characteristics between these two groups. The median time to progression for the 60 patients (45.8%) who had either died or were progressing at last follow-up was 8.25 months (IQR = 3.6–16.3 months). 2. Methods This figure was 9 months for the patients with colon cancer (𝑛= 27) and 7.5 months for the patients with rectal carcinoma (𝑛= 33). The median progression-free survival (PFS) in the 2 groups was not statistically different (𝑃= 0.70). On the other hand, there was a greater preponderance of poor prognostic features such as poorly differentiated carcinoma (49% versus 29%), signet ring morphology (37% versus 8%), and advanced stage (80% versus 69%) in the rectal carcinoma group as compared to patients with disease of the colon. f Median OS for the entire cohort was 19 months (IQR = 12.6–33.6 months). The median OS was similar between the right sided and left sided colonic cancer groups and was also not statistically different between the cohorts of colon cancer and rectal carcinoma (𝑃= 0.58). Kaplan-Meier analysis, however, revealed a trend towards an inferior survival for rectal carcinoma 2 years after initial diagnosis (Figure 3). 3.3. Treatment. 92 patients (70%) were treated with curative intent, while the rest had presented with metastatic disease at diagnosis and received palliative treatment. Half of the patients treated with curative intent had rectal cancer.h The rectal carcinoma group had a higher percentage of patients who completed appropriate therapy (96% versus 65%) (Figure 1). FOLFOX (oxaliplatin, calcium leucovorin, and 5-fluorouracil) was used as first line adjuvant treatment in 87% of patients (𝑛= 54) who received adjuvant therapy (Figure 1). t On univariate analysis signet ring morphology, presence of polyps, appropriate reception of therapy, poorly differenti- ated tumor, and late stage at presentation were found signif- icantly different between the two groups (Table 2). However, on multivariable analysis, only appropriate reception of ther- apy (PR 4.99; 95% CI, 1.21–20.6) and signet ring morphology (PR 2.40; 95% CI, 1.33–4.32) remained significantly associ- ated with rectal cancers as opposed to colon cancer (Table 2). 3.4. Outcome Analysis. At the time of analysis, 61 patients (47%) had been lost to follow-up either during or after completion of treatment of which 36 (59%) were in remission and 22 (41%) had progressive disease. Of the remaining 70 patients (53%) who had maintained regular follow-up, 38 patients (54%) had died, 30 (43%) were in remission, and further 2 (3%) were still undergoing treatment. 4. Discussion The definition of “early onset” CRC varies in the literature. Most have defined “early onset” as <40 years though upper limits ranging from 30 to 50 years have been used [18]. Epidemiological surveys indicate that the incidence of col- orectal cancers is much lower in the developing countries. A higher proportion of patients with colon carcinoma were lost to follow-up when compared with the rectal 4 ISRN Oncology ISRN Oncology ISRN Oncology Curative intent Completed appropriate treatment Yes No 74 patients 44/46 rectal (96%) 30/46 colon (65%) 18 patients underwent surgery alone despite needing neoadjuvant/ adjuvant treatment 2/46 rectal (4%) 16/46 colon (35%) (Neo)adjuvant 41 rectal 21 colon 3 rectal 9 colon 54 out of 62 patients received FOLFOX as the adjuvant regimen 8 out of 62 patients received other adjuvant regimens A total of 30 patients underwent surgery alone 12 appropriately 18 inappropriately (n = 92) Colon (n = 46) Rectum (n = 46) CCRT/chemo = 62 Only surgery = 12 Figure 1: Flowchart of patients treated with curative intent. Completed appropriate treatment Completed appropriate treatment 74 patients 44/46 rectal (96%) 30/46 colon (65%) Figure 1: Flowchart of patients treated with curative intent. Table 2: Univariate and multivariable analysis. Variable Univariate analysis Multivariable analysis PR CI PR CI Received appropriate treatment 5.35 1.30–22.1 4.99 1.21-20.6 Signet ring morphology 2.19 1.29–3.71 2.40 1.33-4.32 Poorly differentiated tumor 1.56 0.94–2.61 — Stage III or IV at presentation 1.37 0.73–2.59 — Presence of polyps 0.21 0.03–1.50 — Table 2: Univariate and multivariable analysis. Conversely, there have been reports that suggest a higher incidence of early onset disease in the developing world [19].hf The results of this meta-analysis and several other studies indicate that sporadic early onset colon cancer is rare in the developed countries. On the other hand, early onset CRC is far more common in third world countries [8, 13]. We report a prevalence of sporadic early onset CRC of 32% at our institution during the study period which is substantially higher than what western data suggests [4]. This is also higher than estimates from other Asian studies [7, 16, 21], although it was comparable to that from India [8, 22].h Conversely, there have been reports that suggest a higher incidence of early onset disease in the developing world [19]. 4. Discussion The exact cause of this difference is not known; however, it can be postulated that the higher proportion of younger patients in the underdeveloped countries can be a reason for this skewed finding [16]. However, more interesting is the hypothesis that sporadic early onset colorectal cancer is a biologically and clinically distinct entity, accounting for its aggressive presentation and poorer survival [16, 17, 20]. There are several potential explanations for this interest- ing observation. First of all this study is an institution based study where selection bias cannot be avoided. Moreover, a tertiary care center such as ours would ensure greater concern In a meta-analysis, the proportion of young CRC patients (less than 40 years) among all CRC patients ranged from 0.4% to 36.5% with an average of 7% [4]. The majority of these studies were conducted in North America and Europe. 5 ISRN Oncology Colorectal carcinoma Colon cancer Rectal cancer 29 26 32 23 24 22 1 1 2 47 49 44 Outcome (%) Died In remission On treatment Lost to follow up Figure 2: Outcome of the study population. 0.00 0.25 0.50 0.75 1.00 Survival proportion 0 20 40 60 80 100 120 Time (months) Colon Rectum Overall survival by diagnosis Figure 3: Median overall survival for colon and rectal carcinomas. Colorectal carcinoma Colon cancer Rectal cancer 29 26 32 23 24 22 1 1 2 47 49 44 Outcome (%) Died In remission On treatment Lost to follow up Figure 2: Outcome of the study population. Colorectal carcinoma Colon cancer Rectal cancer 29 26 32 23 24 22 1 1 2 47 49 44 Outcome (%) Died In remission On treatment Lost to follow up Figure 2: Outcome of the study population. our institution with almost a quarter of young adults in the country (aged 18–25) being current smokers [24]. The fact that data on these lifestyle factors could not be collected in our study secondary to the retrospective nature of data collection highlights one of our limitations. Interestingly, SEER (surveillance, epidemiology, and end results) database analysis from 1998–2002 indicates that there was a higher proportion of patients with early onset CRC in Asians of Indian and Pakistani origin, despite residing in USA for many years [25]. 4. Discussion This finding points to a possible genetic or epigenetic etiology for development of early onset CRC in this specific population bearing in mind the fact that chromosomal instability (CNI) and microsatellite instability (MSI) do occur in sporadic cases of CRC [26, 27]. The established increased propensity of developing a colorectal malignancy in this particular subgroup of patients has led to calls for revision of screening guidelines in the Asian popula- tion to include the high risk younger age groups [13], but current guidelines remain almost identical to those adapted by the developed countries [28]. Figure 2: Outcome of the study population. 0.00 0.25 0.50 0.75 1.00 Survival proportion 0 20 40 60 80 100 120 Time (months) Colon Rectum Overall survival by diagnosis Figure 3: Median overall survival for colon and rectal carcinomas. Overall survival by diagnosis An almost equal prevalence of colon and rectal cancer that we found concurred with the ratio seen in countries which have historically been labeled as “low risk” [13, 29]. A worse stage at presentation, poorer grade of tumor, and an increased prevalence of signet ring morphology in our study population were similar to those reported globally reflecting the aggressive nature of the disease in young CRC patients [4, 30, 31].h The fact that only 80% of patients who were planned for curative therapy received appropriate therapy (surgical/neo- adjuvant/adjuvant) is a matter of concern as it leaves this young population at a higher risk for recurrence in the future. The problem can be attributed to the fact that Pakistan is a resource-limited country with most of the health expendi- ture derived from out-of-pocket payment and the financial burden incurred as a result translates into poor adherence to standard treatment guidelines in most defaulting cases.h Figure 3: Median overall survival for colon and rectal carcinomas. The median OS for our cohort of young CRC patients was 19 months. Studies from neighboring countries with similar socioeconomic status such as Nepal and India have shown a dismal survival for young CRC patients [11, 32]. As we would expect, the survival data is varied globally and some studies have suggested that younger age either had no effect or a posi- tive effect on survival [4, 31, 33–35]. However, this is debatable and needs to be taken in context with the geographical location, tumor biology, and appropriateness of treatment. 4. Discussion and provide better facilities for cancer detection than those available generally in the country. This may result in inflation of the prevalence results derived. Secondly, the presence of a very large proportion of the population which is “young” [6] increases the number of “at-risk” subjects and may be responsible for a larger number of cases. p g A large collection of available data suggests that several dietary and lifestyle factors may significantly contribute to the risk of development of CRC [23]. Of these, physical inactivity, excess body weight, and central deposition of adiposity are established risk factors [23] and probably play an prominent role in the pathogenesis of the disease in our population where the prevalence of these factors is unacceptably high [5]. Westernization of the diet with resultant overconsumption of energy and developing insulin resistance may contribute to the prevalence in the more affluent sector of the country’s population, while the lack of specific macronutrients and certain other dietary ingredients in meals may play a part in the under privileged strata [23]. Smoking early in life, which is another established risk factor for the development of colon cancer, may also play a part in the high incidence observed at It is important to bear in mind that our poor survival results may have been exaggerated to a certain extent sec- ondary to the substandard follow-up of some of our patients and the high lost to follow-up rate witnessed in our study when compared to standards of the West and the well- developed Asian countries. This is a major limitation of all the data that comes out of Pakistan as patients in our country are not supported by the state or insurance. In a country where the gross national income per capita is only $1,050 [36], patients are commonly faced with difficult choices and usually opt not to pursue regular follow-up and investigational visits especially if deemed “cured.” It can be extremely challenging to stay in touch with these patients as 6 6 ISRN Oncology ISRN Oncology a significant percentage do not have telephonic access as well. This leaves us with incomplete data pertaining to our patients and potential exaggeration of the poor survival we report due to missing information on “survivors.” early onset colorectal cancer is high. Authors’ Contribution Muhammad Nauman Zahir did the literature search and drafted the paper. Munira Shabbir-Moosajee and Kulsoom Ghias conceived the research paper, drafted the research protocol, and provided guidance for the drafting of the paper. Eisha Mahpara Azhar and Sobia Rafiq did the data collection and the data entry and helped in drafting the paper. All authors read and approved the final paper. The strengths of our study include the fact that this is the first report of young CRC patients from the country. It is also unique in that it compares the different aspects of the 2 subgroups of CRC including the completion of appropriate treatment and provides us with an estimate of the survival of these patients, data regarding which is extremely scarce from our region. References [1] A. Jemal, F. Bray, M. M. Center, J. Ferlay, E. Ward, and D. Forman, “Global cancer statistics,” CA Cancer Journal for Clini- cians, vol. 61, no. 2, pp. 69–90, 2011. Conflict of Interests The authors declare that there is no conflict of interests regarding the publication of this paper. Abbreviations CRC: Colorectal carcinoma AKUH: Aga Khan University Hospital AJCC: American Joint Committee on Cancer SPSS: Statistical Package for Social Sciences HNPCC: Hereditary nonpolyposis colorectal carcinoma FAP: Familial adenomatous polyposis OS: Overall survival FOLFOX: Oxaliplatin, calcium leucovorin, and 5-fluorouracil IQR: Interquartile range PFS: Progression-free survival PR: Prevalence ratio CI: Confidence interval SEER: Surveillance, epidemiology, and end results CNI: Chromosomal instability MSI: Microsatellite instability. p g j Although the median OS was not statistically different between the two subgroups, analysis revealed a trend towards inferior prognosis for rectal carcinoma 2 years after ini- tial diagnosis. This, despite the fact that a higher number of patients in this same subgroup completed appropriate therapy, raises the possibility of rectal carcinoma being an aggressive subset of the young CRC population. Further weight is added to this hypothesis by studies conducted in the past which have shown significant difference in survival between colon and rectal cancers [38, 39]. A Turkish study in particular documented a 5-year survival rate of 81% for colon cancer which was more than double that for rectal carcinoma (39%) in patients less than 40 years of age [38]. This opens doors to evaluate differences in genetics, biology, severity, and prognosis of rectal cancers from colon cancers in general.h 4. Discussion Bench research is also required to elucidate the genetic, pathophysiological, and molecular characteristics of this high risk population whereby early detection could be made through molecular testing and the management modified accordingly. During the course of the study, an interest developed to compare the subgroups of the disease (colon versus rectal). This idea was instigated by a previous study which concluded that distinct mechanisms of oncogenesis may be responsi- ble for tumors of the rectum [37]. This subgroup analysis revealed that signet ring morphology of the tumor cells and poorly differentiated carcinomas, which have historically been regarded as poor prognostic factors [4, 30, 31], were significantly more prevalent in the rectal group. Another interesting fact that was highlighted was the statistically significant proportion of rectal cancer patients completing appropriate therapy as opposed to patients with colon carci- noma. This difference probably arises from the chronology of treatment for the two subgroups whereby rectal carcinoma patients receive neoadjuvant therapy, whereas patients with colon cancer undergo surgery first giving them a false sense of security of having achieved “cure” and dampening their motivation of pursuing adjuvant treatment.f 5. 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https://openalex.org/W4206835146	https://phsreda.com/e-articles/10348/Action10348-100665.pdf	Russian	null	Propedevtika endokrinnykh zabolevanii (metody issledovaniia bol'nykh s zabolevaniiami endokrinnoi sistemy)	Publishing house Sreda eBooks	2,022	cc-by	26,755	И. В. Терещенко Терещенко И. В. Пропедевтика эндокринных заболеваний (методы исследования больных с заболеваниями эндокринной системы) : монография / И. В. Терещенко. – Чебоксары: ИД «Среда», 2022. – 104 с. Терещенко И. В. Пропедевтика эндокринных заболеваний (методы исследования больных с заболеваниями эндокринной системы) : монография / И. В. Терещенко. – Чебоксары: ИД «Среда», 2022. – 104 с. Т35 DOI 10.31483/a-10348 ISBN 978-5-907411-89-0 Монография Монография УДК 616.4 ББК 54.15 Т35 Автор: Терещенко Ирина Владимировна – д-р мед. наук, почетный профессор ФГБОУ ВО «Пермский государственный медицинский университет им. А.Е. Вагнера». УДК 616.4 ББК 54.15 Т35 Терещенко Ирина Владимировна – д-р мед. наук, почетный профессор ФГБОУ ВО «Пермский государственный медицинский университет им. А.Е. Вагнера». ISBN 978-5-907411-89-0 Постановка эндокринного диагноза – всегда сложная проблема. Без знаний пропедевтики эндокринных заболеваний невозможен правильный диагноз. В первом разделе данного пособия излагается методика опроса жалоб пациентов с эндокринной патологией, правила сбора анамнеза за- болевания и анамнеза жизни больных с эндокринопатиями, также рас- сматриваются особенности опроса по системам органов при гормональ- ных нарушениях. Даются указания, на что должен обратить внимание врач, проводя объективное исследование эндокринного больного. Во вто- ром разделе проанализированы основные эндокринные синдромы, что важно для диагностики и дифференциальной диагностики эндокринных заболеваний. Раздел третий посвящён современным лабораторным и ин- струментальным методам исследования в практической эндокринологии. При подготовке пособия автором использован многолетний клиниче- ский опыт. Знание пропедевтики эндокринных заболеваний поможет в практической работе как начинающему врачу, так и врачу с клиническим опытом избежать диагностических ошибок. DOI 10.31483/a-10348 © Терещенко И. В., 2022 ISBN 978-5-907411-89-0 © ИД «Среда», оформление, 2022 © Терещенко И. В., 2022 © ИД «Среда», оформление, 2022 DOI 10.31483/a-10348 ISBN 978-5-907411-89-0 Содержание Содержание Содержание Список сокращений ................................................................................. 4 Введение ...................................................................................................... 5 Раздел 1. Методика расспроса и осмотра эндокринных больных ....................................................................................................... 6 Жалобы больных с заболеваниями эндокринной системы ................ 6 Изучение анамнеза заболевания у эндокринного больного ............ 14 Опрос по системам органов ................................................................. 15 История жизни больного ...................................................................... 18 Объективное обследование эндокринного больного. Общий осмотр больного с заболеванием эндокринной системы .............................. 19 Раздел 2. Эндокринные синдромы ...................................................... 39 Раздел 3. Лабораторные и инструментальные методы исследования больных при диагностике эндокринной патологии ................................................................................................. 61 Раздел 4. Клинические примеры диагностических ошибок (анализ причин ошибочных диагнозов) ............................................ 80 Приложения ............................................................................................. 93 Тестовые задания для самоконтроля усвоения материала ............... 93 Ситуационные задачи ........................................................................... 96 Ответы на тестовые задания................................................................. 99 Ответы на вопросы ситуационных задач ........................................... 99 Дефиниция избранных медицинских терминов .............................. 102 3 Пропедевтика эндокринных заболеваний Список сокращений АГ – артериальная гипертензия АД – артериальное давление АКТГ – адренокортикотропный гормон АЛТ – аланинаминотрансфераза АСТ – аспартатаминотрансфераза АТ – ТГ – антитела к тиреоглобулину АТ – рТТГ – антитела к рецепторам тиреотропина АТ – ТПО – антитела к тиреопероксидазе ВДКН – врождённая дисфункция коры надпочечников ВЭМ – велоэргометрия ГаммаГТП – гаммаглютамилтранспептидаза Гли НвА1с – гликированный гемоглобин ДГТ – дигидротестостерон ДГЭА – дигидроэпиандростерон ЖКТ – желудочно-кишечный тракт ИМТ – индекс массы тела ЛГ – лютеинизирующий гормон МСГ – меланоцитостимулирующий гормон НАЖБП – неалкогольная жировая болезнь печени ПРЛ – пролактин ПЮД – пубертатно-юношеский диспитуитаризм Св. Т3 – свободный трийодтиронин Св. Т4 – свободный тироксин СД2 – сахарный диабет 2-го типа СПКЯ – синдром поликистозных яичников СТГ – соматотропный гормон ТТГ – тиреотропный гормон УЗИ – ультразвуковое исследование ФСГ – фолликулостимулирующий гормон ХБП – хроническая болезнь почек ХПН – хроническая почечная недостаточность ЦНС – центральная нервная система ЭКГ – электрокардиография Список сокращений И.В. Терещенко И.В. Терещенко Введение Академик В.Г. Баранов называл эндокринологию «высшим пи- лотажем в медицине» и тем самым подчёркивал сложность этой науки. В учебниках по пропедевтике внутренних заболеваний обычно меньше внимания уделяется вопросам обследования эндо- кринного больного, чем пациента с другой терапевтической пато- логией. В монографиях по эндокринным заболеваниям, как пра- вило, методы исследования эндокринной системы излагаются схе- матично. Многие важные детали пропедевтики эндокринных бо- лезней остаются неизвестными для студента, а в результате пробел в знаниях сохраняется и у врача. На практике эндокринологу не- редко приходится сталкиваться с пациентами, у которых не был по- ставлен диагноз эндокринного заболевания в течение многих лет, или, наоборот, с гипердиагностикой эндокринопатий, нередко «ле- чат» не больного, а его анализы. Именно это послужило мотивом написания данного руководства, при составлении которого исполь- зован многолетний клинический опыт автора и уделено внимание важным для диагностики, прежде всего клиническим симптомам эндокринных заболеваний. Ключевые слова: эндокринология; диагностика эндокринных за- болеваний; симптоматика эндокринных заболеваний; пропедев- тика. Key words: endocrinology; diagnostics of endocrine diseases; symp- toms of endocrine diseases; propaedeutics. 5 5 Пропедевтика эндокринных заболеваний алобы больных с заболеваниями эндокринной системы Жалобы больного с заболеванием эндокринной системы зависят прежде всего от гормональных нарушений, а именно, какая эндо- кринная железа поражена, возникла её гипо- или гиперфункция. Однако, можно выделить жалобы, которые встречаются при мно- гих эндокринных заболеваниях (табл. 1). Основные жалобы эндокринологических больных Группы жалоб Проявления Неврологические Головные боли Нарушения сна Расстройства интеллекта, памяти, внимания, эмоциональной сферы Судороги Тремор Астенический симптомокомплекс Жалобы на изменение внешнего вида Нарастание массы тела Похудение (истощение) Отставание в физическом развитии Опережение физического развития Низкорослость Высокорослость Увеличение оконечностей Уменьшение роста Внешние признаки противоположного пола Изменение внешнего вида из-за отёков Половые расстройства У женщин расстройство менструальной функции У мужчин импотенция Бесплодие Снижение либидо или изменение полового влечения Нарушения со стороны органа зрения Снижение остроты зрения или слепота Пучеглазие Двоение в глазах Слезотечение Светобоязнь 6 И.В. Терещенко Окончание таблицы 1 Группы жалоб Проявления Нарушения влечений Жажда Отсутствие аппетита Повышенное чувство голода Отсутствие чувства насыщения Извращение вкуса Прочие жалобы Повышение температуры Повышенная зябкость Кардиальные (сердцебиение, одышка, перебои сердца, ощущение усиленной пульсации и т.д.) Запоры Повышенная потливость, зуд Даже незначительный дефицит или избыток любого гормона в организме, в первую очередь, вызовет изменения функции цен- тральной нервной системы. Головные боли наблюдаются при аденомах гипофиза, гипота- ламо-гипофизарной недостаточности, сахарном диабете, инсули- номах, заболеваниях щитовидной железы, гипогонадизме, патоло- гии надпочечников (гипокортицизме и гормонально-активных опу- холях надпочечников). Патогенетические механизмы возникнове- ния этих цефалгий различны. Поэтому характеристика головных болей будет также отличаться. Встречаясь с жалобами на головную боль, врач должен выяснить, постоянная головная боль или возни- кает периодически. Должен уточнить её локализацию (лоб, виски, темя, затылок, диффузные головные боли по всей голове), расспро- сить о характере головных болей (тупые, острые, распирающие, да- вящие и т.д.), о причинах, вызывающих или усиливающих голов- ную боль, а также, что облегчает или полностью устраняет голов- ные боли. При аденомах гипофиза (акромегалии, пролактиноме, гормо- нально-неактивных опухолях и т. д.) из-за повышения внутриче- репного давления головные боли отличаются постоянством. Не- редко больные рассказывают: «Даже во сне чувствую, как болит голова». При этом приём анальгетиков не приносит облегчения. Как правило, при аденомах гипофиза головные боли тупые, но пе- риодически (при утомлении, напряжении, перегревании и т. д.) обостряются и становятся интенсивными. 7 7 7 Пропедевтика эндокринных заболеваний Цефалгии встречаются при многих (точнее – при всех) заболе- ваниях щитовидной железы. алобы больных с заболеваниями эндокринной системы При тиреотоксикозе они обусловлены гипоксией ЦНС и поэтому непостоянны, возникают при волнении, спешке, утомлении, появляются в душном помещении и в жаре, к которой так чувствительны пациенты с болезнью Грейвса (тирео- токсикозом), при этом интенсивность, локализация, продолжитель- ность головных болей может быть различной. Обычно цефалгии при тиреотоксикозе сопровождаются усилением раздражительно- сти, плаксивости, невозможностью уснуть, прерывистым сном. При гипотиреозе цефалгии тоже обусловлены гипоксией, но кроме того, при дефиците тиреоидных гормонов в организме (осо- бенно при микседеме) развивается отёчность тканей, в том числе, мозга. Из-за внутричерепной гипертензии головные боли приобре- тают сходство с цефалгиями при опухолях гипофиза. Многие эндокринные заболевания характеризуются повыше- нием АД. Это синдром и болезнь Иценко-Кушинга, феохромоци- тома, альдостерома, акромегалия и др. (см. раздел 2 «Синдром эн- докринной артериальной гипертонии»). В этих случаях головные боли зависят от уровня АД, обычно начинаются в затылочной об- ласти, могут сопровождаться шумом в ушах, неприятными ощуще- ниями пульсации в висках. Чем выше АД, тем интенсивнее голов- ная боль у этих больных. Нередко повышение АД может быть проявлением коморбидно- сти гипертонической болезни с эндокринным заболеванием, что важно диагностировать. Такая сочетанная патология встречается особенно часто при СД2, ожирении, климаксе. Головные боли, вызванные латентными гипогликемиями, воз- можны при гипоталамо-гипофизарной недостаточности, аддисоно- вой болезни, гипотиреозе, сахарном диабете, ожирении. По харак- теру эти цефалгии тупые, разлитые, длительные, но непостоянные, зависят от степени снижения уровня глюкозы в крови, могут воз- никать в разное время, в том числе и ночью, сопровождаться ощу- щением дурноты, желанием срочно поесть, подавленным настрое- нием, чувством тоски, а нередко конфликтностью, раздражитель- ностью, агрессией. Помимо головных болей, изменения функции ЦНС при эндо- кринных заболеваниях могут проявляться нарушениями сна. Жа- 8 И.В. Терещенко лобы на бессонницу или прерывистый сон обычно предъявляют па- циентки, страдающие патологическим климаксом. При болезни Грейвса чаще всего невозможно нормальное засыпание. При гипо- тиреозе, наоборот, появляется патологическая сонливость: больной может уснуть в транспорте, на работе, за рулём и даже во время осмотра врача. Нарушение формулы сна (сонливость днём и бес- сонница ночью) возникает у подростков с ПЮД. При морбидном ожирении (синдроме Пиквика) наблюдается выраженная сонли- вость, усиливающаяся после еды. лобы на бессонницу или прерывистый сон обычно предъявляют па- циентки, страдающие патологическим климаксом. При болезни Грейвса чаще всего невозможно нормальное засыпание. При гипо- тиреозе, наоборот, появляется патологическая сонливость: больной может уснуть в транспорте, на работе, за рулём и даже во время осмотра врача. Нарушение формулы сна (сонливость днём и бес- сонница ночью) возникает у подростков с ПЮД. алобы больных с заболеваниями эндокринной системы При морбидном ожирении (синдроме Пиквика) наблюдается выраженная сонли- вость, усиливающаяся после еды. Расстройства памяти, внимания, интеллектуальные затруд- нения, нарушения эмоциональной сферы встречаются при многих (если не при всех) эндокринных заболеваниях. Например, дети с не леченным вовремя врождённым гипотиреозом не поддаются обу- чению в общеобразовательной школе. Жалобы на когнитивные расстройства характерны для пациентов с гипотиреозом, в том числе субклиническим, с болезнью и синдромом Иценко-Кушинга, гипокортицизмом, возникают при длительной декомпенсации са- харного диабета и т. д. Раздражительность, плаксивость – специфи- ческие жалобы пациента с тиреотоксикозом, а подавленное настро- ение, депрессия типичны для гипотиреоза. Пациенты с евнухоидиз- мом отличаются злобностью. Жалобы на судороги мышц лица и конечностей, ощущение пол- зания мурашек, онемение конечностей наблюдаются у больных ги- попаратиреозом, обусловлены гипокальциемией. При сахарном диабете тоже может возникать чувство онемения конечностей, а также боли по ходу периферических нервных стволов. Это признак присоединения осложнения диабета – диабетической сенсорной полинейропатии. Боли в руках, онемение пальцев рук, боли в пояс- нице с иррадиацией в нижние конечности бывает первым проявле- нием акромегалии: нервные корешки сдавливает и раздражает раз- растающаяся костная ткань, что обусловлено гиперсекрецией гор- мона роста опухолью гипофиза. Онемение пальцев кистей и ладоней с ощущением длительной жгучей боли, покалывания наблюдается при синдроме запястного канала (карпальный туннельный синдром), может возникнуть при гипотиреозе, акромегалии, ожирении, сахарном диабете, болезни Педжета, обусловлен сдавлением срединного нерва между ко- 9 9 Пропедевтика эндокринных заболеваний стями, поперечной кистевой связкой и сухожилиями мышц запя- стья. Перкуссия предплечья над местом прохождения срединного нерва, сдавление манжеткой руки при измерении АД вызывают па- рестезии и боли в пальцах и ладонях. Также существуют туннель- ные синдромы ног, шеи, туловища. Ощущения имеют непостоян- ный характер и связаны с деятельностью, нагрузками. стями, поперечной кистевой связкой и сухожилиями мышц запя- стья. Перкуссия предплечья над местом прохождения срединного нерва, сдавление манжеткой руки при измерении АД вызывают па- рестезии и боли в пальцах и ладонях. Также существуют туннель- ные синдромы ног, шеи, туловища. Ощущения имеют непостоян- ный характер и связаны с деятельностью, нагрузками. Астенический симптомокомплекс наблюдается при очень мно- гих эндокринных заболеваниях. Самая выраженная астения вызы- вается снижением калия в крови (так называемая гипокалиемиче- ская слабость). При этом больные не могут без посторонней по- мощи войти в транспорт. Чтобы встать с сидения, они должны опе- реться на руки. Из-за слабости им трудно держать ложку и т. д. Ги- покалиемическая слабость характерна для болезни Иценко–Ку- шинга, глюкостеромы, альдостеромы. При альдостероме слабость может сопровождаться периодическими параличами (миоплегией). р р р ( ) Клинический пример. алобы больных с заболеваниями эндокринной системы Больная В-ва, 47 лет, доцент педагогиче- ского университета. Внезапно по пути на работу почувствовала, что «ноги парализовало». После отдыха движения в ногах восста- новились полностью. В дальнейшем подобные эпизоды повторя- лись часто при ходьбе, подъёмах по лестнице, спешке. Врачами ставился диагноз «истерия». Через 3 года присоединилась АГ, ре- зистентная к антигипертензивной терапии. Госпитализирована в кардиологическое отделение на обследование. При проведении ВЭМ возникла миоплегия. Заподозрена альдостерома. Диагноз подтвердился. После удаления опухоли миоплегии прекратились. Жалоба на слабость может быть ведущей при манифестации са- харного диабета или может служить признаком его декомпенса- ции: при дефиците инсулина мышцы лишаются запасов гликогена. При тиреотоксикозе тоже выражен астенический синдром, так как наблюдается дефицит макроэргов (АТФ), возникает гипотрофия мышечной ткани, а также влияет электролитный дисбаланс. Значи- тельная общая слабость характерна для гипотиреоза, поскольку мышечная ткань при этом заболевании отёчна; в мышечных клет- ках накапливается «невостребованный» креатинфосфат. Жалобы на слабость предъявляют больные аддисоновой болезнью. Из-за ги- перкалиемии, снижения уровня глюкозы в крови, дефицита надпо- чечниковых половых стероидов (анаболиков), ответственных за синтез мышечного белка, слабость при гипокортицизме достигает 10 И.В. Терещенко степени прострации. Например, пациент с аддисоновой болезнью из-за слабости не может самостоятельно без посторонней помощи изменить неудобную позу в постели. Выраженная слабость наблю- дается при гипогонадизме, так как половые гормоны, особенно ан- дрогены, обеспечивают развитие скелетной мускулатуры. Нередко подростки с отставанием в половом развитии не справляются со школьной программой по физкультуре. степени прострации. Например, пациент с аддисоновой болезнью из-за слабости не может самостоятельно без посторонней помощи изменить неудобную позу в постели. Выраженная слабость наблю- дается при гипогонадизме, так как половые гормоны, особенно ан- дрогены, обеспечивают развитие скелетной мускулатуры. Нередко подростки с отставанием в половом развитии не справляются со школьной программой по физкультуре. Жалобы на изменение внешнего вида встречаются при многих эндокринных заболеваниях. Прежде всего, это касается массы тела. Полнота беспокоит больных с метаболическим синдромом, болез- нью (синдромом) Иценко–Кушинга, конституционально-экзоген- ным или послеродовым ожирением, пубертатно-юношеским дис- питуитаризмом, гипотиреозом, инсулиномой. Врач обязан уточ- нить, каков у пациента аппетит, какие лекарства он принимал и принимает, темпы нарастания массы тела, где откладывается жир. Жалобы на похудение (истощение) предъявляют больные ти- реотоксикозом, нервной анорексией, гипоталамо-гипофизарной недостаточностью, аддисоновой болезнью, сахарным диабетом 1-го типа, причём при тиреотоксикозе, сахарном диабете аппетит может быть повышен, но больные тем не менее худеют. При гипо- питуитаризме, гипокортицизме, наоборот, выражена анорексия. Также как проявление интоксикации из-за избытка паратгормона похудение беспокоит больных с гиперпаратиреозом. Жалобы на отставание или опережение сверстников по росту и физическому развитию нередко беспокоят детей или подростков. алобы больных с заболеваниями эндокринной системы Терещенко И.В. Терещенко алобы больных с заболеваниями эндокринной системы Низкорослость встречается при таких эндокринопатиях: при заболе- ваниях, связанных с недостаточностью гормона роста, при гипоти- реозе, особенно врождённом, при длительной декомпенсации сахар- ного диабета 1-го типа у ребёнка, при избытке или недостатке поло- вых гормонов (см. раздел 2 «Синдром низкорослости»). Высокорос- лость может быть проявлением конституциональных особенностей человека или признаком заболеваний – гигантизма, евнухоидизма, болезни Марфана. У взрослых пациентов, несмотря на закрытие зон роста костей, при гиперсекреции СТГ также возможно изменение внешнего облика. Больные акромегалией жалуются на рост оконеч- ностей: увеличение носа, ушей, языка, нижней челюсти, кистей, стоп и т. д. По поводу уменьшения роста в ряде случаев обращаются 11 Пропедевтика эндокринных заболеваний пациенты, страдающие синдромом (болезнью) Иценко–Кушинга, гиперпаратиреозом, остеопорозом. пациенты, страдающие синдромом (болезнью) Иценко–Кушинга, гиперпаратиреозом, остеопорозом. Нередко мотивом обращения к врачу эндокринных больных служит появление внешних признаков противоположного пола. Мужчины могут жаловаться на рост молочных желёз, а женщины на оволосение лица, огрубение кожи, обилие вульгарных угрей, сальности кожи, на залысины на лбу, облысение в области темени. Жалобы на нарушение половой функции бывают ведущими при многих гормональных расстройствах. Именно по этому поводу жен- щины вынуждены обращаться не к эндокринологу, а к гинекологу, а мужчины консультироваться с сексопатологом. Не только при дис- функции гонад, но при любой эндокринной патологии у пациенток может возникнуть расстройство менструальной функции: любое от- клонение от нормы цикличности месячных, продолжительности кро- вотечений, объёма кровопотерь называется аномальным маточным кровотечением. Жалоба пациентки на болезненные месячные тоже указывает на патологию (требуется назначить обследование). У муж- чин при любой гормональной дисфункции может возникнуть жалоба на импотенцию. Нередко пациент об этом умалчивает, необходимо о половой функции его расспросить. Жалобы на нарушение зрения встречаются при сахарном диа- бете, аденомах гипофиза, гипопаратиреозе, тиреотоксикозе. В связи с этим пациенты прежде всего обращаются к окулистам. Вы- яснение характера зрительных нарушений может помочь в поста- новке диагноза. Так, при сахарном диабете снижается острота зре- ния из-за поражения сетчатки – диабетической ретинопатии, а также при возникновении диабетической катаракты. При опухолях гипофиза страдает периферическое зрение. Например, больному трудно перейти улицу из-за отсутствия бокового зрения (латераль- ная гемианопсия). При гипопаратиреозе развивается катаракта на одном или обоих глазах, что снижает остроту зрения, а также у больных гипопаратиреозом может появиться светобоязнь, блефа- роспазм. Глазные жалобы при тиреотоксикозе варьируют: ощуще- ние распирания или песка в глазах, пучеглазие с одной или с двух сторон, двоение в глазах, слезотечение, резь в глазах, отёчность век, покраснение глаз, невозможность сомкнуть веки во время сна и др. Особенно ярко выражены глазные жалобы при аутоиммунной офтальмопатии. 12 И.В. Жалобы на нарушение аппетита. у В одних случаях у больных появляется повышенный аппетит или постоянное чувство голода. Например, при болезни Грейвса, инсуломе, при декомпенсации сахарного диабета, при ожирении. В других случаях аппетит снижен (при гипотиреозе, при кетоацидозе у больных с декомпенсированным сахарным диабетом) или отсут- ствует (при нервной анорексии, гипоталамо-гипофизарной недо- статочности, гиперпаратиреозе). При гипокортицизме возникает повышенная потребность в поваренной соли. Больной не всегда выскажет эту жалобу, но врач, заподозрив аддисонову болезнь, обязан это уточнить. Характерный признак инсуломы – патологи- ческая потребность в сахаре. Обычно больные с этим заболеванием постоянно носят кулёк с сахарным песком и ложку; при ощущении дурноты ложками потребляют сахар. При гипотиреозе аппетит от- сутствует в утренние часы; такие больные с трудом просыпаются после ночного сна и могут никогда не завтракать. Жалобы на повышенную жажду. Повышенной жаждой страдают больные с несахарным диабе- том, причём их жажду утоляет только холодная вода! Жалобы на жажду предъявляют пациенты сахарным диабетом, гиперпарати- реозом, альдостеромой (см. раздел 2 «Инсипидаоный синдром»). Кардиальные жалобы. При многих эндокринопатиях больные предъявляют кардиаль- ные жалобы. Нередко такие жалобы становятся ведущими. Так, ос- новным и часто первым симптомом тиреотоксикоза является по- стоянное сердцебиение, не прекращающееся в покое. Больные ощущают усиленную пульсацию в области сердца, в пятках, вис- ках. Учащенное сердцебиение сопровождается одышкой. Могут присоединяться боли в области сердца. Такая симптоматика вы- нуждает их обращаться в первую очередь не к эндокринологу, а к кардиологу. Кардиальные жалобы наблюдаются при гипотиреозе, сахарном диабете, феохромоцитоме, ожирении. Нарушения ритма сердца также нередко беспокоят больных с этими заболеваниями. Напри- мер, у них возможна фибрилляция предсердий. 13 Пропедевтика эндокринных заболеваний Изучение анамнеза заболевания у эндокринного больного Анамнез заболевания у эндокринного больного собирают по об- щим правилам. Однако имеются особенности, которым следует уделять особое внимание. Прежде всего, надо выяснить, когда стали наблюдаться первые симптомы настоящего заболевания или когда врачи установили первые признаки эндокринопатии. Так, в настоящее время вирильную форму ВДКН могут распознать уже во внутриутробном периоде. Кретинизм клинически проявляется при рождении, а спорадическая форма недостаточности гормона роста – в возрасте ≈ 3-х лет, ожирение чаще всего возникает после 30 лет и т. д. Симптомы многих эндокринопатий становятся яв- ными при эндокринной перестройке организма: в период пуберта- ции, климактерия, во время беременности, кормления грудью и т.д. Важно выяснить причины, вызвавшие эндокринную патологию. Например, гипотиреоз или гипопаратиреоз могут развиться после операции – резекции или экстирпации щитовидной железы, гипо- кортицизм – вследствие туберкулёзного процесса в надпочечниках, эндемический зоб – при дефиците йода в окружающей среде и т.д. Следует оценить, как развивалось заболевание, – остро или по- степенно. Так, сахарный диабет 1-го типа проявляется внезапно, быстро; сахарный диабет 2-го типа нередко выявляется случайно в связи с сопутствующими заболеваниями (гипертонической болез- нью, ИБС и др.) или при профилактическом осмотре, например, за- полнении санаторно-курортной карты. Важно проследить течение заболевания, динамику признаков гормональных нарушений: усиление, ослабление или исчезновение первичных симптомов, появление новых симптомов. Например, первым симптомом была головная боль, затем появилось ожире- ние, яркие стрии на коже живота, бёдер. Такая последовательность характерна для синдрома Иценко–Кушинга. Наоборот, на фоне многолетнего ожирения пациента стали беспокоить головные боли. Это не синдром Иценко–Кушинга; надо подумать, в первую очередь, о конституционально-экзогенном ожирении и присоеди- нении сосудистой патологии. 14 И.В. Терещенко Необходимо выяснить предыдущее лечение, в частности приме- нение гормонов, их дозы, эффективность, переносимость препара- тов, регулярность их приёма, осложнения гормонотерапии – эти сведения при сборе анамнеза заболевания у эндокринного боль- ного особенно важны. Следует расспросить о возможных осложне- ниях заболевания: какие осложнения, чем обусловлены, их дина- мика, лечение, профилактика. Так, у больного сахарным диабетом необходимо выяснять, были ли комы и какие, чем спровоцированы, где оказывалась неотложная медицинская помощь. Важно уточ- нить, не является ли настоящее заболевание рецидивом. Например, может рецидивировать болезнь Грейвса через несколько лет, при этом чувствительность к тиреостатикам нередко изменяется. Опрос по системам органов Эндокринный больной не всегда способен изложить свои жа- лобы и сообщить о развитии заболевания. Чем тяжелее гормональ- ная энцефалопатия, тем затруднительнее выяснить историю его за- болевания. Поэтому опрос по системам органов (и изучение имею- щихся медицинских документов) приобретает особую ценность, позволяет врачу помимо жалоб, которые беспокоят пациента в настоящее время и заставили его обратиться за медицинской помо- щью, выявлять и другие субъективные симптомы заболевания. Опрос эндокринного больного по системам органов проводят по общепринятой схеме. 1. Общее состояние больного: уточняется самочувствие боль- ного, наличие недомогания, слабости, утомляемости, снижения трудоспособности, зябкости, потливости, ознобов, чувства жара, повышения температуры, похудения или увеличения массы тела (уточнить, в какой степени, давно ли, причины). 1. Общее состояние больного: уточняется самочувствие боль- ного, наличие недомогания, слабости, утомляемости, снижения трудоспособности, зябкости, потливости, ознобов, чувства жара, повышения температуры, похудения или увеличения массы тела (уточнить, в какой степени, давно ли, причины). (у р ) 2. Состояние ЦНС и органов чувств: самооценка больного – спокойный, «нервный», раздражительный, вспыльчивый, апатич- ный, общительный или замкнутый. Сон – нормальный или нару- шен (чуткий, тревожный, прерывистый), лёгкость засыпания, сон- ливость или бессонница. Головокружение, шум в голове, обмо- роки, мелькание «мушек» перед глазами. Головные боли: характер, частота, длительность, время появления в течение суток, интенсив- ность, локализация. Память сохранена или ухудшилась. Судороги, онемение конечностей, отдельных участков тела. Зуд общий или 15 Пропедевтика эндокринных заболеваний локализованный, ощущение ползания мурашек. Жалобы на ухуд- шение зрения, слуха. Изменение осязания (ослабление, извраще- ние, отсутствие чувствительности). Извращение или отсутствие обоняния, а также – вкуса. локализованный, ощущение ползания мурашек. Жалобы на ухуд- шение зрения, слуха. Изменение осязания (ослабление, извраще- ние, отсутствие чувствительности). Извращение или отсутствие обоняния, а также – вкуса. 3. Кожа: сухость, повышенная потливость, пигментация (об- щая или отдельных участков), высыпания гнойничков, угревой сыпи, зуда. Появление розовых или багровых, или белесых полос растяжения в области живота, бёдер, ягодиц, молочных желёз, дру- гих участках. Усиленный рост волос на брюшной стенке, по белой линии живота, вокруг сосков, на конечностях. У женщин рост во- лос на лице (рост усов, бороды, бакенбард). Выпадение волос с во- лосистой части головы, ресниц, бровей, тотальное их выпадение; у мужчин – изменение интенсивности их роста. 3. Кожа: сухость, повышенная потливость, пигментация (об- щая или отдельных участков), высыпания гнойничков, угревой сыпи, зуда. Появление розовых или багровых, или белесых полос растяжения в области живота, бёдер, ягодиц, молочных желёз, дру- гих участках. Усиленный рост волос на брюшной стенке, по белой линии живота, вокруг сосков, на конечностях. Опрос по системам органов У женщин рост во- лос на лице (рост усов, бороды, бакенбард). Выпадение волос с во- лосистой части головы, ресниц, бровей, тотальное их выпадение; у мужчин – изменение интенсивности их роста. 4. Опорно-двигательный аппарат: боли в костях конечностей, позвоночника, в плоских костях, суставах, боли в мышцах (с чем связаны), уменьшение мышечной силы, ограничение подвижности в суставах. Уменьшение роста и насколько. 5. Система кровообращения: болевые ощущения в области сердца или за грудиной; их характер, иррадиация, частота появле- ния, интенсивность, продолжительность, условия, при которых они возникают, поведение пациента во время приступа боли, от каких мероприятий успокаиваются боли в сердце. Сердцебиения, пере- бои сердца, ощущения пульсации в разных областях тела и обсто- ятельства их появления. Одышка в покое или при физическом напряжении, периодическая или постоянная. Удушье. Отёки, их локализация, особенности появления, нарастания или ослабления, постоянные или периодически исчезающие, время появления, связь с физическим напряжением, потреблением жидкости. Тя- жесть в правом подреберье. 6. Система дыхания: дыхание через нос свободное или затруд- нённое, «насморк», выделения из носа (количество, характер, за- пах), носовые кровотечения (уточнить из какой половины носа или из обеих), ощущение сухости или болей в горле. Боли в грудной клетке при кашле, дыхании. Одышка, приступы удушья. Кашель и его особенности. Кровохарканье. 7. Система пищеварения: аппетит, насыщаемость, неприятный вкус во рту, потребность приёма пищи ночью. Глотание, прохож- дение пищи через пищевод. Тошнота, рвота и её особенности. Боли 7. Система пищеварения: аппетит, насыщаемость, неприятный вкус во рту, потребность приёма пищи ночью. Глотание, прохож- дение пищи через пищевод. Тошнота, рвота и её особенности. Боли 16 И.В. Терещенко в животе, локализация. Деятельность кишечника – поносы, запоры, характер стула. Боли в левом или правом подреберьях. в животе, локализация. Деятельность кишечника – поносы, запоры, характер стула. Боли в левом или правом подреберьях. 8. Система крови: кровотечения, кровоподтёки (локализация, величина). Обмороки, головокружения. Увеличение лимфатиче- ских узлов, локализация. 8. Система крови: кровотечения, кровоподтёки (локализация, величина). Обмороки, головокружения. Увеличение лимфатиче- ских узлов, локализация. у 9. Мочевыделительная система: особенности мочеиспуска- ния – затруднение, болезненность, примерная частота днём, ночью. Количество и особенности мочи. Боли в пояснице, их характер. Отёки и их особенность. 10. Половая функция: менструации – время появления послед- ней менструации (своевременность), болезненная, обильная, скуд- ная, продолжительность, регулярность, какие интервалы между ме- сячными. Боли в проекции органов малого таза, пояснице, в обла- сти крестца. У мужчин расспросить об эрекциях и эякуляции, ли- бидо, регулярности утренних эрекций. 10. Опрос по системам органов Половая функция: менструации – время появления послед- ней менструации (своевременность), болезненная, обильная, скуд- ная, продолжительность, регулярность, какие интервалы между ме- сячными. Боли в проекции органов малого таза, пояснице, в обла- сти крестца. У мужчин расспросить об эрекциях и эякуляции, ли- бидо, регулярности утренних эрекций. Такова примерная схема опроса по системам органов. Этот опрос очень важен для постановки диагноза и для дифференциаль- ной диагностики. Например, кажется лишним вопрос о насморке эндокринному пациенту. Но этот симптом часто появляется при ги- потиреозе из-за отёков слизистой полости носа. Или другой при- мер – извращение обоняния может быть симптомом аденомы гипо- физа (макроаденомы!). Кровотечения из одной половины носа не- редко обусловлены местными сосудистыми изменениями, но они могут сопровождаться спазмом сосудов гипофиза и гипоталамуса и приводить к гипоталамо-гипофизарной недостаточности. На фоне гормональной дисфункции часто возникает коморбидная па- тология. Опрос по системам органов поможет в её выявлении. 17 Пропедевтика эндокринных заболеваний История жизни больного У эндокринного больного анамнез жизни собирают по общим правилам. Однако, имеются детали, которым надо уделять особое внимание. Так, выясняя место рождения и дальнейшего прожива- ния пациента, надо расспросить, не является ли местность энде- мичной по зобу и была ли там налажена массовая йодная профи- лактика, или являлась зоной экологического бедствия (уточнить время и характер химического или радиационного загрязнения). Не было ли в последующие годы жизни частой смены места житель- ства в разных климатических и временных поясах. Важно учиты- вать возраст родителей при рождении ребёнка. Хромосомные ано- малии – синдром Шерешевского–Тернера и синдром Клайнфель- тера – чаще встречаются у возрастных родителей. Вопрос, как пи- талась мать, вынашивая беременность, принципиален, если у боль- ного ожирение. Большое количество углеводов и жиров в рационе беременной женщины приводит к рождению ребёнка с увеличен- ным количеством преадипоцитов, которые превратятся в жировые клетки позднее. Надо расспросить, как происходило физическое, умственное, половое развитие, не повлияла ли дальнейшая трудо- вая деятельность на эндокринную систему, не было ли контакта с химическими веществами или ионизирующей радиацией. Напри- мер, эти сведения важны для постановки диагноза АИТ. Расспрашивая о перенесённых ранее заболеваниях и травмах (особенно черепно-мозговых травмах, сколько и когда), необхо- димо уточнить, не применялись ли для лечения какие-либо гормо- нальные препараты, их дозы, длительность лечения. Например, са- харный диабет, а также ожирение нередко проявляются после те- рапии глюкокортикоидами. Возможно, применялись медикаменты, не относящиеся к гормонам, но влияющие на эндокринные железы. В качестве иллюстрации можно привести случаи гиперпролактине- мии или лактореи, резвившихся при лечении психотропными сред- ствами (антидепрессантами, нейролептиками, наркотиками, анти- конвульсантами, анестетиками). Следует уточнить, какими видами спорта занимался и не употреблял ли анаболические гормоны. Надо собрать сведения о вредных привычках. Так, курение почти в 100% случаев провоцирует патологию щитовидной железы, зло- употребление алкоголем может вызвать нарушение функции лю- бой эндокринной железы. Важно детально собрать гинекологиче- ский и акушерский анамнез женщин и половой анамнез мужчин. 18 И.В. Терещенко Особое внимание надо уделить опросу о родственниках (семей- ному анамнезу и наследственности), так как ко многим эндокрино- патиям имеется наследственная предрасположенность. Объективное обследование эндокринного больного. Общий осмотр больного с заболеванием эндокринной системы Как и при других заболеваниях, общий осмотр эндокринного больного начинается с оценки состояния. Оно может быть крайне тяжёлым, тяжёлым, средней тяжести и в большинстве случаев удо- влетворительным. Так, при йоддефицитном зобе, компенсирован- ном сахарном диабете, гипогонадизме, манифестации микроаде- номы гипофиза и др. состояние больных вполне удовлетворитель- ное. Состояние средней тяжести может наблюдаться при болезни Грейвса, осложнённой тиреотоксическим сердцем, при гипопара- тиреозе во время приступа тетании, при декомпенсации гипокор- тицизма и т. д. Тяжёлое состояние появляется при осложнении са- харного диабета нефропатией и ХПН 3Б – 5 стадии или при кри- зах – тиреотоксическом, аддисоническом, гиперкальциемическом, феохромоцитомном. Крайне тяжёлое состояние возникает при эн- докринных комах. Положение эндокринного больного может быть активное, пассивное, вынужденное. У большинства больных с эндокринной патологией бывает активное положение. Пассивное положение обусловлено либо развитием эндокринной комы, либо резко выра- женной слабостью, например, при аддисоновой болезни или альдо- стероме (гипокалиемическая слабость). Примером вынужденного положения у больной с большим загрудинным зобом может слу- жить такая поза пациентки: лежит в постели с запрокинутой голо- вой; при этом «ныряющий» зоб перемещается из-за ярёмной вы- резки грудины на переднюю поверхность шеи и меньше сдавливает гортань и трахею. Асфиксия уменьшается. Другим примером вы- нужденного положения является положение больного с левосто- ронней феохромоцитомой: во время и после приёма пищи лежит на правом боку. Наполненный пищей желудок в меньшей степени да- вит на опухоль (обычно большую и мягкую); тем самым предот- вращается симпатоадреналовый криз. 19 Пропедевтика эндокринных заболеваний Сознание эндокринного больного бывает ясным, спутанным (ступор, сопор, например, при тиреотоксическом или аддисониче- ском кризе, при лёгкой гипогликемии). При эндокринных комах больные теряют сознание. Основные эндокринные комы:  гипопитуитарные (их 5 видов: гипогликемическая, гипертермическая, аддисоническая, гипотермическая, смешанная);  диабетические (их 4 вида: две – гипергликемические – кетоацидотическая и гиперосмолярная; гипогликемическая; гиперлактатацидемическая);  тиреотоксическая;  гипотиреоидная;  аддисоническая От избытка или недостатка в организме того или иного гормона зависит выражение лица эндокринного больного. Изменение лица при эндокринных заболеваниях 1 2 Facies mixedematica Бледное или желтушное (чаще субиктеричное), амимичное, у многих больных (особенно у женщин) с ярким, ограниченным «кукольным» румянцем. Кожа лица сухая. Черты лица укрупнённые и отёчные, веки припухшие, глаза маловыразительные, взгляд тупой. Губы отёчные, нередко рот приоткрыт Facies bazedovica Большие, широко открытые, выпученные, немигающие и блестящие глаза, придающие всему лицу выражение как бы застывшего испуга или ужаса; или «гневный» взгляд (встречается чаще у мужчин). Объективное обследование эндокринного больного. Общий осмотр больного с заболеванием эндокринной системы Во многих случаях facies bazedovica позволяет на расстоянии поставить диагноз болезни Грейвса Facies аcromegalica Увеличение надбровных дуг, скуловых костей и подбородка, в результате чего лицо приобретает суровый вид. Мягкие ткани лица разрастаются, гипертрофируются, и это вызывает увеличение носа, ушей, губ. Кожа лица, даже у молодых, утолщается, появляются глубокие морщины. Нередко язык не умещается во рту, рот полуоткрыт. Нижняя челюсть выпирает вперёд (прогнатизм). Увеличиваются промежутки между зубами (диастема) 20 И.В. Терещенко Окончание таблицы 2 1 2 Кушингоид- ное (лунооб- разное) лицо Лицо круглое, багрово-цианотичной окраски – вид «полной» луны, с двойным-тройным подбородком из- за отложения жира Евнухоидное лицо Бледное или субиктеричное лицо, без вторичного оволосения у мужчин. Кожа сухая. Рано появляются морщины, у глаз «гусиные лапки». Выражение лица апатичное Лицо кретина Круглое, с маленькими глазками, седловидным носом. Выражение лица тупое. Рот открыт, язык торчит изо рта. Зубы кривые, так как постоянные зубы прорезаются, когда ещё сохранились молочные зубы Лицо при гипопа- ратиреозе Выражены трофические нарушения, даже у молодых, в том числе и у детей с гипопаратиреозом. Часто развивается катаракта, появляется блефароспазм. Волосы, брови, ресницы выпадают, характерна ранняя седина. Зубы разрушаются (обширный кариес). При смехе из-за спастических сокращений мимической мускулатуры углы рта опускаются вниз, и рот напоминает «пасть рыбы» Лицо при anorexa nervosa Сухая кожа обтягивает скелет лица. Исчезают со щёк жировые комочки Биша, щёки ввалившиеся. Кайма губ бледная, шелушится. Нередко выражен ангулярный стоматит. Глаза с блеском! Окончание таблицы 2 Следует отметить, что при своевременной постановке диагноза и лечении изменения лица могут не исчезнуть полностью. Возраст по внешнему виду может соответствовать паспортному, например, у пациента с компенсированным сахарным диабетом 1-го типа. Больной может выглядеть моложе своих лет (при инфан- тилизме или при церебрально-гипофизарном нанизме и др.) или выглядеть старше (при гипотиреозе, нервной анорексии, pubertas praecox и т. д.). Запах тела больного и выдыхаемого им воздуха в отдельных случаях может приобретать специфические свойства. Так, при ке- тоацидозе от больного сахарным диабетом пахнет ацетоном (под- гнившими яблоками); при акромегалии – резкий неприятный запах 21 Пропедевтика эндокринных заболеваний грязного тела (из-за гиперплазии потовых желёз у больного посто- янное обильное потоотделение). При болезни Грейвса тоже наблю- дается повышенная потливость, при этом потовые железы начи- нают выбрасывать излишки тиреоидных гормонов, что придаёт поту особый запах, не встречающийся у других людей. Телосложение (habitus) больных при эндокринных заболева- ниях может быть правильным и неправильным. Правильное тело- сложение в зависимости от пола больного, гормонального статуса бывает по мужскому типу (ширина плечевого пояса больше тазо- вого) или по женскому типу (больше ширина таза, чем ширина плеч). Известно, телосложение зависит от развития костной, жиро- вой, мышечной ткани, на что влияет гормональная система. При многих эндокринных заболеваниях формируется характерный па- тологический габитус.  Так, при врождённом гипотиреозе (кретинизме) большая го- лова не соответствует размерам туловища, укороченные кривые конечности, большой живот.  При евнухоидизме, наоборот, голова маленькая, длинные ко- нечности.  При евнухоидизме, наоборот, голова маленькая, длинные ко- нечности.  При вирильной форме ВДКН у женщин плечевой пояс прева- лирует над тазовым, чрезмерно развита скелетная мускулатура на фоне дефицита подкожно-жировой клетчатки.  При гипотиреозе изменяются кисти, становятся лопатообраз- ными, кажутся гипертрофированными проксимальные мышцы ко- нечностей (мышцы плеча, бедра).  Можно диагностировать системный остеопороз без рентгено- логического обследования по изменению телосложения: у боль- ного остеопорозом образуется кифоз грудного отдела позвоноч- ника, появляется более или менее выраженный горб, рёберные дуги сближаются с крыльями подвздошных костей, органы брюшной полости выпячивают стенку живота вперёд, туловище наклонено вперёд.  Кушингоидное телосложение развивается при избытке в орга- низме глюкокортикоидов любого генеза: появляется диспластиче- ское ожирение с преимущественным отложением жира в области лица (лунообразное лицо), шеи («бычий загривок») и живота; яго- дицы сглажены. 22 И.В. Терещенко Рост, массу тела, ИМТ необходимо измерять и оценивать, со- ответствуют ли эти показатели норме (см об этом подробнее в раз- деле 2 «Синдром ожирения», «Синдром нарушения роста»). Походка при некоторых эндокринопатиях может изменяться. Окончание таблицы 2 Так, при гиперпаратиреозе становится шаркающей или утиной, при тимоме с миастенией – паралитической, при тиреотоксикозе может быть суетливой. При гиперандрогении у женщин походка теряет лёгкость, становится похожей на мужскую. Кожные покровы изменяются при очень многих эндокринопа- тиях. Изменяется 1. Цвет кожи. Таблица 3 Таблица 3 Эндокринопатия Цвет Патогенез Декомпенсация сахарного диабета Нежный розовый Повышение уровня ГлиНв А1с Декомпенсация гипотиреоза Субиктеричность Избыток каротина в крови Аддисонова болезнь Гиперпигментация Гиперсекреция АКТГ и МСГ и меланина Гипоталамо- гипофизарная недостаточность «Алебастровая» бледность Дефицит АКТГ и МСГ Болезнь (синдром) Иценко–Кушинга Цианотично- багровый, мраморный Истончение дермы, избыток гемоглобина, эритроцитоз Синдром Пиквика Цианотичная Гипертензия в малом круге кровообращения и гипоксемия Следует пояснить, что гемоглобин, связанный с молекулами глюкозы (ГлиНвА1с), прочно соединяется с кислородом. При ги- пергликемии уровень глиНвА1с повышается, иногда в 2–3 раза. Чем более выражена и длительнее декомпенсация сахар- ного диабета, тем выше содержание в крови глиНвА1с. Гликиро- ванный гемоглобин не отдаёт тканям кислород. Поэтому венозная кровь у пациентов с декомпенсированным сахарным диабетом, как и артериальная, богата оксигемоглобином, что придаёт кожным по- кровам нежный розовый оттенок. Это именуют rubeosa diabeticа. 23 Пропедевтика эндокринных заболеваний Этот симптом позволяет даже без лабораторного контроля заподо- зрить декомпенсацию углеводного обмена. При гипотиреозе страдает утилизация витамина А, т. е. каро- тина. Избыточный уровень каротина придаёт кожным покровам желтушный оттенок, иногда интенсивный. Это в отдельных слу- чаях ошибочно трактуют как желтуху. Важно помнить, что каротин не накапливается в слизистых, а окрашивает только кожные по- кровы! Склеры глаз, слизистые полости рта при гипотиреозе не желтеют, остаются светлыми. У пациентов с аддисоновой болезнью дефицит кортизола вызы- вает гиперсекрецию АКТГ. Молекула АКТГ имеет большое сход- ство с МСГ. Избыток меланина у пациентов с гипокортицизмом вызывает гиперпигментацию кожных покровов и слизистых. Ги- перпигментация при аддисоновой болезни особенно выражена на участках, подверженных инсоляции, в местах трения одеждой, в областях физиологической пигментации (ареолы и соски, промеж- ность, анус, складки кожи). При расовой или конституциональной пигментации в слизистых никогда пигмент не откладывается. Чтобы дифференцировать, надо осмотреть дёсны, слизистые поло- сти рта. При первичном гипокортицизме там можно обнаружить пятна пигмента, а красная кайма губ бывает обведена тёмной поло- сой, как будто провели вокруг губ тёмно-коричневым карандашом. При гипокортицизме аутоиммунного генеза можно обнаружить, наряду с гиперпигментацией, участки депигментации – лейко- дерму или витилиго: это сразу подскажет, что у пациента аутоим- мунный генез заболевания. При вторичном гипокортицизме из-за дефицита выработки АКТГ, а значит, и МСГ, развивается «алебаст- ровая» бледность. «Алебастровая» бледность всегда означает де- фицит МСГ. Окончание таблицы 2 При этом могут депигментироваться волосы и даже светлеть цвет радужки. Бледность кожных покровов при анемиях, заболеваниях почек, спазме сосудов и т. д. нельзя сравнить с «але- бастровой» бледностью пациентов с гипоталамо-гипофизарной не- достаточностью. При болезни Пиквика крайняя степень ожирения вызывает та- кое повышение внутрибрюшного давления, что экскурсия лёгких резко ограничивается, что приводит к постоянной гипоксемии и диффузному цианозу. При феохромоцитоме из-за длительного из- бытка катехоламинов расширяются сосуды микроциркуляторного 24 И.В. Терещенко русла, это становится заметным на коже в области суставов, где rete articularis. По той же причине у пациента с феохромоцитомой ла- дони всегда «ледяные» и покрыты липким потом. русла, это становится заметным на коже в области суставов, где rete articularis. По той же причине у пациента с феохромоцитомой ла- дони всегда «ледяные» и покрыты липким потом. р 2. Могут появиться характерные высыпания на коже. 2. Могут появиться характерные высыпания на коже. Важно детальнее описать мигрирующую некролитическую эри- тему при избыточной секреции опухолью глюкагона. Сначала на коже появляется пятно размером менее 1 см, которое превращается в папулу с запавшим центром и чётким краем. Из него образуется везикула, потом эрозия. На месте эрозии возникает корочка или че- шуйка, а после слущивания остаётся пигментация. Весь процесс за- нимает 7 – 10 дней. Кожные покровы приобретают пёстрый вид: элементы сыпи проходят разные стадии развития. Излюбленная ло- кализация сыпи - ноги, паховые области, низ живота, редко лицо. 3. Изменение придатков кожи. А. Выпадение волос, облысение, ломкость волос. Алопеция бывает гнёздной, когда облысение происходит на от- дельных участках волосистой части головы; диффузной – равно- мерно по всей голове, тотальной, если помимо выпадения волос на голове, выпадают брови, ресницы, исчезает половое оволосение. Различают андрогенный тип облысения (залысины на лбу, облысе- ние в области темени). При дефиците половых гормонов теряется половое оволосение. Так, мужчины начинают реже бриться, исче- зает рост волос на теле, на лобке. 25 Таб ния Патогенез ражение; тройства, шай, язвы, алазмы Гликирование тканей, нарушения микроциркуляции жирная Воздействие избытка андрогенов на кожу и придатки Тиреоидные гормоны выделяются в избытк потовыми железами, вызывая зуд тей, стоп, Дефицит тиреоидных гормонов нарушает в коже обмен каротина, обмен белков ия на коже уди и т.д. Катаболический эффект избытка глюкокортикоидов тазы в коже Избыток паратгормона и гиперкальциемия тема, ит Избыток глюкагона. Патогенез не ясен гиэктазии, ения кожи ща ового цвета Выброс опухолью серотонина и других биологически активных веществ Та ость роста волос Патогенез тотальная алопеция, ть волос, Дефицит СТГ и / или других гормонов гипофиза ие, истончение, дение волос Усиление катаболизма тканей из-за избытка Т3 и Т4 иффузная алопеция; волос Тканевая гипоксия из-за дефи Т3 и Т4 падение бровей, ресниц Дефицит паратгормона, кото выполняет трофическую фун гипокальциемия мужскому типу: ысение теменной части ысение головы Избыток андрогена ДГТ, ↑чувствительность к ДГТ волосяных фолликулов, ↑акт 5α редуктазы енность полового Дефицит половых гормонов ласти подмышечных стический признак!) Дефицит надпочечниковых андрогенов-ДГЭА, андростен Пропедевтика эндокринных заболеваний Интересно отметить ещё один характерный симптом гипоти- реоза – это облысение латеральных отделов бровей (только лате- ральных!). Патогенез такой алопеции не ясен, но достаточно заме- тить рост бровей только от носовой части до середины и латераль- ное их облысение, как это подскажет необходимость проверки функции щитовидной железы. Заместительная терапия гипоти- реоза восстанавливает нормальный рост бровей. Б. Особенность роста волос. Б. Особенность роста волос. Таблица 6 Эндокринопатия Особенность роста волос Патогенез Болезнь (синдром) Иценко– Кушинга Низкая граница роста волос на лбу, гирсутизм, гипертрихоз Избыток кортизола и надпочечниковых андрогенов Акромегалия Усиленный рост стержневых волос на лице, конечностях, туловище Избыток СТГ, гипертрофия волосяных фолликулов ВДКН Гирсутизм, усиленный рост волос на конечностях, белой линии живота, на лобке, вокруг сосков Избыток 17α ОН- прогестерона и других андрогенов надпочечников СПКЯ Незначительный гирсутизм (рост усиков), выражен рост волос по белой линии Гиперпродукция андрогенов тека- клетками яичников Андрогенпро- дуцирующие опухоли надпочечника или яичника Гирсутизм и гипертрихоз ярко выражены Значительный избыток опухолевых андрогенов Anorexia nervosa Усиленный рост пушковых волос на конечностях, спине, крестце, одновременно выпадение с головы Как фактор адаптации гиперсекреция анаболических гормонов надпочечников 28 И.В. Терещенко И.В. Терещенко При гормональных расстройствах изменяется влажность кожи. Избыточная потливость – гипергидроз – бывает диффузным и локальным. Наиболее выражен диффузный гипергидроз при бо- лезни Грейвса: даже область голеней, которая у здорового человека и при другой патологии не потеет, при тиреотоксикозе постоянно покрыта потом. Этот симптом очень помогает сориентироваться в дифференциальном диагнозе, прежде всего с НЦД. Диффузный очень обильный гипергидроз наблюдается при акромегалии (не- редко больные вынуждены мыться по нескольку раз в день!). Для пациенток с гиперандрогенией характерен обильный локальный гипергидроз в подмышечных ямках, пот со специфическим андро- генным запахом. При симпатоадреналовых кризах у больного с феохромоцитомой появляется липкий холодный пот на лбу, в об- ласти носогубного треугольника (так называемый «симпатический пот»). У женщин с патологическим климаксом наблюдаются при- ливы жара, сопровождающиеся обильным потом. Чем тяжелее про- текает синдром патологического климакса, тем больше таких при- ливов жара и пота. При гипогликемии из-за повышенного выброса катехоламинов тоже возникает обильное потоотделение (стано- вится мокрым бельё, пододеяльник, подушка). Сухость кожи – характерный симптом гипотиреоза, при этом наблюдается выраженный гиперкератоз в области локтей, стоп, ки- стей, а на голенях шелушение в виде крупы. При гипоталамо-гипо- физарной недостаточности тоже проявляется гипофункция пото- вых желёз и сухость кожи. При гормональных расстройствах изменяется тургор кожи. Так, его снижение может быть признаком обезвоживания при де- компенсации сахарного или несахарного диабета, при аддисоновой болезни. Также быстрое похудение и исчезновение подкожно-жи- рового слоя снижает тургор кожи при болезни Грейвса, гипота- ламо-гипофизарной недостаточности, anorexia nervosa. Повышение тургора кожи, когда кожу невозможно взять в складку, возникает при выраженной гиперпролактинемии. 4. Отёки: 4. Отёки: Встречаются при гипотиреозе, гиперпролактинемии, сахарном диабете, осложнённым диабетической нефропатией, появляются при ХСН у больных с эндокринопатиями (тиреотоксическом сердце, при ожирении). Это – характерный симптом микседемы. 29 Таб Характеристика отёков Интенсивность Консистенция Основные факт патогенеза Выражены при микседеме. При гипотиреозе чаще всего периорбитальные Плотные, не смещаемые; при надавливании ямки нет В тканях накоплен гиалуроновой кис гликозаминоглика муцина Незначительно выражены, постоянные Повышен тургор кожи Задержка жидкост организме из-за избытка ПРЛ Интенсивные, тёплые, бледные Мягкие, смещаемые Гипоальбуминеми снижение онкотического давления Зависят от степени ХСН Умеренно плотные, цианотичные, холодные Ремоделирование миокарда, АГ, НР (стойкая тахикард фибрилляция предсердий) Зависят от степени ХСН Умеренно плотные, цианотичные, холодные Диастолическая дисфункция, дист миокарда, Повыш ОЦК И.В. Терещенко 5. Ногти подвергаются трофическим изменениям при многих эндокринопатиях: при гипотиреозе сухие, ломкие; при гипопарати- реозе могут исчезнуть полностью и не расти, при сахарном диабете нередко появляется грибковое поражение ногтей, при акромегалии быстро растут, утолщены. 5. Ногти подвергаются трофическим изменениям при многих эндокринопатиях: при гипотиреозе сухие, ломкие; при гипопарати- реозе могут исчезнуть полностью и не расти, при сахарном диабете нередко появляется грибковое поражение ногтей, при акромегалии быстро растут, утолщены. р р у , у 6. Мышечная система плохо развита при инфантилизме, гипо- гонадизме. Наоборот, гипертрофия мышц и увеличение мышечной силы характерны для гиперандрогении любого генеза у женщин. При многих эндокринных заболеваниях возникает миопатия, про- являющаяся миалгиями, мышечной скованностью, проксимальной или дистальной слабостью. Так, диабетическая миопатия чаще наблюдается при длительном течении сахарного диабета 1-го типа. Тиреотоксическая миопатия возникает из-за катаболизма мышеч- ной ткани, гипокалиемии. Генерализованная псевдогипертрофия мышц в сочетании со снижением мышечной силы главным образом проксимальных мышц наблюдается при гипотиреозе из-за накоп- ления креатинфосфата в мышцах, который из-за дефицита тирео- идных гормонов не используется. Гипотрофия мышц возникает у больных с гипоталамо-гипофизарной недостаточностью из-за де- фицита белка. Стероидная миопатия характерна для больных с син- дромом Иценко–Кушинга (причина – стероидный катаболизм). При аддисоновой болезни миопатия встречается часто в связи с ги- посекрецией анаболических гормонов надпочечников. Проявления миопатии на фоне лечения обратимы не всегда. 7. Осмотр и пальпация области шеи. 7. Осмотр и пальпация области шеи. 7. Осмотр и пальпация области шеи. При осмотре шеи возможно обнаружить её деформацию зобом или увеличенными лимфоузлами, врождёнными кистами шеи, или из-за наличия послеоперационных рубцов. Необходимо оценить особенности выявленных деформаций. Наличие венозной сети, особенно, с одной стороны, служит косвенным признаком рака щи- товидной железы. 8. Осмотр по системам органов. Проводится обычно. Особое внимание для диагностики и диф- ференциальной диагностики заслуживает осмотр сердечно-сосуди- стой системы. 4. Отёки: Так, обратились две пациентки с предполагаемым диагнозом токсического зоба и со сходными жалобами на нервоз- ность, раздражительность, плаксивость, плохой сон, боли в сердце, сердцебиение, ощущение комка в горле. Врач у обеих пациенток 31 Пропедевтика эндокринных заболеваний проверил пульс в покое и на фоне задержки дыхания. У одной боль- ной пульс лабильный, выражена дыхательная аритмия. У другой – тахикардия стойкая. Уже на поликлиническом приёме в первом случае можно отвергнуть предварительный диагноз тиреотокси- коза, и избавить больную от проведения ненужного и иногда за- тратного обследования. Во втором случае стойкую тахикардию, очевидно, вызывает избыток в организме тиреоидных гормонов, и надо продолжить обследование. проверил пульс в покое и на фоне задержки дыхания. У одной боль- ной пульс лабильный, выражена дыхательная аритмия. У другой – тахикардия стойкая. Уже на поликлиническом приёме в первом случае можно отвергнуть предварительный диагноз тиреотокси- коза, и избавить больную от проведения ненужного и иногда за- тратного обследования. Во втором случае стойкую тахикардию, очевидно, вызывает избыток в организме тиреоидных гормонов, и надо продолжить обследование. д р д д 9. У мужчин можно выявить гинекомастию. Осмотром и паль- пацией оцениваем, имеется на одной стороне или двусторонняя, размеры молочных желёз, наличие изменений ареолы и сосков. Втянутый сосок или его деформация являются тревожным симпто- мом онкологического процесса. Гинекомастия бывает истинной, состоящей из железистой ткани, ложной – из-за отложения жира, смешанной. Истинная гинекомастия всегда бывает обусловлена гормональными нарушениями (избытком эстрогенов, не ПРЛ!). Ги- персекреция ПРЛ любого генеза может вызвать персистирующую лакторею. Ложная гинекомастия возникает, как правило, при ожи- рении, смешанная – чаще всего при эндокринопатиях, сопровожда- ющихся гиперэстрогенией и избыточной массой тела. Пальпация молочных желёз укажет, какая гинекомастия: при истинной – паль- пируется железистая ткань, при ложной – консистенция тестоватая, при смешанной – ощущаются оба компонента. Также оцениваем наличие болезненности при пальпации. Болезненность наблюда- ется при воспалении в молочной железе, особенно интенсивная – при гнойном процессе; возможно, увеличение молочной железы и болезненность являются следствием травмы (гематома может ими- тировать гинекомастию). Следует надавить на сосок и проверить наличие отделяемого, насколько обильное, из одного соска или лакторея двусторонняя, оценить характер – кровь, молозиво, сероз- ная жидкость, гной. Вторичное оволосение мужчин зависит от возраста, националь- ности, наследственности, гормональной патологии. Избыточный рост волос на груди, спине, конечностях может быть врождённым и приобретённым, например, при возникновении семиномы (оце- нивать динамику роста волос). 32 И.В. Терещенко 8. Пальпация эндокринных желёз. 8. Пальпация эндокринных желёз. 8. Пальпация эндокринных желёз. Только щитовидная железа и мужские гонады топографически доступны для пальпации. Остальные эндокринные железы недо- ступны. А. Пальпация щитовидной железы проводится 4 способами. Способ 1 (ориентировочный). Врач фиксирует шею пациента 4 пальцами каждой кисти, а большими пальцами последовательно пальпирует перешеек, правую и левую доли, причём это проводит в покое и при глотании. Этот способ считается ориентировочным, так как осязательных телец в кончиках больших пальцев меньше, чем в остальных пальцах. Фиксировать шею необходимо, иначе па- циент при приближении пальцев врача к шее инстинктивно отпря- нет назад. Важно учитывать, что щитовидная железа анатомически нахо- дится на щитовидном хряще гортани, спаяна с ним и при глотании будет смещаться вместе с ним. Это помогает в дифференциальной диагностике с другими опухолями в проекции щитовидной же- лезы – с лимфоузлами, врождёнными кистами шеи, лейомиомами шеи и т. д. Если при глотании образование остаётся неподвижным, значит, это не ткань щитовидной железы. Пальпируя щитовидную железу, всегда надо оценивать, смещается ли она при глотании. Способ 2. Левая рука врача фиксирует шею, 4 пальцами правой руки скользящими движениями он пальпирует перешеек, каждую долю щитовидной железы. Способ 3. Врач заводит по 4 пальца каждой руки за кивательные мышцы с обеих сторон и расположенные там ткани подаёт вперёд. Если там скрывались узловые образования, кисты щитовидной же- лезы (иногда большого размера), то такой способ пальпации позво- лит их выявить. Способ 4. Врач становится позади пациента и 4 пальцами каж- дой руки пальпирует поочерёдно перешеек и обе доли щитовидной железы. Следует проводить пальпацию щитовидной железы всеми спо- собами в положении пациента стоя, если по состоянию больного это возможно. Лёжа и сидя у обследуемого изменяется топография средостения и щитовидной железы, и её не удастся чётко опреде- лить. Во время пальпации следует следить за реакцией пациента: 33 Пропедевтика эндокринных заболеваний боль, дискомфорт могут указать на патологию (воспаление, онко- логический процесс). Самая интенсивная болезненность наблюда- ется при остром гнойном тиреоидите или при подостром тиреои- дите. боль, дискомфорт могут указать на патологию (воспаление, онко- логический процесс). Самая интенсивная болезненность наблюда- ется при остром гнойном тиреоидите или при подостром тиреои- дите. Проводя пальпацию щитовидной железы, оцениваем: Проводя пальпацию щитовидной железы, оцениваем:  форму, размеры и степень увеличения;  консистенцию, наличие фокальных образований, узлов, кист;  смещаемость, спаянность с соседними тканями шеи.  смещаемость, спаянность с соседними тканями шеи. Нормальную форму щитовидной железы сравнивают с бабоч- кой; её доли почти симметричны. 8. Пальпация эндокринных желёз. Если нет увеличения щитовид- ной железы, то её размеры примерно равны концевой фаланге соб- ственного большого пальца. Встречаются врождённые аномалии щитовидной железы, например, отсутствие одной доли или доли и перешейка. Так, примером может служить юноша К-в, 18 лет. Жа- лоб не предъявлял, при прохождении призывной комиссии выяв- лен «зоб II степени» справа. Визуально справа определялась доля щитовидной железы, пальпацией не удавалось обнаружить пере- шеек и левую долю. Назначена операция, хотя при проведении УЗИ щитовидной железы не выявлено отклонений от нормы един- ственной доли; её объём составлял 18 см3 (т. е. нормальный). Ясно, что в данном случае была врождённая аномалия щитовидной же- лезы, и оперативное вмешательство отменено. Могут встречаться дополнительные щитовидные железы, если во время эмбриогенеза (чаще всего в 5 недель гестации) произошло отделение частички её зачатка. Такие образования могут имитировать узлы, а чаще явля- ются УЗИ-находкой. О степени увеличения щитовидной железы сообщается в разделе 2. В норме при пальпации консистенция щи- товидной железы однородная и эластическая наощупь, при патоло- гии теряет однородность и может становиться плотной в разной степени. Каменистую плотность она приобретает при раке. Конси- стенция неоднородная наблюдается при АИТ, узловом и многоуз- ловом зобе. При тиреотоксикозе даже при лёгкой пальпации в ряде случаев удаётся уловить усиленный кровоток (имитация журча- ния). Нормальная щитовидная железа не спаяна с окружающими тканями, смещаема, подвижна. УЗИ щитовидной железы не заме- няет, а дополняет полученные пальпацией данные о её состоянии и наоборот – пальпация дополняет информацию, полученную при УЗИ ЩЖ. 34 И.В. Терещенко И.В. Терещенко 9. Аускультация щитовидной железы. Из-за богатого кровоснабжения тиреоидной паренхимы, её ана- томической близости к сонным артериям, аорте и другим крупным сосудам в норме над щитовидной железой можно выслушать тоны сердца. Аускультация щитовидной железы необходима, когда про- водим дифференциальную диагностику нейро-циркуляторной дистонии (НЦД) с болезнью Грейвса или с тиреотоксикозом дру- гого генеза. В отличие от НЦД при тиреотоксикозе микроциркуля- торное русло переполнено, кровоток усилен и ускорен, что при аускультации сопровождается появлением не только тонов, но и систолических и диастолических шумов. Для этого, проводя аускультацию щитовидной железы, врач просит пациента глубоко вздохнуть, выдохнуть и замереть, насколько это возможно. Во всех других случаях, включая НЦД, можно выслушать только тоны. 10. Паращитовидные железы пропальпировать невозможно из- за маленького их размера, но для выявления гипопаратиреоза и ги- покальциемии необходимо проверять наличие симптома Хвостека: ударить пальцем или молоточком у мочки уха пациента в месте вы- хода лицевого нерва, иннервирующего мимические мышцы лица. При снижении уровня кальция в крови механическое раздражение лицевого нерва вызовет судорожное подёргивание мышц лица: только угла рта, если гипокальциемия незначительная (положи- тельный симптом Хвостека III); угла рта и крыла носа (положитель- ный симптом Хвостека II) – при умеренном дефиците кальция; и, кроме того, мышц века (положительный симптом Хвостека I), если дефицит кальция значительный. Следует также исключить наличие положительного симптома Труссо: появление судороги кисти («рука акушера») при сдавлении сосудисто-нервного пучка в обла- сти плеча наложением жгута или сдавлением манжеткой (нередко из-за этого больному невозможно измерить АД). О гипокальцие- мии свидетельствует также положительный симптом Люста: при поколачивании молоточком по малоберцовому нерву (ниже го- ловки малоберцовой кости) возникает непроизвольное тыльное сгибание стопы с ротацией ноги снаружи. 35 Пропедевтика эндокринных заболеваний 11. Оценка развития вторичных половых признаков и степени полового созревания. Это особенно важно оценивать у подростков, хотя в ряде слу- чаев возникает необходимость оценить у пациента из других воз- растных групп. Целесообразно использовать шкалу Таннера. Шкала Таннера позволяет насчитывать 5 степеней развития пер- вичных и вторичных половых признаков и представлять формулу полового созревания, которая у мужчин обозначается аббревиату- рой PAFLV (P -pubis; A – axillaries; F – facies; L – larynx; V – voice); у женщин MeMaPA (Me – mensis; Ma – mammae; P -pubis; A – axil- laris). Таблица 8 ц Половое развитие у мужчин в норме Степень Половое развитие Формула полового созревания I Появляется рост волос на лобке вокруг полового члена P0-1A0F0L0V0 II Усиливается рост волос на лобке, начинается рост волос в подмышечных впадинах P1A0-1F0L0V0 III Оволосение усиливается. 9. Аускультация щитовидной железы. Появляются пушковые волосы на лице, начинается формирование кадыка, ломка голоса P1-2A1-2F1L0- 1V0-1 IV Обильный рост лобковых, аксиллярных волос, волос на лице, формируется кадык, голос становится низкого тембра P3A2-3F2-3L1- 2V1-2 V Формирование вторичных половых признаков завершается (гениталии и наружные половые признаки, как у зрелого мужчины) P4A3F3L3V3 Половое развитие у мужчин в норме При 1 степени полового развития половой член, мошонка, те- стикулы имеют такие же размеры и пропорции, как в раннем дет- стве; при II степени полового развития мошонка и яички увеличи- ваются, кожа мошонки утолщается и начинает пигментироваться, лобковые волосы также начинают пигментироваться; при III сте- 36 И.В. Терещенко пени гениталии продолжают увеличиваться, половой член удлиня- ется и утолщается. Сформировавшаяся головка полового члена означает наступление IV стадии полового развития, лобковые во- лосы темнеют, грубеют и становятся вьющимися, кадык выражен, голос мужского тембра. В V стадию половое созревание заверша- ется, все половые признаки, как у зрелого мужчины. пени гениталии продолжают увеличиваться, половой член удлиня- ется и утолщается. Сформировавшаяся головка полового члена означает наступление IV стадии полового развития, лобковые во- лосы темнеют, грубеют и становятся вьющимися, кадык выражен, голос мужского тембра. В V стадию половое созревание заверша- ется, все половые признаки, как у зрелого мужчины. Не должно быть фимоза. У мальчика (особенно у новорожден- ного) необходимо проверить анатомическое расположение отвер- стия мочеиспускательного канала, исключить гипоспадию. Таблица 9 ц Половое развитие у женщин в норме Степень Половое развитие Формула полового созревания I Появляется уплотнение в области ареол молочных желёз в виде «пуговок», иногда слегка болезненных Me0Ma0-1P0A0 II Молочные железы начинают увеличиваться и выступать над поверхностью грудной клетки. Диаметр ареол увеличивается. У некоторых девочек появляются месячные (овуляции пока нет). Начинается оволосение на лобке, вырастают единичные волосы в подмышечных впадинах Me10-15 Ma1- 2P1A1 III Менархе проявляется в 100% случаев, месячные ещё могут быть нерегулярными. Усиливается лобковое и аксиллярное оволосение. Молочные железы, ареолы выступают в виде конуса. Ареолы и соски начинают окрашиваться MeMa2-3P2-3A2- 3 IV Наружные половые признаки достигают полного развития. Месячные становятся цикличными. Овуляторные циклы могут чередоваться с ановуляторными MeMa3P3A3 V Половое созревание завершено MeMa3-4P3A3 Половое развитие у женщин в норме 37 Пропедевтика эндокринных заболеваний Стадии полового развития не имеют точного совпадения с хро- нологическим возрастом и значительно варьируют. Важно под- черкнуть, у девочек в норме всегда пубертация начинается с фор- мирования молочных желёз, а не с роста волос на лобке или в под- мышечных ямках! Изменение такой последовательности позволяет подозревать ВДКН или другую патологию! 9. Аускультация щитовидной железы. Молочные железы сле- дует пальпировать и проверять наличие лактореи у небеременных и не кормящих женщин, при этом учитывать односторонняя лакто- рея или двусторонняя, какие выделения (молоко, серозное отделя- емое, кровь, гной), скудные или обильные. Секрецию молока вы- зывает ПРЛ, рост молочных желёз зависит от эстрогении (секреции эстрадиола и эстрона). Иногда лакторею можно наблюдать у муж- чин (это всегда патология!). У женщин редко возможно развитие гигантских молочных желёз (гипермастия, гигантомастия, макро- мастия), т.е. чрезмерная гипертрофия соединительной ткани мо- лочных желёз. Генез гипермастии пока не ясен. Очевидно, чувстви- тельность рецепторов тканей молочной железы к ростовым факто- рам патологически высокая, что и приводит к чрезмерному росту молочных желёз. Наиболее часто встречающаяся патология молоч- ных желёз – мастодиния. Мастодиния – это субъективно неприят- ные ощущения в области молочных желёз, болезненность при пальпации, при прикосновении одежды, ощущение распирания, отёчности. Таких женщин необходимо обследовать, прежде всего исключать онкопатологию молочных желёз и половой сферы! 38 38 И.В. Терещенко Раздел 2. ЭНДОКРИННЫЕ СИНДРОМЫ Основные патологические синдромы, обусловленные гормо- нальными нарушениями, следующие. 1. Ожирения. 2. Синдром дефицита массы тела. 3. Нарушения роста: А. Высокорослости; Б. Низкорослости. 4. Увеличения щитовидной железы. 4. Увеличения щитовидной железы. 5. Патологии уровня глюкозы в крови: А. Гипергликемии; Б. Ги- погликемии. 6. Эндокринной артериальной гипертензии. 7. Инсипидарный синдром. 8. Синдром гирсутизма и вирилизма. Синдром ожирения й Ожирение – синдром, характеризующийся избыточным отложе- нием и хроническим латентным воспалением жировой ткани в ор- ганизме. В норме жировая ткань составляет 15–20% от общей массы тела. О наличии ожирения и его степени можно судить по ИМТ кг/м2. Для расчёта ИМТ надо массу тела (кг) разделить на рост (м2). ц Оценка ИМТ ИМТ кг/м2 Оценка ИМТ кг/м2 Оценка < 19 Дефицит массы тела 20–24,9 Нормальная масса тела 25–29,9 Избыточная масса тела 30,0–34,9 Ожирение I степени 35,0–39,9 Ожирение II степени 40 и более Морбидное ожирение Также ожирение относят к морбидному, если ИМТ >35 кг/м2 и имеются серьёзные осложнения, связанные с ожирением. Выде- ляют абдоминальное и глютеофеморальное ожирение; при абдоми- нальном – существенно чаще возникают осложнения заболевания. По соотношению окружности талии (ОТ см) к окружности бёдер (ОБ см) можно определить тип ожирения и количество висцераль- ного жира. 39 Пропедевтика эндокринных заболеваний Абдоминальное (тип яблока, висцеральный, верхний, андроидный тип) Глютеофеморальное (тип груши, нижний, гиноидный тип) ОТ/ОБ у мужчин ≥ 0,9–1,0 ОТ/ОБ у женщин ≥ 0,8–0,82 ОТ/ОБ у мужчин < 0,9 ОТ/ОБ у женщин < 0,8 Риск развития осложнений ожирения очень высокий, если ОТ у мужчин ≥ 102 см, у женщин ≥ 88 см, причём это касается всех органов и систем. К осложнениям ожирения относят сахарный диабет 2-го типа (СД2), артериальную гипертензию (АГ), атеросклероз и сер- дечно-сосудистые заболевания, синдром ночного апноэ, НАЖБП, ЖКБ, нарушения опорно-двигательного аппарата, репродуктивные расстройства, некоторые локализации онкологических заболеваний. Появился термин «метаболически здоровое ожирение», что подразу- мевает более благоприятный профиль воспаления в жировой ткани, меньший объём висцерального депо жира, менее выраженную ин- фильтрацию жировой ткани макрофагами, меньший размер адипоци- тов, т. е. риск осложнений ожирения отсутствует или меньше, чем при метаболически нездоровом ожирении. Не изучен отдалённый прогноз метаболически здорового ожирения, насколько часто и когда здоро- вое ожирение переходит в нездоровое. Пока сомнительно существо- вание «здорового» ожирения. Кроме того, ожирение бывает диспластическим, когда жировые отложения в одних местах исчезают, в других – появляются. Так при кушингоидном ожирении становятся худыми конечности, яго- дицы, но появляется жировая ткань на туловище, животе, лице. При ожирении Барракер-Симмондса истощается верхняя часть тела, исчезают с лица жировые комочки Биша, и развивается мас- сивное ожирение нижней части туловища и нижних конечностей. Болезнь Маделунга – диспластическое ожирение, когда разраста- ние жира происходит на шее. Встречается (редко) болезненное ожирение, которое называется «болезнь Деркума». Синдром дефицита массы тела Синдром дефицита массы тела Дефицитом массы тела является снижение ИМТ ≤ 18,5 кг/м2; при ИМТ ≤ 16 кг/м2 наблюдается истощение, именуемое кахексией. Нередко снижение массы тела, вызванное эндокринопатиями, яв- ляется первым, а иногда единственным или основным симптомом заболевания эндокринной системы. 40 Табли ости синдрома дефицита массы тела ндокринных заболеваниях нижения массы тела Генез ыстро прогрессирует, идратации больного, жировая ткань, возникает мышечной ткани и дефицит ыть повышенный аппетит Катаболизм из-за абсолютного дефицита инсулина. Гипергликем вызывает полиурию и обезвожива яется первым симптомом нижения массы тела зависит Уменьшается количество на гипотрофия мышц. нов в крови может При этом аппетит овышен. Встречается икоза – марантическая, ей Гиперсекреция тиреоидных гормо вызывает катаболизм азвивается и нарастает похудение происходит за чезает аппетит, отмечается Избыток паратгормона и гиперкальциемия обусловливают выраженную интоксикацию, котор вызывает анорексию и поражение ЖКТ х гормо ливают ю, котор ажение Окончание т ижения массы тела Генез развивается истощение лезни Шиена дефицит меренным Степень истощения зависит п всего от дефицита СТГ, в мен степени – от дефицита други тропных гормонов (АКТГ, П ыстро, сопровождается ышенной потребностью в й пище. Выражены исчезает подкожный и ечная ткань как скелетная, руется Дефицит надпочечниковых минералокортикоидов приво дегидратации, дефицит глюкокортикоидов нарушает углеводный обмен, вызывает гликогена в клетках и тканях исчезновение жира, при дефи надпочечниковых анаболико невозможен синтез мышечно приёма пищи развивается массы тела, кахексия и ндокринной системы оз) Возникает вследствие расстр регуляции секреции дофамин серотонина вследствие психологического стресса И.В. Терещенко Синдром дефицита массы тела может иметь причины не гормо- нальные. Наиболее частые негормональные причины – конститу- циональное снижение массы тела (проявление неспецифической дисплазии соединительной ткани), алиментарное истощение, ка- хексия при онкологических заболеваниях, при синдроме нарушен- ного всасывания. Необходимо проводить дифференциальную диа- гностику эндокринопатий, сопровождающихся похудением, не только между собой, но и с другой патологией. Нарушения роста: А. Высокорослость Нормы роста значительно варьируют, зависят от расы, нацио- нальности, места проживания, особенностей питания, образа жизни. Для Уральского региона характерны колебания показателей роста у мужчин от 162 до 193 см, у женщин от 150 до 184 см. Рост каждого человека запрограммирован генетическим кодом, реализа- ция кода зависит не только от условий жизни, но и от перенесённых заболеваний, в первую очередь, от гормональных нарушений в дет- ском возрасте и в пубертатном периоде. Примерами конституцио- нальной высокорослости соотечественников являются Ф.И. Шаля- пин (рост 195 см), композитор С.В. Рахманинов (198 см), царь Пётр I (204 см), император Николай I (205 см). Таблица 12 Гормоны, влияющие на рост До начала полового созревания В период пубертации Гормон Степень влияния Гормон Степень влияния СТГ + + + СТГ + Тироксин + + Андрогены + + + Тормозят СТГ Инсулин + + Эстрогены + + Тормозят СТГ Гормоны тимуса + Тироксин + Гормоны, влияющие на рост До начала полового созревания В период пубертации Гормон Степень влияния Гормон Степень влияния СТГ + + + СТГ + Тироксин + + Андрогены + + + Тормозят СТГ Инсулин + + Эстрогены + + Тормозят СТГ Гормоны тимуса + Тироксин + Кроме того, рост тормозят глюкокортикоиды. Нарушения роста закладываются в детском и подростковом возрасте, но сохраня- ются пожизненно и проявляются высокорослостью и низкоросло- стью. Гормоны, влияющие на рост Гормоны, влияющие на рост Кроме того, рост тормозят глюкокортикоиды. Нарушения роста закладываются в детском и подростковом возрасте, но сохраня- ются пожизненно и проявляются высокорослостью и низкоросло- стью. 43 Пропедевтика эндокринных заболеваний Рис. 1 Высокорослость может достигать субгигантизма или гиган- тизма. При субгигантизме рост не превышает 2 м, при гигантизме – более 2 м. При пропорциональной высокорослости равномерно растут конечности, голова, туловище. Так, если опухоль гипофиза, продуцирующая СТГ (соматотропинома), развивается в детском или подростковом возрасте, возникает гипофизарный гигантизм: рост пациента, длина его конечностей, размер кистей и стоп, объём головы – всё пропорционально растёт и приобретает большие раз- меры. При непропорциональной высокорослости формируется дисгармоничное телосложение. Например, при евнухоидизме (бо- лезни Клайнфельтера) из-за дефицита половых гормонов запазды- вает закрытие зон роста, в первую очередь, трубчатых костей. Верхние и нижние конечности становятся длинными – паукообраз- ными. Синостоз костей черепа наступает значительно быстрее, и голова смотрится маленькой, а туловище как будто приставлено к голове от другого человека. Болезнь Марфана – тоже пример не- пропорциональной высокорослости: это генетическая неполноцен- ность соединительной ткани, главным образом коллагена. Нарушения роста: А. Высокорослость Харак- терна арахнодактилия («паучьи пальцы»), высокий рост, истонче- ние подкожной клетчатки, долихоцефалия, «птичье» лицо, ворон- кообразная или килевидная грудная клетка, искривлённый позво- ночник, тонкие рёбра и т.д. Конституциональная высокорослость обычно пропорциональная, но тоже может наблюдаться дисгармо- ния. Так, рост Петра I достигал 204 см, а размер его обуви не пре- вышал ≈ 38, размер кистей тоже был маленький, носил камзол 48-го размера, но отличался необыкновенной силой. 44 И.В. Терещенко И.В. Терещенко Б. Низкорослость Выделяют субнанизм и наниз. Нет точной градации, какой рост относить к нанизму и субнанизму. Для Уральского региона рост мужчин ≤ 155 см – субнанизм, ≤ 145 см – нанизм, соответственно у женщин – ≤ 145 см и ≤ 135 см. Известно много причин низкорос- лости. Важно их установить, чтобы своевременно начать лечение. Низкорослость, особенно для подростка и особенно для мальчика – всегда тяжёлый отрицательный психологический фактор. Причины низкорослости могут быть как эндокринными, так и не эндокрин- ными (основные причины представлены ниже). Рис. 2 Рис. 2 В первую очередь, сбор анамнеза поможет в дифференциальной диагностике причин низкорослости. При конституциональной низ- корослости обычно оба родителя низкого роста, при этом у ре- бёнка, кроме низкорослости, нет других симптомов патологии. При гипофизарном нанизме больные рождаются с нормальной длиной и весом, задержка проявляется после 2–3 лет, слабо формируется мышечная система, нарушается развитие и смена зубов, возникает половой инфантилизм. При отсутствии гормонального лечения больные приобретают своеобразный старческий вид. Если низко- рослость вызвана опухолями гипоталамуса, возникают характер- ные головные боли, ожирение, офтальмологические нарушения. фф Тиреоидные гормоны стимулируют рост и дифференцировку тканей, рост и развитие организма. Их дефицит в раннем (включая внутриутробный период) возрасте становится причиной задержки роста. При этом формируется кретинизм и непропорциональная 45 Пропедевтика эндокринных заболеваний низкорослость: большая голова из-за позднего закрытия родничков черепа, короткие и кривые конечности, увеличенный живот. Изме- няются черты лица: нос квадратный седловидный, с западением спинки, далеко расставленные друг от друга глаза, большой язык. При этом физическое недоразвитие всегда сопровождается ум- ственным и половым недоразвитием. В тяжёлых случаях выра- жены миксематозные отёки. В настоящее время, когда проводится УЗИ-скрининг плода и постнатальная проверка ТТГ для диагно- стики врождённого гипотиреоза, заместительная терапия тиреоид- ными гормонами начинается своевременно, врачу приходится всё реже встречаться с этой причиной низкорослости. Pubertas praecox любой этиологии всегда сопровождается гипер- секрецией половых гормонов, которые вызывают преждевремен- ное закрытие зон роста. Из-за избытка половых гормонов в орга- низме сначала темпы роста значительно ускоряются, затем рост прекращается полностью. Как правило, возникает субнанизм. Pubertas tarda разного генеза тоже может приводить к низкорос- лости из-за дефицита половых гормонов в организме. При консти- туциональной причине pubertas tarda (наиболее частая причина) за- держка роста и полового развития может быть временной, впослед- ствии формирование происходит нормально, достигается генетиче- ски запрограммированный окончательный рост, который легко рассчитать: у мальчика (рост отца + рост матери + 12,5) : 2; у девочки (рост отца + рост матери – 12,5) : 2. у мальчика (рост отца + рост матери + 12,5) : 2; у девочки (рост отца + рост матери – 12,5) : 2. При Pubertas tarda другого генеза (вследствие гамартомы, дру- гих опухолей гипоталамуса, деструкции гипофиза, повреждения гонад из-за травм, радиации, химикатов, тяжёлых соматических за- болеваний, например, болезни Крона) прогноз всегда неблагопри- ятный. Низкорослость бывает стойкой из-за отсутствия стимулиру- ющего влияния на рост половых гормонов, но при дефиците поло- вых гормонов синостоз невозможен, и в дальнейшем может раз- виться высокорослость. Низкорослость может быть обусловлена избытком в организме ребёнка кортизола при болезни и синдроме Иценко–Кушинга, в ре- зультате длительного лечения глюкокортикоидами. Избыток кор- тизола нарушает образование белковой матрицы кости и усиливает 46 И.В. Терещенко выход кальция из костей. Деформация позвонков («рыбьи по- звонки») также способствует уменьшению роста, и не только у де- тей и подростков, но и у взрослых. выход кальция из костей. Деформация позвонков («рыбьи по- звонки») также способствует уменьшению роста, и не только у де- тей и подростков, но и у взрослых. Сахарный диабет 1-го типа у ребёнка всегда негативно влияет на процессы роста, и хотя его окончательный рост может оказаться нормальным, но обычно ниже расчётных показателей. Частая де- компенсация углеводного обмена приводит к низкорослости, по- скольку инсулин влияет на все виды обмена, оказывает анаболиче- ское действие, подавляет катаболизм. У детей с тяжёлым течением сахарного диабета 1-го типа, длительно не получавших адекватной терапии инсулином, может развиться синдром Мориака или син- дром Нобекура. Клиническая картина синдрома Мориака характе- ризуется задержкой роста и полового развития, кушингоидным пе- рераспределением подкожного жира, гепатомегалией. Клиниче- ская картина синдрома Нобекура подобная, но нет избыточной подкожной жировой клетчатки, наоборот, наблюдается понижен- ная масса тела. В настоящее время в клинической практике эти син- дромы практически не встречаются. Хромосомный и генетический генез низкорослости заподозрить бывает несложно: как правило, нанизм, а чаще субнанизм сочета- ется с пороками развития, дисэмбриональными стигмами. В первую очередь, это касается синдрома Шерешевского-Тернера, при котором генотип X0, отставание в росте проявляется с раннего детства, телосложение неправильное (плечевой пояс шире тазо- вого, широко расставленные соски на бочкообразной грудной клетке, крыловидные складки кожи в области шеи, низко посажен- ные уши, низкая линия роста волос на затылке, укороченная шея, готическое нёбо и другие стигмы). Низкорослость и другие прояв- ления синдрома Шерешевского–Тернера имеют сходство с симп- томатикой синдрома Нунан, при котором кариотип нормальный; синдром обычно носит семейный характер, может встречаться также у мальчиков. К генетическим причинам низкорослости (нанизма) относится хондродистрофия (диафизарная аплазия, ахондроплазия). у мальчика (рост отца + рост матери + 12,5) : 2; у девочки (рост отца + рост матери – 12,5) : 2. Из-за ге- нетических мутаций возникает неспособность рецепторов хряща зон роста трубчатых костей, главным образом бедренных и плече- вых, воспринимать инсулиноподобный фактор роста 1 (ИФР-1). При этом рост конечностей невозможен, они остаются короткими, размер туловища достигает нормы. 47 Пропедевтика эндокринных заболеваний Синдром увеличения щитовидной железы Увеличение размеров щитовидной железы (зоб) оценивают по степеням. По классификации ВОЗ, зоб бывает I и II степени. Зоб I степени визуально не определяется, но при пальпации размеры щитовидной железы превышают концевую фалангу собственного большого пальца руки. Если щитовидная железа видна при осмотре области шеи, а пальпаторно больше нормы, то это зоб II степени (независимо от размеров!). Более удачна классификация зоба, раз- работанная отечественным эндокринологом О.В. Николаевым: он предложил различать 5 степеней размеров щитовидной железы: IV и V степени – это гигантский зоб, занимающий всю область шеи, а также он может занимать и загрудинное пространство. Для постановки правильного диагноза важно оценить форму щитовидной железы: она может быть правильной и неправильной. При диффузном йоддефицитном зобе её форма правильная. Непра- вильная форма встречается при тиреоидном раке, узловом и мно- гоузловом зобе, врождённой аномалии щитовидной железы (нали- чии одной доли) и т. д. Следует проверить, смещается ли зоб при проглатывании слюны. Если не смещается, то это либо лимфоузлы, либо кисты шеи, другие опухоли, например, лейомиома, но не щи- товидная железа, которая прикреплена к щитовидному хрящу гор- тани и вместе с ним смещается вверх–вниз при глотании. Можно заметить пульсацию щитовидной железы. Это бывает при болезни Грейвса из-за усиленного кровенаполнения зоба. Передаточная пульсация наблюдается при зобе, частично опущенном за югуляр- ную вырезку; при этом создаётся анатомическая близость к дуге аорты и крупным сосудам, отходящим от дуги аорты. Визуально и пальпаторно надо выявлять наличие узлов, оценивать их количе- ство, консистенцию, форму, размеры, смещаемость при глотании. ф р у р р р Возможна болезненность щитовидной железы при пальпации. Это особенно характерно для острого (гнойного) или подострого тиреоидита. Покраснение кожных покровов над щитовидной желе- зой возникает только при остром тиреоидите. При подостром ти- реоидите, несмотря на выраженный острый воспалительный про- цесс в щитовидной железе, гиперемия над её проекцией не появля- ется. Раковые узлы в щитовидной железе обычно безболезненны. Болезненность в области щитовидной железы может быть обуслов- лена не тиреоидной патологией, а проявлением тиреоалгии, т. е. за 48 И.В. Терещенко счёт иррадиации со стороны других органов: нередко из-за шей- ного остеохондроза, гастро-эзофагеальной рефлюксной болезни в результате заброса кислого содержимого желудка в пищевод и ро- товую полость, при раке пищевода, гортани, лёгкого и т. д. Пациент при этом уверен, что «болит именно щитовидная железа». Необхо- димо проводить дифференциальную диагностику источника боли. Возможна и сочетанная патология, вызывающая боль. Синдром увеличения щитовидной железы счёт иррадиации со стороны других органов: нередко из-за шей- ного остеохондроза, гастро-эзофагеальной рефлюксной болезни в результате заброса кислого содержимого желудка в пищевод и ро- товую полость, при раке пищевода, гортани, лёгкого и т. д. Пациент при этом уверен, что «болит именно щитовидная железа». Необхо- димо проводить дифференциальную диагностику источника боли. Возможна и сочетанная патология, вызывающая боль. Осматривая переднюю поверхность шеи, можно обнаружить из- менения кожи над щитовидной железой: послеоперационные или иные рубцы, покраснение, венозную сеть, пигментацию. Пигмен- тация рубцов на шее, кожи шеи может наблюдаться при синдроме Шмита, т.е. аутоиммунном полиэндокринном синдроме типа 2, т.е. при сочетании лимфоцитарного зоба с гипокортицизмом аутоим- мунного генеза (самый частый вариант аутоиммунного полиэндо- кринного синдрома). Надо проверить смещаемость зоба, оценить, не страдают ли со- седние органы шеи (нет ли осиплости голоса или афонии, не про- явилась ли анизокория, асфиксия, дисфагия) – всё это признаки прорастания рака щитовидной железы в соседние органы. Необходимо обратить особое внимание на опасность пальпа- ции щитовидной железы у больных с болезнью Грейвса: грубая пальпация может вызвать резкое повышение уровня тиреоидных гормонов в крови, что приводит к остановке сердца и внезапной смерти пациента, а в лучшем случае – к развитию тиреотоксиче- ского криза. 49 Табли актеристика щитовидной железы при тиреоидной патологии й Особенности изменений в щитовидной железе зы Возникает диффузное или узловое увеличение щитовидной железы, при этом она безболезненная, смещаемая, не спаяна с соседними органами шеи, при диффузном зобе сохраняет правильную форму («бабочки»), узлы округлые или овальные, окружены ободком ител Форма щитовидной железы неправильная, консистенция неоднородная за счёт фиброза и псевдоузлов, размеры могут быть увеличены, например при лимфоцитарном зобе Хашимото, или уменьшены за счёт деструкции и атрофии её паренхимы. Болезненности нет, но больные нередко испытывают дискомфорт в области щитовидной железы. Соседние органы шеи не страдают. При пальпации железа смещаема зы ТТГ Размеры щитовидной железы увеличены, могут варьировать в короткие отрезки времени за счёт изменения её кровенаполнения. Если коллатерали заполняются кровью - увеличиваются обе доли и перешеек, возникает журчание, выслушиваются систолический и диастолический шумы. Болезненности нет ия могут фии её ы. леза ровать ью - ние, мы. Окончание та Особенности изменений в щитовидной железе Размеры щитовидной железы быстро увеличиваются. Выражены все признаки острого воспаления. Кожа кра Если формируется абсцесс, определяется флюктуация проекцией. Болезненность резкая Характерна иррадиация боли в челюсть, зубы, горло, у При пальпации щитовидная железа резко болезненна, о покраснения кожи над ней нет. Синдром увеличения щитовидной железы Воспаление может захв ткань щитовидной железы не всю целиком, а участкам причём ежедневно патологический процесс в тиреоидн паренхиме может мигрировать Обнаруживается узловое образование в щитовидной ж его размеры обычно небольшие (чаще это УЗИ-находк запущенных случаях раковый узел плотный (каменист плотности), не смещаемый, безболезненный, может по лимфоаденопатия на шее и нарушаться функция сосед органов шеи Пропедевтика эндокринных заболеваний Патология уровня глюкозы в крови: А. Синдром гипергликемии В норме уровень глюкозы натощак в капиллярной крови не дол- жен превышать 5,5 ммоль/л, в венозной 6,1 ммоль/л, у беременной женщины < 5,1 ммоль/л; через 2 часа после перорального глюкозо- толерантного теста соответственно в капиллярной и венозной < 7,8 ммоль/л, у беременных < 8,5 ммоль/л. Состояние хронической гипергликемии называется сахарным диабетом. Известно много причин хронической гипергликемии. Гипергликемия развивается в том случае, если у больного имеется абсолютный или относитель- ный дефицит инсулина, или возникает избыток контринсулярных гормонов: СТГ, кортизола, катехоламинов, тиреоидных гормонов, глюкагона и др. Гипергликемия наблюдается при многих эндо- кринных и генетических заболеваниях. ц Сахарный диабет при эндокринных заболеваниях Эндокринопатия Гормональное нарушение Эндокринопатия Гормональное нарушение Сахарный диабет 1-го типа Абсолютный дефицит инсулина Синдром (болезнь) Иценко– Кушинга Избыток кортизола Сахарный диабет 2-го типа Относитель- ный дефицит инсулина Феохромо- цитома Избыток адреналина и норадре- налина Акромегалия Избыток СТГ Глюкагонома Избыток глюкагона Болезнь Грейвса Избыток тиреоидных гормонов Альдостерома Избыток альдостерона Сахарный диабет при эндокринных заболеваниях Клинически гипергликемия, независимо от генеза, проявляется слабостью, учащенным мочеиспусканием, сухостью во рту, жаж- дой, симптомами дегидратации, иногда повышенным аппетитом. Чем выше гипергликемия, тем более выражены эти симптомы. Сле- дует отметить, что в большинстве случаев синдром гипергликемии стойкий и сохраняется после излечения эндокринного заболевания, вызвавшего нарушение углеводного обмена. Например, после хи- 52 И.В. Терещенко рургического лечения опухолей – кортикостеромы, феохромоци- томы, глюкагономы, соматотропиномы, випомы, после излечения тиреотоксикоза. Пропедевтика эндокринных заболеваний Пропедевтика эндокринных заболеваний Для дифференциальной диагностики причин гипогликемий важно учесть:  для инсулиномы или гиперплазии островков поджелудочной железы (незидиобластомы) характерны тощаковые гипогликемии и гипогликемии на фоне голода;  для инсулиномы или гиперплазии островков поджелудочной железы (незидиобластомы) характерны тощаковые гипогликемии и гипогликемии на фоне голода;  при гипопитуитаризме, гипотиреозе, гипокортицизме могут быть гипогликемии тощаковые и постпрандиальные, но, как пра- вило, без гипогликемических ком;  при гипопитуитаризме, гипотиреозе, гипокортицизме могут быть гипогликемии тощаковые и постпрандиальные, но, как пра- вило, без гипогликемических ком;  функциональные гипогликемии при гепатитах, циррозе пе- чени, НАЖБП, ХПН, синдроме нарушенного всасывания, болезнях оперированного желудка гипогликемии развиваются после еды, то- щаковые не характерны;  функциональные гипогликемии при гепатитах, циррозе пе- чени, НАЖБП, ХПН, синдроме нарушенного всасывания, болезнях оперированного желудка гипогликемии развиваются после еды, то- щаковые не характерны;  тяжёлая сердечная недостаточность, тяжёлая ХПН или печёноч- ная недостаточность могут протекать как с тощаковыми, так и с пост- прандиальными гипогликемиями, но без гипогликемических ком.  тяжёлая сердечная недостаточность, тяжёлая ХПН или печёноч- ная недостаточность могут протекать как с тощаковыми, так и с пост- прандиальными гипогликемиями, но без гипогликемических ком. Б. Синдром гипогликемии р Гипогликемия – это снижение уровня глюкозы в крови < 3,5 ммоль/л. Гипогликемии могут быть скрытыми. Клиническая симптоматика гипогликемии становится явной при уровне глике- мии < 2,8 ммоль/л. Тяжёлые гипогликемии, сопровождающиеся выключением сознания (гипогликемической комой), вызывает вы- раженный избыток инсулина (вследствие передозировки инсулина при лечении сахарного диабета при недостаточном поступлении углеводов или при значительных, непропорциональных тратах ка- лорий), а также массивная секреция инсулина при инсулиноме). Скрытые гипогликемии наблюдаются при гипоталамо-гипофизар- ной недостаточности, аддисоновой болезни, гипотиреозе. Клини- ческие проявления гипогликемии характеризуются появлением внезапной раздражительности и слабости, головной боли, агрес- сивности, шаткости походки, нарушения зрения, дизартрии, повы- шенной потливости, желания немедленно что-угодно съесть. Не- редко невозможно измерением уровня глюкозы в крови подтвер- дить гипогликемию, так как гипогикемия – стресс, и в организме быстро срабатывают защитные механизмы – гиперсекреция ка- техоламинов и глюкокортикоидов. Не случайно, приступ гипогли- кемии сопровождается симптомами гиперкатехолемии: обильной потливостью, бледностью, мидриазом, тремором, тахикардией, подъёмом АД. По этой симптоматике можно сделать заключение, что пациент перенёс гипогликемию, даже если при измерении уровня глюкозы в крови её уровень уже окажется нормальным. Тем не менее, подозревая скрытые гипогликемии следует попытаться их выявить исследованием глюкозы крови. Низкий уровень глю- козы может появляться не только при гормональных нарушениях, но и при некоторых терапевтических заболеваниях (гепатитах, цир- розе печени, НАЖБП, ХПН, синдроме нарушенного всасывания, демпинг-синдроме), при отравлении алкоголем, при неадекватных физических нагрузках, ваготонии и т. д. 53 Синдром эндокринной артериальной гипертензии Артериальная гипертензия развивается при многих эндокрино- патиях, именуется вторичной эндокринной гипертензией. Нередко у больного с гормональной патологией из-за нарушения липидного обмена, дисэлектролитных расстройств, дислипидемии и гиперхо- лестеринемии, инсулинорезистентности возникает коморбидность с эссенциальной гипертензией. Коморбидность, как правило, со- здаёт трудности в лечении артериальной гипертензии и ухудшает прогноз. Жалобы больных с эндокринной гипертензией анало- гичны жалобам при повышении АД другого генеза; основная жа- лоба – головные боли, особенно в затылке. Эндокринные артери- альные гипертензии могут быть постоянными и транзиторными, преимущественно систолическими или диастолическими, или си- столо-диастолическими, иметь кризовое течение. Ниже представ- лены патогенетические механизмы и особенности течения артери- альных гипертензий при разных эндокринопатиях. 54 я повыше толичес е давлен я я к Табли ристика артериальной гипертензии ри эндокринных заболеваниях териальной гипертензии Характеристика артериальной гипертензии той стенки Стойкая, преимущественно диастолическая артериальная гипертензия икоидов Стойкая систолическая и диастолическая артериальная гипертензия, резистентная к гипотензивной терапии ме натрия и воды из-за Умеренное и непостоянное повыше артериального давления го выброса вследствие х гормонов и катехоламинов Систолическая артериальная гипертензия, снижение диастоличес давления, высокое пульсовое давле удистой стенки, осклероза и нередко – ртонической болезни Стойкая главным образом диастолическая артериальная гипертензия, рефрактерная к гипотензивной терапии з-за нефрокальциноза и Стойкая систолическая и диастолическая артериальная гипертензия. Резистентность к гипотензивной терапии Окончани териальной гипертензии Характеристика артер гипертензии ропатия Стойкая систолическая и диастолическая артериальн гипертензия. лезнь нередко предшествует диабета. Артериальная ается на фоне диабета и опатией или проявлением опатии Стойкая систолическая и диастолическая артериальн гипертензия Чаще систолическое артер давление в норме. Стойкая диастолическая гипертензи пульсовое артериальное да Возможны гипертонически еналина и норадреналина Гипертонические кризы на нормального или стойко по артериального давления ункция гипоталамуса Изменчивый уровень артер давления, наряду с вегетат нарушениями И.В. Терещенко Артериальная гипертензия может быть медикаментозной в ре- зультате применения гормональных препаратов, особенно часто при лечении глюкокортикоидами, симпатомиметиками или из-за использования гормональных контрацептивов. Артериальная гипертензия нередко является первым симпто- мом эндокринопатии, например, опухоли надпочечника. Молодой возраст пациента, сразу возникшая резистентность к многокомпо- нентной медикаментозной антигипертензивной терапии должны насторожить и назначать обследование. Особенности проявлений артериальной гипертензии следует выявлять. Так, при феохромо- цитоме криз развивается даже при незначительном сдавлении опу- холи: при наклонах, чтобы обуться, после еды из-за переполнен- ного пищей желудка и т. д. Синдром эндокринной артериальной гипертензии Если имеются характерные внешние проявления эндокринопатий, то они помогают в постановке пра- вильного диагноза: при феохромоцитоме и параганглиоме – при- знаки симпатикотонии (блеск глаз, тахикардия, холодные, потные ладони и стопы, расширенная микроциркуляторная сеть в области суставов (мраморность кожи над суставами), при синдроме Ку- шинга – ожирение, матронизм, гирсутизм, при синдроме Конна – полиурия, гипокалиемическая слабость, при тиреотоксикозе и ги- потиреозе – характерные признаки. Важна ранняя диагностика ге- неза эндокринной артериальной гипертензии, поскольку своевре- менное лечение может навсегда избавить больного от повышения артериального давления и, наоборот, при длительном существова- нии опухоли артериальная гипертензия может сохраниться и после оперативного лечения. Инсипидарные синдромы Инсипидарные синдромы – это патологические состояния, ха- рактеризующиеся повышенной жаждой и повышенным выделе- нием жидкости почками. Наиболее ярко инсипидарный синдром проявляется при несахарном диабете как гипофизарным, так и по- чечным, и характеризуется низким удельным весом мочи (всегда относительная плотность мочи ≤ 1007). Кроме того, инсипидарный синдром развивается при сахарном диабете, гиперпаратиреозе, пи- елонефрите, ХПН. Встречается психогенная полидипсия, приобре- тающая «маску» несахарного диабета. Ниже представлена симпто- матика заболеваний, протекающих с инсипидарным синдромом. 57 Т особенности их проявлений Гиперпаратиреоз Пиелонефрит, ХПН Псих пол > 2–5 л 2–3 л От нор до 7–8 Мутная из-за солей кальция (как молоко) Обычный, могут быть хлопья Обычн Гипостенурия Гипостенурия Зависи от объ выпит Любая вода (горячая тоже) Любая жидкость Вода ( всякие Отсутствует Плохой В норм т но о 7–8 быч авис т объ ыпит ода сяки нор И.В. Терещенко И.В. Терещенко Синдром гирсутизма и вирилизма Гирсутизм – это рост пушковых или стержневых волос у жен- щин на лице в виде усов, бороды, бакенбард. Гирсутизм может быть конституциональным, передаваться как от отца, так и от ма- тери. Также гирсутизм появляется при гиперандрогении любого ге- неза. Гипертрихоз – это повышенный рост волос на лице, конечно- стях, туловище. Появление гипертрихоза у взрослого мужчины всегда должно настораживать об опухоли, продуцирующей андро- гены (семиноме или опухоли надпочечника). ( у ) Вирилизм (от латинского термина virilis – мужской) – появление мужских половых признаков у женщин: увеличения клитора, гипо- трофии молочных желёз, малых половых губ, матки, яичников, нарушения месячных (в конечном итоге аменореи), ановулятор- ного бесплодия. Снижается тембр голоса, гортань перестраивается по мужскому типу. Моторика становится мужеподобной, теряется женственная походка. Кроме гирсутизма, появляется угревая бо- лезнь, повышенная сальность кожи. Кожа становится грубой, жир- ной, а на лице в виде «апельсиновой корочки». Возможно облысе- ние по мужскому типу: залысины со лба, выпадение волос с те- мени, тотальное с волосистой части головы. Помимо изменения внешнего вида, может нарушиться половая ориентация и либидо. Вирилизм (от латинского термина virilis – мужской) – появление мужских половых признаков у женщин: увеличения клитора, гипо- трофии молочных желёз, малых половых губ, матки, яичников, нарушения месячных (в конечном итоге аменореи), ановулятор- ного бесплодия. Снижается тембр голоса, гортань перестраивается по мужскому типу. Моторика становится мужеподобной, теряется женственная походка. Кроме гирсутизма, появляется угревая бо- лезнь, повышенная сальность кожи. Кожа становится грубой, жир- ной, а на лице в виде «апельсиновой корочки». Возможно облысе- ние по мужскому типу: залысины со лба, выпадение волос с те- мени, тотальное с волосистой части головы. Помимо изменения внешнего вида, может нарушиться половая ориентация и либидо. Вирильный синдром всегда свидетельствует о выраженной ги- персекреции андрогенов в организме женщины, точнее – либо о па- тологии надпочечников, либо – яичников. Надпочечниковые фрак- ции андрогенов – это ДГЭА, андростендион, 17 ОН-прогестерон, при овариальной гиперандрогении возникает главным образом ги- персекреция тестостерона (преимущественно – свободного тесто- стерона). ру р Вирильный синдром всегда свидетельствует о выраженной ги- персекреции андрогенов в организме женщины, точнее – либо о па- тологии надпочечников, либо – яичников. Надпочечниковые фрак- ции андрогенов – это ДГЭА, андростендион, 17 ОН-прогестерон, при овариальной гиперандрогении возникает главным образом ги- персекреция тестостерона (преимущественно – свободного тесто- стерона). Рис. 3 При функциональной овариальной гиперандрогении гирсутизм и гипертрихоз обычно незначительные, нет увеличения клитора. Синдром гирсутизма и вирилизма При функциональной надпочечниковой гиперандрогении, даже при ВДКН НФ, проявляется не только гирсутизм и гипертрихоз, но 59 Пропедевтика эндокринных заболеваний теряется женственный фенотип (плечевой пояс становится шире бедренного). Андрогены надпочечников влияют на развитие ске- летной мышечной системы. Из-за этого пациенты с надпочечнико- вой гиперандрогенией обычно отличаются успехами в спорте. теряется женственный фенотип (плечевой пояс становится шире бедренного). Андрогены надпочечников влияют на развитие ске- летной мышечной системы. Из-за этого пациенты с надпочечнико- вой гиперандрогенией обычно отличаются успехами в спорте. р р у р Вирилизация может развиться при приёме стероидных лекар- ственных препаратов – андрогенов, стероидных мочегонных (ве- рощпирона), гормональных контрацептивов и даже глюкокортико- идов. Гирсутизм и нарушение менструальной функции встречается при опухолях гипофиза – акромегалии, пролактиноме, кортикотро- пиноме, гормонально неактивной аденоме гипофиза. При климаксе нередко возникает компенсаторная гиперсекреция надпочечнико- вых андрогенов, что может сопровождаться незначительным гир- сутизмом. Синдром гипогонадизма у пациентов зрелого возраста Гипогонадизм в зрелом возрасте может возникнуть вследствие па- тологии гипоталамо-гипофизарной системы или гонад, характеризу- ется снижением либидо и гипоплазией вторичных половых призна- ков, наружных гениталий, а также гипоплазией и гипофункцией го- над. Всегда необходимо выяснять генез гипогонадизма. Иногда этот синдром сопровождается развитием ожирения, по поводу которого больные, в первую очередь, обращаются к врачу. В качестве клиниче- ского примера может служить следующее наблюдение: больной Ф-в Ю.П., 40 лет, обратился с жалобой на прогрессиру- ющее увеличение веса, которое отметил в последние 3 года не- смотря на прежнее питание и образ жизни. Ежегодно проходил диспансеризацию, рекомендовано «меньше есть и больше дви- гаться», что не давало эффекта. При расспросе оказалось, что у па- циента отсутствует либидо, нет утренних эрекций, прекратилась сексуальная жизнь. Имеет двоих детей, младшему 5 лет. При осмотре установлено равномерное ожирение I степени, смешанная гинекомастия II степени. Выявлена гиперпролактинемия: уровень ПРЛ 1400 нг/мл при норме до 18 нг/мл. Проведена МРТ гипофоза и выявлена макроаденома размером 4,6 см в диаметре. Таким обра- зом, у больного обнаружена пролактинома, гиперпролактинемиче- ский гипогонадизм. Часто встречается вариант овариального гипогонадизма у моло- дых женщин – синдром преждевременного уменьшения фоллику- лярного запаса яичников, что приводит к гипоэстрогении. При этом в первую очередь страдает менструальная функция. Особенно ча- сто этот синдром развивается у вегетарианок, у женщин, страдаю- щих дефицитом железа и малокровием, при гипотиреозе, в том числе субклиническом, при дефиците массы тела. 60 И.В. Терещенко Раздел 3. ЛАБОРАТОРНЫЕ И ИНСТРУМЕНТАЛЬНЫЕ МЕТОДЫ ИССЛЕДОВАНИЯ БОЛЬНЫХ ПРИ ДИАГНОСТИКЕ ЭНДОКРИННОЙ ПАТОЛОГИИ Рассмотрим применяемые в практической работе врачей лабо- раторные и инструментальные методы исследования больных с эн- докринной патологией. Их выбор зависит от характера заболева- ния. Помимо общеклинического обследования (общий анализ крови и мочи, биохимический анализ крови, включающий исследо- вание уровня в крови глюкозы, белка, АЛТ, АСТ, гаммаГТП, липи- дограммы, билирубина, мочевой кислоты, по показаниям электро- литов - натрия, калия, кальция, фосфора, магния, ферритина, цинка, проведение флюорографии), больные с эндокринной патологией нуждаются в гормональном контроле. В настоящее время разра- ботаны методики определения всех известных гормонов в биоло- гических жидкостях. В практической работе наиболее часто возни- кает необходимость исследовать следующие гормоны (таблица), причём забор крови производят в утренние часы натощак. Таблица 17 61 ц Исследование гормонов в крови Место выработки гормона Гормоны Гипофиз СТГ, АКТГ, ПРЛ, макропролактин, ТТГ, ЛГ, ФСГ Щитовидная железа. Тиреоциты Светлые клетки Трийодтиронин (св. Т3); тироксин (св. Т4) Кальцитонин Околощитовидные железы Паратгормон Поджелудочная железа Инсулин, С-пептид, глюкагон Надпочечники Минералокортикоидные гормоны (альдостерон) Глюкокортикоидные гормоны (кортизол) Андрогены (ДГЭА, андростендион, 17 α ОН-п-н) Гонады Тестостерон, св. тестостерон, эстрадиол, прогестерон, при беременности – эстриол Жировая ткань Лептин, адипонектин ЖКТ Гастрин, грелин Печень и почки Витамин Д Исследование гормонов в крови Исследование гормонов в крови 61 Пропедевтика эндокринных заболеваний В практике эндокринолога нередко возникает необходимость исследования инсулиноподобного фактора роста-1 (ИПРФ-1), или соматомедина С, важнейшего эндокринного посредника действия СТГ. Контролировать уровень ИПРФ-1 следует для выяснения причин нарушения роста, для выявления нейроэндокринных опу- холей (инсулиномы, глюкагономы, випомы и лр.). Для диагностики АИТ и гипотиреоза исследуют блокирующие антитела АТ-ТПО, АТ-ТГ; при диагностике тиреотоксикоза – стимулирующие анти- тела АТ-рТТГ. Проверяют онкомаркёры щитовидной железы – АТ- ТГ и тиреоглобулин (они являются также показателями деструк- тивного процесса в щитовидной железе). Для диагностики медул- лярного рака щитовидной железы необходимо исследовать кальци- тонин: повышение его уровня укажет на медуллярный рак щито- видной железы. С целью выявления аутоиммунного сахарного диабета 1-го типа и LADA-диабета проверяют антитела к антигенам бета-клеток под- желудочной железы: аутоантитела к глутаматдекарбоксилазе (АТ- GAD), инсулину, тирозинфосфатазе. Некоторые гормоны информативнее исследовать не в крови, а в моче. Так, из-за высокой вариабельности физиологической секре- ции кортизола его следует проверять не в крови, а в суточной моче. За последнее время стали определять свободный кортизол в слюне, собранной вечером в 23.00 часа в специальную пробирку со шва- бом, впитывающим слюну. Шваб – ватный тампон, находящийся в пробирке, помещают в ротовую полость на 2–3 минуты. После до- статочного пропитывания слюной шваб без усилий зубами возвра- щается в пробирку. Раздел 3. ЛАБОРАТОРНЫЕ И ИНСТРУМЕНТАЛЬНЫЕ МЕТОДЫ ИССЛЕДОВАНИЯ БОЛЬНЫХ ПРИ ДИАГНОСТИКЕ ЭНДОКРИННОЙ ПАТОЛОГИИ В течение 30 минут до сбора слюны нельзя упо- треблять пищу, питьё (кроме простой воды), чистить зубы. Про- бирка со швабом после сбора слюны помещается в холодильник (не замораживать) и может храниться до 7 дней. Такое исследование кортизола в слюне целесообразно провести двухкратно: в нашей стране у практически здоровых людей уровень кортизола в слюне в 23.00 часа в среднем составляет 4,5 нмоль/л. р В суточной моче определяют катехоламины (мета / нормета- нефрины). При обследовании половой системы в комплексе с определе- нием половых гормонов в ряде случаев возникает необходимость проверять следующие биологически активные вещества в крови (таблица), причём эти исследования используются в практике не только эндокринологами, но также гинекологами, урологами, анд- рологами, онкологами и другими специалистами. 62 Табли вня биологически активных веществ в крови ьной оценки состояния половой системы Показания к исследованию том сти ХГЧ яет жёлтое 12 недель  диагностика беременности, контроль за её течением в ранн сроках;  онкомаркер при опухолях трофобласта (пузырном заносе, хорионкарциноме) и опухолях эмбрионального зачатка – при тератоме, (у мужчин при тератоме яичка, семиноме) тор роста β  диагностика и лечение бесплодия у мужчин и женщин;  подготовка пациенток к ЭКО;  оценка овариального резерва яичников (при истощении резерва уровень АМГ снижается);  диагностика СПКЯ (АМГ повышен);  диагностика гранулёзоклеточных опухолей яичников (АМГ повышен) мирующий уется в в ников. При тывает Маркер овариального резерва яичников и сперматогенеза у мужчин (при обеднении уровень снижается). Оценка эффективности ЭКО. Диагностика гранулёзоклеточных и муцинозных опухолей яичников (повышен). Диагностика нарушений полового развития. Обследование супружеских пар по поводу бесплодия оциты  позволяет судить о количестве связанных с белком, т.е. неактивных андрогенов и / или эстрогенов;  позволяет рассчитать уровень свободного тестостерона тка – оме) щин; щении ков енеза у ухолей я м, т.е. ерона Пропедевтика эндокринных заболеваний Результаты гормонального исследования не могут быть веду- щими в постановке диагноза! Клиническая симптоматика всегда играет решающую роль в диагностике. Можно привести немало примеров грубых диагностических ошибок, когда это правило врач нарушает и «лечит анализ», а не больного. Так, снижение уровня ТТГ в крови интерпретируется как болезнь Грейвса, хотя у паци- ента нет ведущего клинического симптома – стойкой тахикардии, ему назначается тирозол. Пониженный уровень ТТГ нередко воз- никает при деструктивном процессе в щитовидной железе, при снижении выработки ТТГ гипофизом, при лечении глюкокортико- идами, при подостром тиреоидите и т. д. Также часто применяются больным агонисты дофамина (достинекс, агалатес и другие), если содержание в крови ПРЛ оказалось хотя бы ничтожно повышен- ным. Раздел 3. ЛАБОРАТОРНЫЕ И ИНСТРУМЕНТАЛЬНЫЕ МЕТОДЫ ИССЛЕДОВАНИЯ БОЛЬНЫХ ПРИ ДИАГНОСТИКЕ ЭНДОКРИННОЙ ПАТОЛОГИИ В результате «лечения» уровень ПРЛ становится ниже нормы или совсем не определяется и может проявиться деструкция ткани аденогипофиза. Для определения гормонов используют главным образом метод иммуноферментного анализа. В зависимости от того, какие наборы реактивов использованы для определения того или иного гормона, показатели норм варьируют (нормы должны всегда указываться в результатах анализа). У менструирующих женщин анализ на гона- дотропины, половые гормоны следует производить на 3–5 дни мен- струального цикла, на прогестерон – на 22–23 дни. АМГ и ингибин В проверяют на 3-й день менструального цикла. Эти сроки игнори- руются, если нарушена менструальная функция. Функциональные гормональные пробы  Повышение уровня кальцитонина – гормона, вырабатывае- мого светлыми клетками щитовидной железы, является признаком медуллярного рака щитовидной железы.  Повышение уровня кальцитонина – гормона, вырабатывае- мого светлыми клетками щитовидной железы, является признаком медуллярного рака щитовидной железы.  Проба с тироджином. Проба с тироджином проводится для проверки радикальности хирургического лечения папиллярной или фолликулярной карциномы щитовидной железы. Тироджин (тиро- ген) – синтетический тиреотропный гормон – вводят для стимуля- ции существующих в организме после операции раковых клеток щитовидной железы (клетки дифференцированного рака щитовид- ной железы сохраняют способность отвечать на стимуляцию ТТГ, значит, и тироджином), вследствие этого возрастает уровень в крови тиреоглобулина. Тироджин вводят внутримышечно дважды по 0,9 мг с интервалом в 24 часа. Затем для выявления локализации очагов рака проводят сцинтиграфию всего тела с йодом-123.  Проба с тироджином. Проба с тироджином проводится для проверки радикальности хирургического лечения папиллярной или фолликулярной карциномы щитовидной железы. Тироджин (тиро- ген) – синтетический тиреотропный гормон – вводят для стимуля- ции существующих в организме после операции раковых клеток щитовидной железы (клетки дифференцированного рака щитовид- ной железы сохраняют способность отвечать на стимуляцию ТТГ, значит, и тироджином), вследствие этого возрастает уровень в крови тиреоглобулина. Тироджин вводят внутримышечно дважды по 0,9 мг с интервалом в 24 часа. Затем для выявления локализации очагов рака проводят сцинтиграфию всего тела с йодом-123. Функциональные гормональные пробы Однократное определение уровня гормонов может быть мало- информативным. Поэтому кроме определения базального уровня гормонов, используют различные нагрузочные тесты и контроли- руют динамику секреции гормонов.  Малая проба с дексаметазоном помогает выявлять эндоген- ный гиперкортицизм. Дексаметазон в дозе 1 мг больной принимает между 23.00–24.00 часами. На следующее утро между 8.00–9.00 ча- сами определяют уровень кортизола в сыворотке крови. У здоро- вых людей дексаметазон подавляет секрецию АКТГ, следова- тельно, и кортизола. У пациентов с эндогенным гиперкортицизмом в ответ на низкую дозу дексаметазона подавления секреции АКТГ 64 И.В. Терещенко не происходит. Использование более высоких доз дексаметазона не улучшает диагностических возможностей теста. не происходит. Использование более высоких доз дексаметазона не улучшает диагностических возможностей теста.  При подозрении на инсулиному проводят пробу с голоданием продолжительностью до 72 часов. Больному дают ужин. Утром сразу после пробуждения определяют в венозной крови глюкозу, инсулин, С-пептид, проинсулин (можно ограничиться исследова- нием глюкозы и инсулина). Затем исключается любая пища (пить воду разрешено). Каждые 4 часа глюкометром проверяют уровень глюкозы в капиллярной крови. При появлении симптомов гипогли- кемии, при снижении гликемии ≤ 2,7 ммоль/л повторяют определе- ние в венозной крови глюкозы, инсулина, С-пептида, проинсулина, а голодание прекращают. Оценивают коэффициент отношения ин- сулина к глюкозе. У здоровых людей он всегда ниже 0,4, в то время как при инсулиноме всегда выше этого показателя; уровень С-пеп- тида и проинсулина тоже выше нормы. р у р  Проба с подавлением СТГ глюкозой: гипергликемия после приёма глюкозы подавляет секрецию соматолиберина, и уровень СТГ в крови снижается. При соматотропиноме этого достигнуть не удаётся. Пациенту дают принять внутрь натощак 100 г глюкозы. Пробы крови для определения СТГ берут до приёма глюкозы, за- тем через 0,5, 1, 2, 3 часа после приёма глюкозы (целесообразно брать дополнительно пробы крови через 4 и 5 часов). У здоровых лиц в течение 2 часов уровень СТГ снижается до < 2,0 нг/мл; на 4–5-ом часу может наблюдаться «феномен отдачи», т.е. уровень СТГ повышается и может превышать 7,0 нг/мл. При соматотропи- номе отсутствует подавление секреции СТГ. Однако иногда бывает недостаточное подавление СТГ или парадоксальная реакция – сти- муляция СТГ глюкозой. В этих и всех других случаях повышения уровня СТГ в крови (физическая нагрузка, стресс, избыток белка в рационе, продолжительное голодание, нервная анорексия, синдром нарушенного всасывания) нагрузка глюкозой может дать ложнопо- ложительный результат. Приходится повторять пробу или выби- рать другую нагрузочную пробу. 65 Пропедевтика эндокринных заболеваний Онкомаркёры щитовидной железы Онкомаркёры щитовидной железы  Повышение уровня в крови АТ-ТГ и тиреоглобулина может наблюдаться при раке щитовидной железы. Контроль гликемии При сахарном диабете или подозрении на нарушения углевод- ного обмена всегда проводят многократный контроль гликемии. Важно проверить уровень глюкозы в крови утром натощак. Из- вестно, первым этапом нарушения углеводного обмена при риске СД2 является утренняя гипергликемия. Это называется наруше- нием гликемии натощак. Следует контролировать уровень глю- козы и после еды – постпрандиальную гипергликемию. Целесооб- разно осуществлять контроль глюкозы через 1 и 2 часа после при- ёма пищи, а также периодически в 3 часа ночи. Больные с явным диабетом должны систематически проводить самоконтроль глике- мии собственным глюкометром и вести дневник, а врач должен при каждой встрече с пациентом просматривать записанные резуль- таты измерения уровня глюкозы и корректировать инсулинотера- пию и другую сахароснижающую терапию. Примерная форма ве- дения дневника гликемии такова: Д В У П Дата Время Уровень глюкозы Примечания: когда и что ел, лекарства, самочувствие Дата Время Уровень глюкозы Примечания: когда и что ел, лекарства, самочувствие 66 И.В. Терещенко Тест толерантности к глюкозе д Необходимо проводить тест толерантности к глюкозе в сле- дующих случаях:  сомнительные результаты контроля гликемии натощак, в том числе при беременности;  случайно выявленная глюкозурия;  случайно выявленная глюкозурия;  вынашивание беременности;  появление клинических симптомов сахарного диабета. Таблица 19 Таблица 19 Интерпретация теста Интерпретация теста Диагностический уровень содержания глюкозы (ммоль/л) Диагноз Момент взятия крови Цельная венозная кровь Цельная капиллярная кровь Плазма венозной крови Норма Натощак 3,3–5,5 3,3–5,5 4,0–6,1 Через 2 часа после после нагрузки глюкозой ≤ 6,7 ≤ 7,8 ≤ 7,8 Нарушение толерантно- сти к глю- козе Натощак Через 2 часа после нагрузки глюкозой ≤ 6,1 6,7–10,0 ≤ 6,1 7,8–11,1 ≤ 7,0 7,8–11,1 Сахарный диабет Натощак Через 2 часа после нагрузки глюкозой ≥ 6,1 ≥ 10,0 ≥ 6,1 ≥ 11,1 ≥ 7,0 ≥ 11,1 Таблица 20 Пороговые значения глюкозы венозной плазмы для диагностики гестационного СД Глюкоза венозной плазмы ммоль/л Натощак ≥5,1, но < 7,0 После приёма 75 г глюкозы Через1 час ≥10,0 Через 2 часа ≥8,5 Пороговые значения глюкозы венозной плазмы 67 Пропедевтика эндокринных заболеваний Исследуется только уровень глюкозы в венозной плазме. Иссле- дование проб цельной, капиллярной крови не целесообразно. По результатам перорального теста толерантности к глюкозе с 75 г глюкозы для установления гестационного СД достаточно хотя бы одного значения уровня глюкозы венозной плазмы из трёх, которое было бы равным или выше порогового. При аномальном значении в исходном измерении нагрузка глюкозой не проводится; при по- лучении аномальных значений во второй точке, третье измерение не требуется. Контроль гликемии Если глюкоза венозной плазмы натощак < 5,1 ммоль/л и через 1 час в ходе перорального теста толерантно- сти к глюкозе < 10,0 ммоль/л, а через 2 часа ≥ 7,8ммоль/л и < 8,5 ммоль/л (что соответствует нарушенной толерантности к глюкозе у небеременных), то для беременных это будет вариантом нормы. Т б 21 Таблица 21 Пороговые значения глюкозы венозной плазмы для диагностики манифестного (впервые выявленного) СД во время беременности Манифестный (впервые выявленный) СД у беременных Глюкоза венозной плазмы натощак ≥7,0 ммоль/л Глюкоза венозной плазмы вне зависимости от вре- мени суток и приёма пищи при наличии симпто- мов гипергликемии ≥ 11,1 ммоль/л Гли НвА1с ≥6,5% Если аномальные значения были получены впервые и нет симп- томов гипергликемии, то предварительный диагноз манифестного СД во время беременности должен быть подтверждён уровнем глюкозы венозной плазмы натощак или Гли НвА1с. Исследование инсулинорезистентности Для дифференциальной диагностики СД 1-го и СД 2-го типа ис- следуют уровень С-пептида в крови и инсулинорезистентность. Ги- перинсулинемия и инсулинорезистентность характерны для СД 2-го типа; инсулинорезистентность не наблюдается при СД 1-го типа. С-пептид – это часть молекулы проинсулина, которая оста- ётся после отделения аминокислотной цепочки инсулина. В отли- чие от инсулина, который является нестойким соединением, быстро разрушается инсулиназой печени и протеолитическими 68 И.В. Терещенко ферментами почек и других тканей (в среднем циркулирует в кро- вотоке ≈ 4 минуты), С-пептид – стойкое соединение, в кровотоке не разрушается, экскретируется почкой в неизменённом виде. По- вышение содержания в крови С-пептида свидетельствует о гипе- ринсулинемии. ферментами почек и других тканей (в среднем циркулирует в кро- вотоке ≈ 4 минуты), С-пептид – стойкое соединение, в кровотоке не разрушается, экскретируется почкой в неизменённом виде. По- вышение содержания в крови С-пептида свидетельствует о гипе- ринсулинемии. Для определения инсулинорезистентности рассчитывают ин- декс HOMO IR и индекс CARO. Индекс HOMO IR исследуют нато- щак и рассчитывают по формуле [глюкоза (ммоль/л) х инсулин (мкМЕ)] : 22,5. Его значения выше 2,86 свидетельствуют о наличии инсулинорезистентности. Индекс CARO – это отношение глюкозы (ммоль/л) к инсулину в плазме (мкМЕ/мл). Значения этого индекса менее 0,33 указывают на инсулинорезистентность. Контроль гликемии в режиме реального времени и Flash-мониторирование гликемии Даже при ежедневном проведении самоконтроля гликемии мо- гут оставаться не диагностированными её резкие колебания или скрытые гипогликемии, а это всегда приводит к осложнениям диа- бета. В последнее время в клиническую практику введены новые способы контроля гликемии – непрерывное мониторирование гли- кемии в режиме реального времени и Flesh-мониторирование глю- козы. Непрерывное мониторирование содержания глюкозы в меж- клеточной жидкости подкожной клетчатки проводится сенсором в течение нескольких дней непрерывно. Больной избавляется от ча- стых проколов пальцев. Информация выводится на экран прибора каждые 5–10 минут, а также выводятся прогнозирующие сигналы тревоги гипо- или гипергликемии; вычерчивается график глике- мии. Непрерывное мониторирование особенно важно проводить больным, получающим интенсивную инсулинотерапию. Препят- ствиями для использования непрерывного мониторирования глю- козы являются необходимость установки сенсора каждую неделю, местные реакции на адгезивные материалы, боль и дискомфорт в месте установки сенсора, частые сигналы тревоги (особенно в ноч- ное время), необходимость параллельного измерения глюкозы глюкометром для калибровки сенсора, высокая стоимость метода. Flesh-мониторирование глюкозы не имеет этих недостатков. Си- стема Flesh-мониторинга глюкозы состоит из сканера и одноразо- 69 Пропедевтика эндокринных заболеваний вого датчика, который устанавливают на коже. Датчик представ- ляет собой круглую пластиковую «таблетку» диаметром 3 см и тол- щиной 4 мм, который устанавливается с помощью специального аппликатора на любую удобную часть тела, чаще всего на заднюю поверхность плеча (там его сложнее всего задеть и сместить). От пластикового корпуса в межклеточное пространство кожи прони- кает тонкий чувствительный «волосок»-датчик. Он не ощущается при повседневном использовании, установка совершенно безболез- ненна. Корпус датчика прочно приклеивается специальным гипо- аллергенным пластырем, который наносится заранее. Срок исполь- зования сенсора допустим до 14 дней. Его калибровка не требуется. вого датчика, который устанавливают на коже. Датчик представ- ляет собой круглую пластиковую «таблетку» диаметром 3 см и тол- щиной 4 мм, который устанавливается с помощью специального аппликатора на любую удобную часть тела, чаще всего на заднюю поверхность плеча (там его сложнее всего задеть и сместить). От пластикового корпуса в межклеточное пространство кожи прони- кает тонкий чувствительный «волосок»-датчик. Он не ощущается при повседневном использовании, установка совершенно безболез- ненна. Корпус датчика прочно приклеивается специальным гипо- аллергенным пластырем, который наносится заранее. Срок исполь- зования сенсора допустим до 14 дней. Его калибровка не требуется. УЗИ щитовидной железы и паращитовидных желёз УЗИ щитовидной железы и паращитовидных желёз УЗИ щитовидной железы необходимо проводить прежде всего для дифференциальной диагностики зоба с другими образовани- ями в области шеи, для исключения онкологического процесса, для диагностики АИТ, болезни Грейвса, диффузного, узлового и мно- гоузлового зоба, острого и подострого тиреоидита. УЗИ помогает в оценке динамики заболеваний и эффективности лечения. С помо- щью УЗИ можно определить локализацию щитовидной железы (в норме располагается в области щитовидного хряща гортани); мо- жет быть частично или полностью опущена за грудину, оценить её форму, размеры каждой доли и перешейка, эхоструктуру (однород- ная или неоднородная), эхогенность (нормальная, повышенная, по- ниженная), наличие узлов, измерить размеры каждого узла, изу- чить их особенности – локализацию, форму, капсулу («хало-обо- док»), отложение извести и даже содержимое узла (коллоид или об- разование плотное). Важно проверять, прорастает узел (т.е. рак щи- товидной железы) капсулу, проникает в соседние органы или пока процесс ограниченный, а также выявлять лимфоузлы, их струк- туру, количество, расположение на шее, являются или не являются увеличенные лимфоузлы метастазами или имеют нормальную структуру. УЗИ щитовидной железы стал популярным методом не только во врачебной практике, но и у населения. Необходимо учесть, что даже кратковременное воздействие ультразвуком на тиреоидную паренхиму не безразлично для тиреоцитов. Поэтому проводить УЗИ щитовидной железы надо только по строгим показаниям, а не ради пустого «интереса». УЗИ щитовидной железы не может заме- 70 И.В. Терещенко нить её пальпации. Ошибочные заключения УЗИ щитовидной же- лезы специалистами могут допускаться. Поэтому клиницист дол- жен сопоставлять и анализировать результаты пальпации и резуль- таты УЗИ щитовидной железы. УЗИ органов шеи в отдельных случаях позволяет обнаружить опухоль (или опухоли) паращитовидных желёз и заподозрить пер- вичный гиперпаратиреоз. Паратиромы вплотную примыкают к до- лям ЩЖ, но находятся за её пределами и имеют иные эхографиче- ские характеристики, чем остальные узловые образования в щито- видной железе, располагаются обычно позади доли щитовидной железы, могут деформировать её контур, обычно имеют овальную форму, окружены собственной капсулой, контуры ровные, чёткие, васкуляризация смешанная, размеры паратиром в норме < 1 см – ≈ 2 см. УЗИ загрудинного пространства является ориентировоч- ным методом диагностики тимомы. УЗИ орбит Узи орбит производят для:  диагностики эндокринной офтальмопатии;  планирования лечения офтальмопатии в зависимости от того, уже имеется фиброзирование глазодвигательных мышц и ретро- бульбарных тканей или ещё не наступило;  планирования лечения офтальмопатии в зависимости от того, уже имеется фиброзирование глазодвигательных мышц и ретро- бульбарных тканей или ещё не наступило;  диагностики патологии стекловидного тела у больных сахар- ным диабетом: выявление помутнения после перенесённого в анамнезе кровоизлияния в стекловидное тело, наличия сгустков крови при недавно происшедшем гемофтальме, для поиска де- струкции, шварт, экссудата в стекловидном теле, определения осо- бенностей их локализации, плотности;  диагностики патологии стекловидного тела у больных сахар- ным диабетом: выявление помутнения после перенесённого в анамнезе кровоизлияния в стекловидное тело, наличия сгустков крови при недавно происшедшем гемофтальме, для поиска де- струкции, шварт, экссудата в стекловидном теле, определения осо- бенностей их локализации, плотности;  дифференциальной диагностики эндокринной офтальмопатии с опухолью орбиты при одностороннем экзофтальме.  дифференциальной диагностики эндокринной офтальмопатии с опухолью орбиты при одностороннем экзофтальме. УЗИ надпочечников Если нет опухоли надпочечников, это недостаточно информа- тивный метод, поскольку из-за маленького объёма они не всегда визуализируются. Но при подозрении на опухоль надпочечника необходимо начинать диагностику с проведения УЗИ надпочечни- ков: метод позволит определить локализацию и размеры опухоли, односторонний или двусторонний процесс, подскажет, какие гор- моны надо, в первую очередь, проверять. Так, альдостерома, как правило, имеет маленькие размеры, и не всегда её можно эхогра- фически выявить. Кортикостерома и кортикобластома обычно 71 71 Пропедевтика эндокринных заболеваний средних размеров, опухоли односторонние, деформируют надпо- чечник. Феохромоцитома и феохромобластома достигают крупных размеров, часто бывают двусторонними. Нередко хромаффинные аденомы имеют аберрантную локализацию. Поэтому необходимо проводить таким пациентам УЗИ органов брюшной полости и за- брюшинного пространства. УЗИ органов малого таза и молочных желёз УЗИ органов малого таза необходимо проводить каждой паци- ентке, независимо от вида эндокринопатии. Это особенно важно при гирсутизме или вирильном синдроме. Следует оценить раз- меры яичников, их форму, толщину капсулы, соотношение стромы и фолликулярного слоя, количество фолликулов (в норме их не бо- лее 7 в срезе), их расположение (диффузное или по периферии в виде «чёток»), наличие доминантного фолликула или жёлтого тела. Важно оценить толщину эндометрия (М-эхо), размеры матки и шейки матки, что зависит от гормонального фона. Одним из веду- щих диагностических признаков СПКЯ признана поликистозная морфология яичников по УЗИ. Поликистозную морфологию яич- ников по УЗИ часто путают с мультифолликулярными яичниками, хотя это разная патология, и требуется разное лечение. Мультифол- ликулярные яичники развиваются вследствие длительной ановуля- ции. Мультифолликулярные яичники не возникают при физиоло- гической ановуляции, причины которой – детский, подростковый, старческий возраст, беременность, кормление грудью и т.д. Чаще всего мультифолликулярные яичники формируются при дисфунк- ции гипоталамо-гипофизарной системы из-за гиперсекреции ФСГ и / или эстрогенов. Избыток ФСГ и эстрогенов стимулирует увели- чение сразу множества фолликулов (10 и >, в норме не более 7). Как правило, мультифолликулярные яичники – случайная находка при проведении УЗИ. 72 И.В. Терещенко Таблица 22 Таблица 22 Морфология яичников по УЗИ при СПКЯ и при мультифолликулярных яичниках Особенности яичников по УЗИ СПКЯ Мультифолликулярные яичники Размеры Увеличены (возможно одностороннее увеличение) Увеличены Форма Округлая: «раздутые шары» Неправильная, причудливая Капсула Утолщена Обычной толщины Строма Гиперплазирована Без изменений Количество фолликулов Увеличено Увеличено Размеры фолликулов Мелкие, антральные Варьируют, нередко становятся крупными кистами Расположение фолликулов в яичнике По периферии в виде «чёток» Диффузное, беспорядочное Признаки овуляции Обычно отсутствуют Часто отсутствуют Морфология яичников по УЗИ при СПКЯ и при мультифолликулярных яичниках С помощью УЗИ можно диагностировать гипогонадизм (син- дром преждевременного истощения яичников), а также выявлять гормонально активные опухоли яичника, в частности – арренобла- стому, текому и другие. УЗИ панкреас УЗИ панкреас незаменимо при поиске нейроэндокринных опу- холей – глюкагономы, инсулиномы, соматостатиномы, ВИПомы, гастриномы, карциноида, гормонально неактивные образования. Перечисленные опухоли часто локализуются именно в поджелу- дочной железе, хотя могут иметь и другие локализации. Топиче- ская диагностика нейроэндокринных опухолей (НЭО) очень сложна. Размеры таких опухолей значительно варьируют. Их гор- мональная активность не зависит от размеров опухоли. Образова- ния малых размеров сложно выявлять не только с помощью УЗИ, но и другими диагностическими методами. Топическую диагно- стику НЭО обычно начинают с УЗИ. Если не удалось верифициро- вать диагноз, то необходимо направить пациента на эндоскопиче- ское УЗИ (эндосонографию). В настоящее время этот диагностиче- ский метод признан приоритетным, нередко единственным, даю- щим возможность распознавать и очень мелкие НЭО поджелудоч- ной железы (даже < 1,5 см), а также мультифокальные поражения панкреас. УЗИ органов мошонки УЗИ органов мошонки УЗИ органов мошонки – это информативный метод диагностики гормональной патологии мужских гонад, позволяет оценить их форму, размеры, структуру, состояние придатков яичек. С помо- щью УЗИ можно диагностировать крипторхизм, определить сте- пень половой зрелости яичек. Метод незаменим для оценки темпа полового созревания (раннее или позднее), для диагностики муж- ского гипогонадизма или опухолей яичек или придатков. Нередко, наряду с УЗИ органов мошонки, необходимо одновременно прове- сти УЗИ простаты, например, при признаках гипогонадизма. УЗИ молочных желёз Метод безопасный, позволяющий обследовать женщину в дина- мике и беременную, и кормящую, выявлять диффузную или узло- вую мастопатию, воспалительные или нагноительные процессы в молочной железе, наличие кист, опухоли. Иногда вместо УЗИ мо- лочных желёз назначают маммографию. Однако, оба метода допол- няют друг друга, и нельзя ограничиться только маммографией (тем более при этом неизбежно гамма-облучение молочных желёз). 73 Пропедевтика эндокринных заболеваний УЗИ-метод определения костного возраста ребёнка В последние годы стали применять УЗИ-метод определения костного, т. е. биологического возраста ребёнка. Для этого прово- дят УЗИ тыльной поверхности левого запястья, пясти, дистальных эпифизов локтевой и лучевой костей и выявляют ядра окостенения. Расшифровка аналогична оценке результатов костного возраста по рентгенограммам кисти, но ребёнок не подвергается нежелатель- ному гамма-облучению. 74 И.В. Терещенко Магнитно-резонансная томография в диагностике эндокринной патологии Магнитно-резонансная томография гипофиза (МРТ) с контра- стированием позволяет выявить микро- или макроаденому гипо- физа, опухоли гипоталамуса, оценить направление роста опухоли (вниз, кзади, вверх, кпереди), отношение к зрительному перекре- сту, к кавернозным синусам, к сосудам, помогает обнаружить пу- стое турецкое седло, а также врождённые образования – кисту кар- мана Ратке, установить апоплексию в аденому гипофиза. Важно отметить, что ценность КТ гипофиза для диагностики аденом, особенно микроаденом, недостаточная. Необходимо де- лать МРТ! Наоборот, КТ брюшной полости более информативный метод диагностики эндокринных опухолей, чем МРТ. МРТ мягких тканей шеи, щитовидной железы, околощитовид- ных желёз, загрудинного пространства широко используется во врачебной практике. МРТ обладает наибольшей контрастностью мягких тканей среди всех методов лучевой диагностики, поэтому даёт информацию о структурах шеи, помогает выявлять объёмные образования размером до 1 – 2 мм, позволяет оценить характер кон- туров образования, наличие капсулы, её интактность или прерыви- стость, уточнить прорастание опухоли в окружающие структуры, смещение соседних органов или их сдавление образованием извне, обнаружить лимфоузлы шеи, судить об их количестве, размерах, структуре (нормальное строение или метастазы). Необходимо проводить МРТ загрудинного пространства для ди- агностики загрудинного зоба, выявления аденомы паращитовид- ных желёз при атипичной их локализации (нередко паратиромы располагаются в тимусе), а также для диагностики тимомы. Симптомы миастении являются абсолютным показанием к про- ведению МРТ загрудинного пространства и средостения для кон- троля тимуса. Размеры, форма тимуса, интенсивность МРТ-сиг- нала зависят от возраста: чем старше возраст пациента, тем интен- сивность сигнала более выражена, чем у детей, подростков, моло- дых людей из-за жировой инфильтрации вилочковой железы в ре- зультате возрастной её инволюции. При визуализации с помощью МРТ в норме тимус имеет треугольную или двудольчатую форму, 75 Пропедевтика эндокринных заболеваний толщина доли у взрослых не должна превышать 13 мм. При гипер- плазии вилочковой железы её форма сохраняется, но увеличива- ются размеры долей. Округлые, инкапсулированные, с чёткими контурами образования в тимусе являются доброкачественными. Злокачественные тимомы имеют неровный контур, кистозные и некротические компоненты, кальцинаты; они обычно сопровожда- ются лимфоаденопатией в средостении. МРТ орбит помогает в диагностике эндокринной офтальмопа- тии и в планировании её лечения в зависимости от того, имеется фиброзирование глазных мышц и ретробульбарных тканей или ещё не наступило. По сравнению с УЗИ орбит метод более точный. Компьютерная томография в диагностике эндокринной патологии Компьютерная томография –КТ (предпочтительно мультис- пиральная КТ) – проводится для диагностики патологии органов брюшной полости, забрюшинного пространства, почек, дыхатель- ной и костной систем. КТ органов малого таза предпочтительнее проводить при обнаружении полостных образований, заполненных жидкостными субстанциями. Если предполагаем патологию в мяг- ких структурах малого таза, надо назначать МРТ. ру ур Компьютерная томография брюшной полости необходима для поиска эндокринных опухолей, например, феохромоцитомы, если её локализация атипичная, или для поиска нейроэндокринных опу- холей ЖКТ. КТ денситометрия – позволяет установить плотность костной ткани, её структуру, риск остеопороза. Проводят КТ-денситомет- рию позвоночника, шейки бедренной кости. Анализируют два по- казателя: T-показатель и Z-показатель. T-показатель является ре- зультатом сравнения плотности костной ткани пациента и эталон- ного показателя; норма -1 и выше. В диапазоне от −1 до −2,5 озна- чает низкую минеральную плотность, т.е. остеопению; показатель Z – результат сравнения плотности костной ткани пациента со средним показателем его возрастной группы. Z-показатель менее 2,5 – означает высокий риск переломов, т.е. остеопороз. При слиш- ком высоком или низком Z-показателе необходимо назначить до- полнительное обследование, в первую очередь, рентгенографию костей. Примечание: УЗИ-денситометрия из-за недостаточной точно- сти в настоящее время не должна использоваться для диагностики остеопороза; может применяться только для скрининга. 76 И.В. Терещенко Рентгенологические методы исследования актуальны и в насто- ящее время. Рентгенологические методы исследования актуальны и в насто- ящее время. Рентгенографию костей следует производить для диагностики остеопороза и патологических переломов у больных с остеопорозом, особенно в результате гиперпаратиреоза. В первую очередь надо назначать рентгенографию поясничного отдела и кистей. На пояснич- ный отдел позвоночника приходится наибольшая нагрузка; поэтому спонтанные переломы чаще всего образуются в этом отделе. При остеопорозе может возникнуть лизис дистальных фаланг пальцев рук, что можно обнаружить на рентгенограмме кистей. Рентгенография кисти в ладонной поверхности для определе- ния костного возраста ребёнка или подростка. О костном возрасте судят по наличию ядер окостенения в костях запястья и по остеогенезу костей предплечья. Таблица 23 Определение костного возраста по рентгенограммам кисти и эпифизов предплечья Наименование кости Сроки костного созревания (возраст, годы) Трёхгранная 2–4 Полулунная 3–6 Трапециевидная 4,5–6 Ладьевидная 5–6 Гороховидная 12–13 Сесамовидные 13–14 Синостоз в 1-й пястной кости 13–15 В фалангах 15–18 Определение костного возраста по рентгенограммам кисти и эпифизов предплечья Рентгенография загрудинного пространства применяется для выявления тимомегалии. Рентгенография загрудинного пространства применяется для выявления тимомегалии. Рентгеноскопия пищевода с густым барием назначается для вы- яснения прорастания рака щитовидной железы в пищевод и для ди- агностики сдавления пищевода извне большим зобом или узловым зобом. Сцинтиграфия щитовидной железы с технецием 99 раньше широко использовалась для поиска эктопированной ткани или ме- тастазов дифференцированного рака. В настоящее время всё 77 Пропедевтика эндокринных заболеваний больше вытесняется другими методами исследования (УЗИ, МРТ- без лучевой нагрузки). больше вытесняется другими методами исследования (УЗИ, МРТ- без лучевой нагрузки). Сцинтиграфия паращитовидных желёз с технетрилом приме- няется и сейчас для топической диагностики паратиром, хотя дру- гие методы диагностики становятся предпочтительнее: облучение паращитовидных желёз всегда необходимо избегать! Позитронно-эмиссионная КТ (ПЭТ-КТ) ПЭТ-КТ – метод, позволяющий диагностировать те опухоли, которые невозможно выявить другими средствами визуализации, такими как УЗИ, КТ, МРТ. ПЭТ-КТ всего тела необходимо проводить больному раком щи- товидной железы после оперативного лечения через ≈ 2 месяца для установления наличия и локализации региональных и отдалённых метастазов, для оценки радикальности выполненной операции, ре- шения вопроса о необходимости радиойодтерапии. р р р ПЭТ-КТ незаменима для диагностики нейроэндокринных опу- холей, в том числе соматостатиномы. Морфологические методы исследования Тонкоигольная пункционная биопсия (ТПБ) щитовидной железы необходима для дифференциальной диагностики при узловых обра- зованиях. Целесообразно пунктировать узел под контролем УЗИ, чтобы материал для гистологического исследования был получен именно из узла, а не из соседнего здорового участка щитовидной же- лезы. Исследование полученных мазков проводят врачи-цитологи. Для морфологического диагноза используется признанная во всём мире классификация The Bethesda System for Reporting Thyroid Cyto- pathology, насчитывающая 6 цитологических заключений. 1. Недиагностический или неудовлетворительный пунктат (только кистозная жидкость, затемнён кровью и другое. 2. Доброкачественный узел (коллоидный зоб, зоб Хашимото, подострый тиреоидит). 3. Атипия неопределённого значения или фолликулярные изме- нения неопределённого значения. 4. Фолликулярная неоплазия или подозрение на фолликуляр- ную неоплазию. 78 И.В. Терещенко 5. Подозрение на рак / подозрение на злокачественность / подо- зрение на папиллярный рак / подозрение на медуллярный рак / по- дозрение на метастазы в щитовидную железу / подозрение на дим- фому / другое. 5. Подозрение на рак / подозрение на злокачественность / подо- зрение на папиллярный рак / подозрение на медуллярный рак / по- дозрение на метастазы в щитовидную железу / подозрение на дим- фому / другое. ф у ру 6. Рак / папиллярный рак / плохо дифференцированный рак / ме- дуллярный рак / недифференцированный рак (анапластический) / плоскоклеточный рак / метастазы в щитовидную железу / неходж- кинская лимфома / другое. 6. Рак / папиллярный рак / плохо дифференцированный рак / ме- дуллярный рак / недифференцированный рак (анапластический) / плоскоклеточный рак / метастазы в щитовидную железу / неходж- кинская лимфома / другое. ф дру Тонкоигольная пункционная биопсия (ТПБ) околощитовидной железы применяется в клинической практике. Однако, в последние годы изменилось отношение к ТПБ узла, подозрительного на опу- холь околощитовидной железы, поскольку обычно паратиромы ло- кализуются позади доли щитовидной железы, т.е., пунктируя пара- тирому, неизбежно приходится травмировать паренхиму щитовид- ной железы и в полученный мазок часто попадают транспортные клетки из ткани или узлов щитовидной железы, приводя к ошибоч- ному заключению. Нередко при ТПБ не удаётся получить доста- точный клеточный материал из околощитовидной железы. Обнару- жена особенность ткани аденом паращитовидной железы – отве- чать очень значительным фиброзом опухоли после ТПБ, образовы- вать спайки с окружающими тканями, что в дальнейшем затруд- няет оперативное вмешательство. ТПБ надпочечника в последние годы стали применять при опухолях, наиболее часто – при инци- денталомах. Молекулярно-генетические методы Исследование полового хроматина является скрининг-тестом в диагностике хромосомных аномалий – синдрома Щерешевского- Тернера и синдрома Клайнфельтера. Более точный метод диагно- стики этих и других хромосомных заболеваний – кариотипирова- ние, т.е. определение хромосомного набора. При подозрении на генетическое заболевание с точечной мутацией проводят поиск му- таций. Например, при ВДКН определяют мутации гена CYP 21В. 79 Пропедевтика эндокринных заболеваний Пример 1 Пример 1 Больная Г-ва, 57 лет, обратилась на консультацию в мае 2017 года с жалобами на облысение, рост бороды и усов (бреется ежедневно), рост волос на спине, по задней поверхности бёдер, во- круг ануса, огрубение голоса. Анамнез заболевания: признаки вирилизации появились в 2011 году в возрасте 54 лет, что было расценено врачами как кли- мактерическая алопеция. Осенью 2013 г. было проведено гормо- нальное обследование. Уровень тестостерона в крови оказался 261 нг/дл (норма 5–103 нг/дл), свободный тестостерон 54 пг/л (норма 0–4,1 пг/л). Неоднократно повторяли контроль уровня те- стостерона. В течение года гипертестостеронемия была стойкой: 241–424 нг/дл. В ноябре 2014 года госпитализирована в эндокри- нологическое отделение для обследования. Установлено: содержа- ние в крови тестостерона 424 нг/дл, 17ОН-прогесторона 11,08 нмоль/л (норма <2,1 нмоль/л), андростендиона 1,5 нг/мл (норма 0,4 – 4,1 нг/мл), дигидротестостерона 334 пг/мл (норма 24–450 пг/мл, эстрадиола 45,72 пг/мл (норма 0 – 41 пг/мл), корти- зола 9,2 мкг/дл (норма 5 – 25 мкг/дл), ДГЭА 27,4 мкг/дл (норма 35–430 мкг/дл), альдостерона 21,6 нг/дл (норма 2,56–44,5 нг/дл), ренина 18,6 мМЕ/л (норма 4,4 – 46,9 мМЕ/л), ТТГ 2,27 мМЕ/мл (норма 0,4–2,0 мМЕ/мл). Проведено УЗИ органов малого таза: об- наружена интрамуральная миома матки (диаметр 15 мм); правый яичник расположен обычно, размеры 32 х 23 х 17 мм; неоднород- ной эхоструктуры, средней эхогенности, без антральных фоллику- лов. Левый яичник расположен вдоль левого ребра матки, размеры 15х7 мм, однородной эхоструктуры, обычной эхогенности, без ан- тральных фолликулов. Заключение: ЭХО-признаки миомы матки. Увеличение размеров правого яичника. В результате проведённого обследования заподозрена андростерома. р у р д д д р др р С целью визуализации опухоли дважды с интервалом в полгода проводилась мультиспиральная компьютерная томография (МКТ) надпочечников: надпочечники расположены обычно, с обеих сто- рон несколько увеличены, с выпуклыми волнистыми контурами, однородной структуры. Справа между ножек расположено округ- 80 И.В. Терещенко лое образование до 14 мм в диаметре с чёткими контурами, жиро- вой плотности (− 87–92 Hu), контраст не копит. Параадреналовая клетчатка не изменена. Заключение: гиперплазия надпочечников. Образование в правом надпочечнике (липома). лое образование до 14 мм в диаметре с чёткими контурами, жиро- вой плотности (− 87–92 Hu), контраст не копит. Параадреналовая клетчатка не изменена. Заключение: гиперплазия надпочечников. Образование в правом надпочечнике (липома). рез полгода повторная КТ надпочечников динамики не выявила Через полгода повторная КТ надпочечников динамики не выявила. Больной проведен тест толерантности к глюкозе: гликемия натощак 5,6 ммоль/л, через 2 часа после приёма внутрь 75 г глю- козы – 9,6 ммоль/л. Пример 1 Несмотря на несомненные МРТ-признаки липомы правого надпочечника, поставлен окончательный клинический диагноз: ан- дростерома правого надпочечника. Сахарный диабет 2-го типа впервые выявленный. Больная переведена в хирургическое отделение, в декабре 2014 г. произведена лапароскопическая правосторонняя адренал- эктомия. Представлено следующее описание макропрепарата: ку- сок ткани размером 4 х 2 х 1,6 см, на разрезе корковое вещество 1 мм, мозговое – 2 мм; обширное кровоизлияние в ткань надпочеч- ника; гистологическое строение удалённой ткани следующее: в ткани надпочечника имеется скопление светлых и тёмных клеток округлой формы, содержащих липиды в цитоплазме. Через месяц после операции состояние не изменилось; уровень тестостерона в крови оказался 225 нг/дл, кортизола 13,7 мкг/дл. В дальнейшем вирилизация прогрессировала, гипертестостеронемия сохранялась в пределах 241–245 нг/дл. Проведено повторное УЗИ органов малого таза – без отрицательной динамики; сохраняется увеличение правого яичника. МСКТ органов брюшной полости: объёмных образований в брюшной полости не выявлено. Гипер- плазия левого надпочечника. Пациентке предложена адреналэкто- мия левого надпочечника, от которой она отказалась и до мая 2017 года к врачам не обращалась. В апреле 2017 года по собственной инициативе проверила уро- вень тестостерона в крови, который составил 305 нг/дл, что для больной явилось поводом обратиться на консультацию: надпочеч- никовая причина гиперандрогении была сразу отвергнута, заподо- зрена вирилизирующая опухоль яичника. Больная направлена на МСКТ органов малого таза: в матке инволютивные изменения. Ле- вый яичник чётко не визуализируется. В структуре правого яич- 81 Пропедевтика эндокринных заболеваний ника определяется образование до 16–18 мм, интенсивно накапли- вающее контраст, с пиком нарастания плотности в венозную фазу, с последующим частичным вымыванием в отсроченную фазу. ника определяется образование до 16–18 мм, интенсивно накапли- вающее контраст, с пиком нарастания плотности в венозную фазу, с последующим частичным вымыванием в отсроченную фазу. Заключение: гиперваскулярное образование правого яичника Анамнез жизни: росла здоровой. Успешно окончила школу и университет. Работала по инженерной специальности до выхода на пенсию в 55 лет. Редко болела простудными заболеваниями. Ме- сячные с 13 лет, прекратились в 2012 году. Замужем, имеет двух взрослых здоровых дочерей. В 40 лет выявлена артериальная ги- пертензия. Более 15 лет принимает антигипертензивные препа- раты. Наследственность отягощена по материнской линии по арте- риальной гипертензии и сахарному диабету. Объективный статус: фенотип маскулинный. Снижены жиро- вые отложения вокруг тазового пояса. Моторика, походка муж- ские. Голос низкий с мужскими обертонами (барифония). Алопе- ция по мужскому типу. Выражен гирсутизм и гипертрихоз. Щито- видная железа не увеличена. Молочные железы гипотрофичны, вя- лые, вокруг ареол обильный рост волос. Со стороны внутренних органов патологии не выявлено. АД 135/80. Гинекологический статус: клиторомегалия. Пример 1 Слизистые промеж- ности атрофичные, цианотичные. Малые половые губы гипоплазиро- ваны. Матка маленьких размеров. Яичники не пальпируются. Таким образом у пациентки выраженная симптоматика вирили- зации и дефеминизации. Клинический диагноз: вирилизирующая опухоль правого яич- ника. Гипертоническая болезнь II стадии, риск 2. р р Лечение: больной предложено оперативное лечение, на что она не сразу согласилась, ещё проверила дважды уровень тестостерона (258 нг/дл, 232 нг/дл). Произведена лапароскопическая двусторон- няя аднексэктомия 15.12.2017 года. Оказалось, яичник слева инво- лютивный, 1,5 х 1,5 х 0,7 см, плотно-эластичный, на разрезе серый, с мелкими единичными бордовыми вкраплениями. Правый яичник 2–3–2 см, на его латеральной поверхности располагается желези- сто-кистозная структура бордового цвета, без капсулы, диаметром 1,5–2 см, на разрезе пёстрая (серо-жёлтая с очагами красного и ко- ричневого цвета), неправильной формы. Гистология: опухоль из крупных полигональных клеток, сходных с гранулёзо-лютеино- выми клетками, напоминает клетки жёлтого тела, в виде диффуз- ных тяжей, полей, гнёзд, с участками кистозной дегенерации; сосу- дистая сеть в опухоли хорошо развита. Заключение: стероидно-ли- пидноклеточная неклассифицируемая опухоль правого яичника; 82 И.В. Терещенко очаговый (узловой) гипертекоз левого яичника (окраска гематокси- лин-эозин; гистохимическое исследование не проводилось). очаговый (узловой) гипертекоз левого яичника (окраска гематокси- лин-эозин; гистохимическое исследование не проводилось). Результаты лечения: через месяц после операции уровень те- стостерона в крови нормализовался (< 20 нг/мл), на голове появи- лись редкие волосы, исчезла барифония. Однако стали беспокоить частые приливы жара. Уровень ФСГ оказался соответствующим норме менопаузального периода (33,7 мМЕ/мл; норма для женщин постменопаузального периода до 133,4 мМЕ/мл), содержание в крови эстрадиола < 10 пг/мл. Через 3 месяца стала чувствовать себя хорошо, приливы прекратились спонтанно. Проводится системати- чески контроль и самоконтроль гликемии: углеводный обмен в норме. Осмотрена ещё раз через два года (в III-2020 г.): исчезли все симптомы вирилизации, появились густые волосы на голове. Обсуждение: Представлен клинический пример нескольких диагностических ошибок у одной пациентки, что привело к несвоевременному рас- познаванию и оперативному лечению опухоли яичника. Из-за диа- гностической ошибки больная перенесла калечащую, совершенно необоснованную, ненужную операцию – одностороннюю адренал- эктомию (случайно избежала тотальной адреналэктомии). Разберём диагностические ошибки. Разберём диагностические ошибки. 1. Начальные симптомы вирилизации врачи отнесли к проявле- ниям климакса, хотя климакс может сопровождаться гирсутизмом, но не дефеминизацией и не вирилизмом. Следовало назначить больной гормональное и эхографическое обследование, что не было сделано. 2. Не учтены симптомы, которые опровергают надпочечнико- вый генез гиперандрогении: 2. Не учтены симптомы, которые опровергают надпочечнико- вый генез гиперандрогении:  андростерома чаще возникает в молодом возрасте; вирилизи- рующие опухоли яичника – обычно в климактерическом периоде;  результаты дважды проведённого МСКТ надпочечников кли- ницисты оценили неправильно. Локализация опухоли между нож- ками надпочечника не характерна для андростеромы, которая ис- ходит из сетчатого слоя, т. е. располагается в самом надпочечнике, а не между его ножек, имеет плотную соединительнотканную кап- сулу, не бывает двусторонней, активно накапливает контраст (опу- холь у больной не накапливала ультравист). Было недопустимо предлагать больной удаление второго надпочечника; 83 Пропедевтика эндокринных заболеваний  несмотря на полное клиническое сходство андростеромы и ви- рилизирующих опухолей яичника, возникающие гормональные сдвиги отличаются кардинально, и это помогает в дифференциаль- ной диагностике. Ясно, вирилизацию и дефиминизацию у наблю- даемой пациентки создавала овариальная гипертестостеронемия. Гормональные сдвиги у неё прямо указывали на овариальный, а не надпочечниковый генез гипертестостеронемии, поскольку содер- жание в крови надпочечниковых фракций андрогенов не было уве- личенным, а ДГЭА (важный показатель надпочечниковой гипе- рандрогении!) даже снижен. При вирилизирующих опухолях яич- ников может быть смешанная гиперсекреция тестостерона и эстро- генов (эстрадиола и эстрона). Очевидно, такой вариант был у пред- ставленной больной, поскольку уровень эстрадиола первоначально у неё был слегка повышен. Таблица 24 Гормональные сдвиги при вирилизирующих опухолях надпочечника и яичника Гормон Андростерома Вирилизирующая опухоль яичника Тестостерон Стойкое и высокое превышение * Стойкое и высокое превышение * Свободный тестостерон Умеренное повышение Стойкое и высокое превышение * ДГЭА Значительное и стойкое повышение (возрастает в 10 раз и >) В норме, бывает незначительно выше нормы * 17αОН- прогестерон Значительное и стойкое повышение В норме или незначительно повышен * Андростендион Значительное и стойкое повышение В норме * Дигидро- тестостерон В норме или повышен В норме или повышен Эстрадиол Всегда снижен В норме, может быть повышен * Примечание: * – изменения уровня гормонов в крови у наблюда- емой больной. Гормональные сдвиги при вирилизирующих опухолях надпочечника и яичника 84 И.В. Терещенко 3. Обсуждение: Для визуализации андрогенсекретирующей опухоли необхо- димо проводить и УЗИ, и КТ (МРТ) надпочечников и органов ма- лого таза. Следует отметить, что у наблюдаемой больной до адре- налэктомии не проводили КТ органов малого таза, хотя эхографи- чески правый яичник был увеличен, причём опухоль не просмат- ривалась. 3. Для визуализации андрогенсекретирующей опухоли необхо- димо проводить и УЗИ, и КТ (МРТ) надпочечников и органов ма- лого таза. Следует отметить, что у наблюдаемой больной до адре- налэктомии не проводили КТ органов малого таза, хотя эхографи- чески правый яичник был увеличен, причём опухоль не просмат- ривалась. р 4. Была ошибочно расценена причина гиперплазии надпочечни- ков. Известно, что при гипертонической болезни и при длительном применении гипотензивных препаратов может развиваться вторич- ный гиперальдостеронизм и гиперплазия надпочечников. Этим объ- ясняется двусторонняя гиперплазия обоих надпочечников у наблюда- емой пациентки (гипертоническая болезнь у неё более 15 лет). ( р у ) 5. Липому надпочечника у больной можно справедливо отне- сти к инциденталоме (не случайно, при проведении МСКТ надпо- чечников образование не накапливало контраст), а так как диаметр липомы не превышал 1,5 см, то показаний к адреналэктомии не было. 6. Вирилизирующие опухоли яичников могут образовываться из разных его элементов. Нередко морфологический диагноз можно поставить только путём проведения гистохимии, что боль- ной не было сделано. Поэтому, к сожалению, не поставлен точный морфологический диагноз. Однако, выраженность вирилизации и быстрота её прогрессирования не зависят от гистологического строения опухоли яичника, а только от её андрогенсекреторной ак- тивности. Топическая диагностика опухолей яичников всегда пред- ставляет большие трудности, поскольку такие опухоли имеют ма- лые размеры, могут локализоваться в области ворот яичника (это затрудняет визуализацию при проведении УЗИ). Они могут чётко не определяться при УЗИ яичников, как это было у наблюдаемой пациентки. В таких случаях должно настораживать одностороннее увеличение яичника, что не было учтено. Нередко у них отсут- ствует капсула; они бывают двусторонними или рецидивировать после операции; всегда имеется риск их малигнизации, а значит, оперативное лечение должно производиться как можно раньше. 6. Вирилизирующие опухоли яичников могут образовываться из разных его элементов. Нередко морфологический диагноз можно поставить только путём проведения гистохимии, что боль- ной не было сделано. Поэтому, к сожалению, не поставлен точный морфологический диагноз. Однако, выраженность вирилизации и быстрота её прогрессирования не зависят от гистологического строения опухоли яичника, а только от её андрогенсекреторной ак- тивности. Топическая диагностика опухолей яичников всегда пред- ставляет большие трудности, поскольку такие опухоли имеют ма- лые размеры, могут локализоваться в области ворот яичника (это затрудняет визуализацию при проведении УЗИ). Обсуждение: Они могут чётко не определяться при УЗИ яичников, как это было у наблюдаемой пациентки. В таких случаях должно настораживать одностороннее увеличение яичника, что не было учтено. Нередко у них отсут- ствует капсула; они бывают двусторонними или рецидивировать после операции; всегда имеется риск их малигнизации, а значит, оперативное лечение должно производиться как можно раньше. 7. Известно, гипертекоз характеризуется инсулинорезистентно- стью. Выраженность стромального гипертекоза непосредственно связана с тем, насколько значительна инсулинорезистеность: тека- 85 Пропедевтика эндокринных заболеваний клетки имеют рецепторы к инсулину, а инсулин способствует вы- работке ими андрогенов. Снижение чувствительности клеток к ин- сулину становится причиной накопления глюкозы в крови, которая не способна проникнуть в клетки, что проявилось у пациентки. Важно отметить, что после оперативного вмешательства у неё нор- мализовался углеводный обмен, т. е. нарушение теста толерантно- сти к глюкозе было, очевидно, проявлением гипертекоза, а диагноз «сахарный диабет 2-го типа» выставлен ошибочно. клетки имеют рецепторы к инсулину, а инсулин способствует вы- работке ими андрогенов. Снижение чувствительности клеток к ин- сулину становится причиной накопления глюкозы в крови, которая не способна проникнуть в клетки, что проявилось у пациентки. Важно отметить, что после оперативного вмешательства у неё нор- мализовался углеводный обмен, т. е. нарушение теста толерантно- сти к глюкозе было, очевидно, проявлением гипертекоза, а диагноз «сахарный диабет 2-го типа» выставлен ошибочно. Пример 2 Больная С-ва, 36 лет, направлена на консультацию с подозре- нием на первичный гиперпаратиреоз. Беспокоят боли в пояснице и коленных суставах. Ранее проведена рентгенологическая денсито- метрия поясничного отдела позвоночника, лучезапястных суста- вов: установлена остеопения. Проверен уровень в крови паратгор- мона: 195 пг/мл (норма до 75 пг/мл). Также было сделано УЗИ щи- товидной железы и околощитовидных желёз: патологии щитовид- ной железы не выявлено; установлено увеличение нижних около- щитовидных желёз справа и слева; их форма овальная, контуры ровные, границы чёткие, размеры 1,09 см. Был поставлен диагноз: двусторонние нижние паратиромы; первичный гиперпаратиреоз; гиперпаратиреоидная остердистрофия. Запланировано оператив- ное лечение. Пациентке назначено дообследование. Уровень паратгормона оказался 127 пг/мл, уровень витамина Д 25-ОН 6,4 нг/мл (мини- мальная норма 30 нг/мл, максимальная – 100 нг/мл), содержание сывороточного кальция 1,9 ммоль/л (норма 2,15–2,7 ммоль/л,), ионизированного кальция 0,9 ммоль/л (норма 1,13–1,31 ммоль/л), фосфора 1,84 ммоль/л (норма 0,74–1,52 ммоль/л). При осмотре вы- является симптом Хвостека (III). Для компенсации дефицита витамина Д3 назначен аквадетрим по 10 капель утром и на ночь в течение месяца с последующим кон- тролем уровня витамина Д 25-ОН в крови. Содержание витамина Д 25-ОН в крови нормализовалось: 34 нг/мл, исчезли парестезии, не определялся симптом Хвостека, меньше стали беспокоить боли в позвоночнике, исчезла гипокальциемия. После устранения дефи- цита витамина Д3 проверен уровень паратгормона: 71 нг/мл. Про- ведена МСКТ области щитовидной железы: паратиромы не обна- 86 И.В. Терещенко ружены. Сделано заключение: рекомендованное оперативное лече- ние не требуется; принимать аквадетрим в профилактической дозе – 6 капель на ночь регулярно, контролируя его уровень каж- дые 3–4 месяца. ружены. Сделано заключение: рекомендованное оперативное лече- ние не требуется; принимать аквадетрим в профилактической дозе – 6 капель на ночь регулярно, контролируя его уровень каж- дые 3–4 месяца. Обсуждение: Известно, длительный дефицит витамина Д может приводить к гиперплазии одной, двух, всех околощитовидных желёз из-за сни- жения тормозящего действия витамина Д на деление клеток пара- щитовидных желёз. Такое же действие вызывает гипокальциемия. У наблюдаемой пациентки было умеренное повышение паратгор- мона в крови на фоне выраженного снижения витамина Д3 и каль- ция. После нормализации их уровней гиперпаратиреоз устранён и исчезла гиперплазия паращитовидных желёз, т. е. предваритель- ный диагноз оказался ошибочным из-за недостаточного обследова- ния больной, при этом было недопустимо планировать оператив- ное лечение до завершения обследования. Кроме того, при подо- зрении на множественные паратиромы было необходимо исклю- чать МЭН 1 или 2 и также назначать необходимое обследование. Пример 3 Больная А-ва, 39 лет, обратилась к терапевту в связи с появле- нием слабости и апатии за последние 3 – 4 месяца. Назначено об- щеклиническое обследование, в том числе проверен уровень ТТГ, который оказался 0,01 мМЕ/мл. Больной назначен тирозол в дозе 30 мг/день, который она принимала 2 месяца. На фоне лечения са- мочувствие не только не улучшилось, а наоборот, ухудшилось, усилилась слабость, возникла постоянная сонливость, заторможен- ность. Впервые проверен уровень АТ-рТТГ, св. Т4. АТ-рТТГ ока- зались в норме, уровень св.Т4 снижен: 0,45 нг/дл (норма 0,7– 1,48 нг/дл). Тирозол отменён, рекомендовано обратиться на кон- сультацию к эндокринологу. При осмотре обращала внимание отёчность и субиктеричность лица, сухость кожных покровов, гиперкератоз локтей, брадикардия (ЧСС. 49 уд’), снижение тонуса мышц брюшной стенки, запоры, т.е. была клиническая симптоматика гипотиреоза. Дифференциаль- ный диагноз проводился между медикаментозным гипотиреозом и гипотиреозом, вызванным деструктивным АИТ. Назначено обсле- дование для исключения АИТ. Уровень АТ-ТПО > 1000 МЕ/мл (норма 0–5,6 МЕ/мл), АТ-ТГ > 800 МЕ/мл (норма 0–4,11 МЕ/мл), 87 Пропедевтика эндокринных заболеваний содержание тироглобулина 186,0 нг/мл (норма < 60,0 нг/мл). Про- ведено УЗИ щитовидной железы: объём значительно уменьшен (3,7 см3 при норме у женщин до 18 см3); гипоэхогенной структуры с преобладанием участков склероза стромы, снижение кровотока. Заключение: АИТ, атрофический вариант. Обсуждение: Таким образом, недомогание, слабость, апатия, которые изна- чально беспокоили больную, были проявлением гипотиреоза; из-за деструктивного процесса в щитовидной железе возникло снижение уровня ТТГ, которое ошибочно расценили как тиреотоксикоз. В ре- зультате допущена грубейшая ошибка – назначены тиреостатики в больших дозах, хотя не проведено необходимое обследование: не сделан контроль АТ-рТТГ, св. Т3, св. Т4, АТ-ТПО, АТ-ТГ, не оце- нено состояние сердечно-сосудистой системы. После дообследова- ния больной назначена заместительная терапия эутироксом, подо- брана его адекватная доза, достигнут эутиреоз. Пример 4 Обсуждение: В представленном случае допущены две диагностические ошибки. В представленном случае допущены две диагностические ошибки. 1. Гипердиагностика аденомы гипофиза и применение до- стинекса без показаний. При наличии кисты кармана Ратке из-за повышения давления в полости турецкого седла может возникать сдавление ножки гипофиза, что вызывает транзиторную, незначи- тельную функциональную гиперпролактинемию, нет необходимо- сти назначения достинекса, бесплодие не обусловлено ничтожным повышением уровня ПРЛ. 2. Длительно нераспознанный и не леченный СПКЯ. Главная ошибка допущена врачами, проводящими УЗИ органов ма- лого таза: были пропущены характерные морфологические признаки СПКЯ. Несвоевременно проведено гормональное обследование. Пример 4 Больная Ч-ва, 34 лет, обследуется по поводу первичного беспло- дия в течение 12 лет. Замужем 12 лет, беременности не возникали. Муж здоров. Месячные начались в 15 лет, всегда нерегулярные по типу опсоменореи. Других жалоб нет. Раньше ничем не болела. Не- однократно осматривалась гинекологом: гениталии без отклонений от нормы. УЗИ органов малого таза также проводилось много раз, патологии не отмечено. Гормональное обследование впервые про- ведено 3 года назад и выявлена незначительная гиперпролактине- мия (ПРЛ 40,0 нг/мл; норма 4,23–26,5 нг/мл) и тенденция к гипо- эстрогении (Э2 25,0 пг/мл; норма 21,0–251 пг/мл). Сделана МРТ ги- пофиза; обнаружена киста кармана Ратке размером ≈ 2 мм в диа- метре. Гинекологом назначено лечение достинексом в дозе 0,5 мг каждые 7 дней. Регулярный приём достинекса не устранил опсоме- норею и бесплодие. Уровень ПРЛ колебался от 0 до 36,0 нг/мл, ги- поэстрогения сохранялась. Направлена на консультацию к эндо- кринологу для согласования терапии. Проверен уровень ЛГ и ФСГ, соответственно ЛГ 14,0 мМЕ/мл при норме 2,39–6,6 мМЕ/мл, ФСГ 2,6 мМЕ/мл при норме 3,03–8,08 мМЕ/мл. Соотношение ЛГ/ФСГ нарушено, превышает норму в 5,4 раза (норма ЛГ/ФСГ 1–2). Выра- жены признаки недифференцированной дисплазии соединитель- 88 И.В. Терещенко ной ткани: плоскостопие, кифосколиоз грудного отдела позвоноч- ника, миопия, аномалия прикуса, гипермобильность пальцев рук. Имеется дефицит массы тела, что, очевидно, тоже можно отнести к симптомам дисплазии соединительной ткани. Предположитель- ный диагноз – СПКЯ. Направлена на повторное УЗИ органов ма- лого таза к специалисту-консультанту, который обнаружил несо- мненные эхографические признаки СПКЯ: яичники округлой формы, умеренно увеличены, их капсулы утолщены, строма высо- кой эхогенности, гиперплазирована; фолликулов > 10, располо- жены на периферии по типу «чёток». В паренхиме яичников мно- жество кист размером 3–8 мм. Доминантный фолликул и жёлтое тело отсутствуют. Выражена гипоплазия эндометрия. ной ткани: плоскостопие, кифосколиоз грудного отдела позвоноч- ника, миопия, аномалия прикуса, гипермобильность пальцев рук. Имеется дефицит массы тела, что, очевидно, тоже можно отнести к симптомам дисплазии соединительной ткани. Предположитель- ный диагноз – СПКЯ. Направлена на повторное УЗИ органов ма- лого таза к специалисту-консультанту, который обнаружил несо- мненные эхографические признаки СПКЯ: яичники округлой формы, умеренно увеличены, их капсулы утолщены, строма высо- кой эхогенности, гиперплазирована; фолликулов > 10, располо- жены на периферии по типу «чёток». В паренхиме яичников мно- жество кист размером 3–8 мм. Доминантный фолликул и жёлтое тело отсутствуют. Выражена гипоплазия эндометрия. Клинический диагноз: СПКЯ. Нарушение менструальной функ- ции. Ановуляторное первичное бесплодие. Недифференцирован- ная дисплазия соединительной ткани. Киста кармана Ратке. Функ- циональная транзиторная гиперпролактинемия. Назначена ярина плюс. Достинекс отменён. Назначена ярина плюс. Достинекс отменён. Пример 5 Призывник Б-н, 18 лет, деревенский житель, окончил 11 классов сельской школы. Считает себя здоровым. Врачебная призывная ко- миссия обнаружила у него высокую артериальную гипертензию: АД 200/95. Другой патологии не выявлено. Общий анализ мочи в норме. Мать умерла в возрасте 30 лет от гипертонического криза. Отец здоров. Других детей в семье не было. Направлен на лечение 89 Пропедевтика эндокринных заболеваний артериальной гипертензии в сельскую больницу по месту житель- ства. Дана отсрочка от службы в армии на год. Из стационара вы- писан с диагнозом гипертоническая болезнь, II стадия, 3 степень по АД, риск 2, дана рекомендация ежедневно принимать эналаприл по 20 мг 2 раза в день. Лечение не проводил. Через 5–6 месяцев почув- ствовал слабость, головные боли, похудел. К врачам не обращался. Из-за недомогания бросил учёбу в колледже. Ещё через 6 месяцев был вызван на призывную комиссию. АД оказалось 200/120. Направлен в клиническую больницу в кардиологическое отделение с диагнозом «Гипертоническая болезнь, злокачественное течение». При осмотре обнаружено опухолевидное образование в животе справа, пальпация опухоли вызывала ещё больший подъём АД. Сделано УЗИ органов брюшной полости и подтверждено наличие опухоли, занимающей всю правую половину брюшной полости, деформирующей правую почку и прорастающей в печень. Слева найдена опухоль, размером ≈ 5–6 см, исходящая из левого надпо- чечника. Назначено проверить суточную экскрецию мета / норме- танефринов, уровень альдостерона и ренина и общеклиническое обследование, КТ органов грудной и брюшной полости. Однако провести обследование не успели, больной скончался. Клиниче- ский диагноз совпал с патологоанатомическим: феохромобластома с прорастанием в соседние органы – печень и почку – справа, феохромоцитома слева. Эндокринная артериальная гипертензия. Об Обсуждение: у При обнаружении повышенного АД у подростка необходимо в первую очередь исключать симптоматическую гипертензию – по- чечную, реноваскулярную, а затем эндокринную. В представлен- ном случае врачами призывной комиссии допущена диагностиче- ская и тактическая ошибки – высокая артериальная гипертензия расценена как гипертоническая болезнь. Безусловно, гипертониче- ская болезнь может начинаться в подростковом возрасте, что наблюдается редко и, как правило, у больного подростка сначала течёт как вегетососудистая дистония по гипертоническому типу. В данном случае АД 200/95 было случайной находкой. Больной нуж- дался в обследовании не в условиях сельской больницы, а в кли- нике. Нормальный результат анализа мочи позволял исключить по- чечную гипертензию, т.е. следовало заподозрить эндокринную ги- пертензию. Известно, опухоли мозгового слоя надпочечников не- редко бывают двусторонними, а из-за богатого кровоснабжения – 90 И.В. Терещенко растут быстро и достигают больших размеров, нередко малигнизи- руются, причём быстро становятся неоперабельными. Кризовое те- чение заболевания встречается чаще. Однако, наблюдаются бес- симптомные формы феохромоцитомы или формы с постоянно вы- сокой гипертонией. У наблюдаемого больного симпатоадренало- вых кризов не было. Феохромоцитома – наследственное заболева- ние. Очевидно, мать больного тоже имела феохромоцитому, но протекающую с кризами (скончалась от гипертонического криза в 30 лет!). Пример 6 Мать привела на консультацию к эндокринологу подростка 15 лет 7 месяцев. Жалуется на постоянные головные боли, наруше- ние сна, постоянную слабость, прогрессирующее увеличение массы тела, прекращение роста. С 13 лет начал полнеть, «вырос живот, прекратился линейный рост, и даже рост как будто умень- шается, не стало памяти, не может соображать, плохо успевает в школе, хотя долго учит уроки», отмечает выраженную слабость, из-за которой на уроках физкультуры «ничего не делает». Посте- пенно присоединялись остальные перечисленные выше симптомы. К педиатру обращались неоднократно, но болезни не обнаружены (заключение – «такой рост»). Чтобы не полнеть, пытался голодать, но живот продолжал увеличиваться. Мать обратила внимание на появление «разрывов» кожи живота, груди, спины» и решила про- консультировать сына с эндокринологом. Родился первым ребён- ком в срок с массой 3200 г, длиной 50 см. Вскармливался грудью до 1 года, 4 мес. Рос и развивался нормально до 12 лет. Травм не было, редко болел простудными заболеваниями. Успешно учился в начальной школе, затем успеваемость снизилась. Наследствен- ность не отягощена, эндокринными болезнями родственники не бо- лели. Состояние удовлетворительное. Телосложение кушингоидное. Рост 146 см, вес 89 кг (средние для региона возрастные показатели 166 см и 64 кг). Кожные покровы «мраморные», яркие атрофиче- ские стрии в области ягодиц, бёдер, груди, живота. Тоны сердца приглушены, АД 140/95 мм рт. ст. Формула полового созревания (по Таннеру): наружные половые органы имеют такие же размеры и пропорции, как в раннем детстве; P0 A0 L0 V0 F0. 91 Пропедевтика эндокринных заболеваний Предварительный диагноз: синдром Кушинга (кортикосте- рома?). Проведена МРТ почек и надпочечников: в левом надпочеч- нике выявлено опухолевое образование, округлой формы, с ров- ными контурами, неоднородной структуры, диаметром ≈ 18 мм. Мать обратилась для дальнейшего обследования и лечения сына в один из медицинских эндокринологических учреждений г. Москвы, где диагноз кортикостеромы был подтверждён, больной прооперирован. Повторно был осмотрен через 5 лет: практически здоров. Кли- нических и гормональных признаков гиперкортицизма нет. Сохра- няется низкорослость: рост 146 см. Успешно учится в педагогиче- ском университете. Приложения Приложения Обсуждение: Опухоль коры надпочечника у наблюдаемого подростка выяв- лена несвоевременно, очевидно, через ≈ 3 года с начала заболева- ния. Гормональная активность аденомы у наблюдаемого подростка была высокой, в связи с чем развился синдром Кушинга и главное – гиперсекреция глюкокортикоидов затормозила рост и половое раз- витие. Избыток кортизола подавляет секрецию СТГ. Белковая мат- рица костей из-за стероидного катаболизма нарушается, и кальций не может удерживаться в костной ткани. Этому способствует также торможение процессов гидроксилирования кальциферола. Глюко- стерома может препятствовать выработке гонадотропинов, вызы- вать атрофию гонад. Процессы катаболизма и гипераммониемия в ЦНС приводят к хронической интоксикации мозга и когнитивным нарушениям. Не возникает сомнения, что у больного была гипока- лиемия и гипокалиемическая слабость, что препятствовало заня- тиям физкультурой. Синдром Кушинга (особенно болезнь Кушинга) редко встреча- ется у детей и подростков. Ожирение ошибочно было расценено как алиментарное. Подобная диагностическая ошибка в клиниче- ской практике встречается нередко. 92 И.В. Терещенко Тестовые задания для самоконтроля усвоения материала Тест 1. Какие заболевания эндокринной системы сопровожда- ются ожирением? 1. Акромегалия, аддисонова болезнь, СПКЯ, сахарный диабет 2-го типа. 1. Акромегалия, аддисонова болезнь, СПКЯ, сахарный диабет 2-го типа. 2. Болезнь Грейвса, нервная анорексия, глюкостерома, сахар- ный диабет 1-го типа. 3. Болезнь (синдром) Иценко-Кушинга, йоддефицитный зоб, ВДКН, СПКЯ. 4. Сахарный диабет 2-го типа, болезнь Иценко-Кушинга, гипо- тиреоз, болезнь Пиквика. 5. Всё перечисленное верно. 5. Всё перечисленное верно. Тест 2. При каких эндокринопатиях встречается диффузная алопеция? При: 1. Гипотиреозе, гипоталамо-гипофизарной недостаточности, ак- ромегалии, альдостероме. 2. Гипотиреозе, гипоталамо-гипофизарной недостаточности, гипопаратиреозе, вирилизирующих опухолях яичника. 3. Нервной анорексии, аддисоновой болезни, болезни Пиквика, ВДКН. 4. СПКЯ, несахарном диабете, сахарном диабете, болезни Грейвса. 5. Всё перечисленное верно. 5. Всё перечисленное верно. Тест 3. При каких эндокринных заболеваниях больные жалу- ются на похудение? 1. Аддисонова болезнь, болезнь Грейвса, нервная анорексия, са- харный диабет 1-го типа. 2. Гипотиреоз, ВДКН, сахарный диабет 2-го типа, гипопаратиреоз 2. Гипотиреоз, ВДКН, сахарный диабет 2-го типа, гипопаратиреоз. 3. Те же заболевания, что в п. 1, и ещё при гиперпаратиреозе. 3. Те же заболевания, что в п. 1, и ещё при гиперпаратиреозе. 4. Всё перечисленное верно. 5. Всё перечисленное верно, кроме сахарного диабета 2-го типа Тест 4. Эндокринная артериальная ипертензия бывает при сле- дующих заболеваниях: 1. Болезнь (синдром) Иценко–Кушинга, альдостерома, феохро- моцитома. 2. Сахарный диабет 1-го и 2-го типа, болезнь Грейвса. 93 Пропедевтика эндокринных заболеваний 3. Гипотиреоз, гиперпаратиреоз, акромегалия. 3. Гипотиреоз, гиперпаратиреоз, акромегалия. 3. Гипотиреоз, гиперпаратиреоз, акромегалия. 4. Всё неправильно. 4. Всё неправильно. 5.Всё правильно. 5.Всё правильно. 5.Всё правильно. р Тест 5. Следующие эндокринопатии могут осложняться ко- мами и кризами, кроме: Тест 5. Следующие эндокринопатии могут осложняться ко- мами и кризами, кроме: 1. Сахарный диабет, болезнь Грейвса, гипотиреоз, альдосте- рома, ВДКН. 2. Гипоталамо-гипофизарная недостаточность, аддисонова бо- лезнь, сахарный диабет 2-го типа, феохромоцитома. 3. Сахарный диабет 1-го типа, болезнь Грейвса, аддисонова бо- лезнь. 4. Аддисонова болезнь, гипотиреоз, гипоталамо-гипофизарная недостаточность, болезнь Грейвса. 4. Аддисонова болезнь, гипотиреоз, гипоталамо-гипофизарная недостаточность, болезнь Грейвса. 5. Сахарный диабет, гипоталамо-гипофизарная недостаточ- ность, аддисонова болезнь, СПКЯ, ВДКН. Тест 6. Повышенный уровень глюкозы натощак и после приёма пищи возможен при следующих эндокринных заболеваниях, кроме: 1 С й б 1 2 б Г й Тест 6. Повышенный уровень глюкозы натощак и после приёма пищи возможен при следующих эндокринных заболеваниях, кроме: щ р у щ р , р 1. Сахарный диабет 1-го и 2-го типа, болезнь Грейвса, аддисо- нова болезнь. 1. Сахарный диабет 1-го и 2-го типа, болезнь Грейвса, аддисо- нова болезнь. 2. Феохромоцитома, синдром Иценко–Кушинга, альдостерома. 3. Акромегалия, глюкагонома, тиреотоксикоз. 4. Сахарный диабет, болезнь Иценко–Кушинга, акромегалия. 5. Сахарный диабет, кортикостерома, феохромоцитома. Тест 7. Инсипидарный синдром возможен при: 1. Сахарном диабете, несахарном диабете, тиреотоксикозе. 2. Сахарном диабете, несахарном диабете, гиперпаратиреозе. 3. Несахарном диабете, аддисоновой болезни, феохромоцитоме 4. Синдроме Иценко–Кушинга, болезни Пиквика, несахарном диабете. 5. Андростероме, сахарном диабете, гипоталамо-гипофизарной недостаточности. Тест 8. Вирильный синдром бывает при следующих эндокрино- патиях: 1. СПКЯ. 2. Андрогенсекретирующих опухолях яичника. 3 ВДКН 2. Андрогенсекретирующих опухолях яичника. 2. Андрогенсекретирующих опухолях яичника. 3. ВДКН. 3. ВДКН. 4. Андростероме. 5. Всё верно. 94 И.В. Терещенко И.В. Терещенко Тест 9. Синдром высокорослости возникает при таких эндо- кринопатиях: 1. Гипофизарном гигантизме. 2. Pubertas praecox. 3. Болезни Марфана. 4. Евнухоидизме. 5. Всё верно, кроме п. 2. Тест 10. Какие гормоны целесообразно проверить в первую оче- редь при синдроме Иценко–Кушинга? 1. АКТГ, кортизол. 2. СТГ. 3. АКТГ, альдостерон, кортизол, 17 ОН-прогестерон. 4. ДГЭА. 4. ДГЭА. 5. ТТГ. 5. ТТГ. Тест 11. Выбрать необходимый минимум лабораторно-ин- струментального обследования при впервые выявленном пальпа- цией узловом зобе: 1. ТТГ, св. Т3, св. Т4, АТ-ТПО. 2. То же, что в п. 1 и УЗИ щитовидной железы. 3. То же, что в п. 2 и онкомаркеры –АТ-ТГ и тироглобулин. 4. То же, что в п. 3 сделать МРТ загрудинного пространства. 5. Достаточно сделать УЗИ загрудинного пространства. Тест 12. Взрослый пациент жалуется на снижение роста. 3. Гипотиреоз, гиперпаратиреоз, акромегалия. Ка- кие эндокринные заболевания могут быть? 1. Синдром Иценко–Кушинга. 2. Гиперпаратиреоз. 3. Длительный не леченный гипогонадизм. 4. Всё верно. 5. Всё неправильно. 95 Пропедевтика эндокринных заболеваний Ситуационные задачи Задача 1. Больная В-ва, 50 лет, обратилась к терапевту с жало- бами на резкую слабость, сонливость днём, постоянную зябкость, отёчность лица, конечностей, запоры. Примерно год назад насту- пила менопауза. Почти одновременно проявились вышеперечис- ленные жалобы. Причину болезни указать не может. Родилась в Перми, росла здоровой. После окончания техникума 20 лет рабо- тала на заводе контролёром-рентгенологом. В настоящее время ра- ботает кладовщиком. Замужем, имеет трёх дочерей. Условия жизни удовлетворительные. Выглядит старше своих лет. Лицо субиктеричное, одутловатое с кукольным румянцем. Выражены периорбитальные отёки. Взгляд тупой. Диффузное поредение волос. На передней поверхности шеи визуально определяется узловое образование размером 4 х 4 см. Пальпация щитовидной железы подтвердила наличие этого узла и ещё обнаружены множественные узловые образования в диаметре ≈ 1,0–1,5 см. Кожные покровы сухие, гиперкератоз локтей, стоп; шелушение голеней. Кисти рук «лопатообразные». На голенях плотные отёки: при нажиме ямки не остаются. Пульс 52 удара в минуту. Тоны сердца глухие. Голос хриплый. Речь замедлена. В лёгких ослабленное дыхание. Пупочная грыжа размером ≈ 3 см. Снижен тонус брюшных мышц. Живот выпячен. Все отделы тол- стого кишечника забиты содержимым. Стула не было 4 суток. Вопросы: сть ли у пациентки симптомы эндокринной патологии? 1. Есть ли у пациентки симптомы эндокринной патологии? 2. Если да, то перечислите, какие. Укажите, патологию какой эндокринной железы необходимо исключать в первую очередь. 3. Исследование каких гормонов подтвердит диагноз? Задача 2. Больная А-ва, 32 лет. Жалуется на постоянные голов- ные боли, увеличение массы тела за полгода на 12 кг, изменение внешнего вида (лицо стало круглым и красным, тонкие руки и ноги, толстый живот), отсутствие месячных 5-й месяц. При осмотре вы- явлено диспластическое ожирение I степени, лунообразное лицо, мраморная сухая кожа, много багровых полос растяжения на жи- воте, молочных железах, бёдрах, АГ до 180/100, 96 И.В. Терещенко Вопросы: Вопросы: р 1. Перечислить симптомы и синдромы, указывающие на эндо- кринное заболевание. 1. Перечислить симптомы и синдромы, указывающие на эндо- кринное заболевание. р 2. Составить план обследования. р 2. Составить план обследования. Задача 3. Больная 22 лет, студентка, госпитализирована в эндо- кринологическое отделение для подготовки к оперативному лече- нию с диагнозом «Многоузловой токсический зоб II степени». Около года назад почувствовала недомогание. Ухудшился аппетит, постепенно стала худеть, периодически возникала лихорадка > 38,5˚, профузные поты, одышка, дискомфорт за грудиной, при- ступы сердцебиения. Зоб обнаружен ≈ 3 месяца назад. Ситуационные задачи Лечащий врач установила при пальпации области щитовидной железы следующее: над югулярной вырезкой определяется желе- зистое образование, уходящее в переднее средостение, при глота- нии неподвижное, не смещающееся, боковые доли щитовидной же- лезы не определяются. Предполагаемый диагноз токсического многоузлового зоба снят. Вопросы: Вопросы: р 1. Какие особенности опухоли не характерны для образований из щитовидной железы? 1. Какие особенности опухоли не характерны для образований из щитовидной железы? 2. Составить план обследования больной. Задача 4. Больная К-ч, 76 лет, обратилась на консультацию к окулисту по поводу пучеглазия, двоения в глазах, слезотечения при ярком освещении. Эти симптомы появились полгода назад без ви- димой причины. В течение 11 лет больна гипотиреозом, получает заместительное лечение эутироксом в дозе 75–88 мкг/сутки под контролем ТТГ. Последнее УЗИ щитовидной железы сделано 3 ме- сяца назад, выявлена атрофия и фиброз тиреоидной паренхимы: объём щитовидной железы 3,0 см3. Окулистом пациентка направ- лена на КТ орбит: установлено утолщение глазных мышц, выра- женная их отёчность. Направлена к эндокринологу с подозрением на аутоиммунную офтальмопатию. Вопросы: 1. Какое обследование необходимо провести для диагностики причин глазных нарушений? 1. Какое обследование необходимо провести для диагностики причин глазных нарушений? 97 Пропедевтика эндокринных заболеваний 2. Какое соматическое и гормональное обследование надо назначить, исходя из предполагаемого диагноза и учитывая воз- можность необходимого лечения медикаментами в старческом воз- расте? Задача 5. При проведении очередной диспансеризации у со- трудницы Ю-й, 33 лет, русской по национальности, врачи обратили внимание на потемнение кожных покровов. Выражен симптом «грязных ладоней и локтей»; в полости рта на слизистой щёк пиг- ментные пятна, АД 85/55, хотя год назад артериальной гипотонии и пигментации не было. Жалуется только на слабость, объясняя её переутомлением. Направлена на консультацию к эндокринологу. В Задача 5. При проведении очередной диспансеризации у со- трудницы Ю-й, 33 лет, русской по национальности, врачи обратили внимание на потемнение кожных покровов. Выражен симптом «грязных ладоней и локтей»; в полости рта на слизистой щёк пиг- ментные пятна, АД 85/55, хотя год назад артериальной гипотонии и пигментации не было. Жалуется только на слабость, объясняя её переутомлением. Направлена на консультацию к эндокринологу. Вопросы: 1. Какую патологию может заподозрить эндокринолог? Обосно- вать. 1. Какую патологию может заподозрить эндокринолог? Обосно- вать. 2. Какое обследование должен провести эндокринолог? 3. Какие отклонения от нормы могут обнаружить в общем ана- лизе крови, мочи и других анализах, проведённых при диспансери- зации? Что может показать ЭКГ? 4. Ещё с какими специалистами надо проконсультировать паци- ентку? Задача 6. Ситуационные задачи У здоровой женщины 22 лет родился первый ребёнок с весом 3650 г, длиной 53 см (рост матери 165 см, отца 164 см), мужского пола, но с недоразвитыми мошонкой, половым членом, с гипоспадией и с крипторхизмом. Результат пренатальной диагно- стики пола по УЗИ плода – мальчик. Формирование скелета, внут- ренних органов без патологии. Новорожденный осмотрен уроло- гом: рекомендована консультация педиатра-эндокринолога и врача-генетика. Проверен хромосомный набор: ХХ – женский пол. Получен результат перинатального контроля уровня 17 ОН-проге- стерона: 48 нг/мл; норма у новорожденных – не выше 35 нг/мл. Вопросы: Задача 6. У здоровой женщины 22 лет родился первый ребёнок с весом 3650 г, длиной 53 см (рост матери 165 см, отца 164 см), мужского пола, но с недоразвитыми мошонкой, половым членом, с гипоспадией и с крипторхизмом. Результат пренатальной диагно- стики пола по УЗИ плода – мальчик. Формирование скелета, внут- ренних органов без патологии. Новорожденный осмотрен уроло- гом: рекомендована консультация педиатра-эндокринолога и врача-генетика. Проверен хромосомный набор: ХХ – женский пол. Получен результат перинатального контроля уровня 17 ОН-проге- стерона: 48 нг/мл; норма у новорожденных – не выше 35 нг/мл. Вопросы: 1. Какой пол ребёнка? р 2. Какие диагностические ошибки допущены врачами при веде- нии беременности? 2. Какие диагностические ошибки допущены врачами при веде- нии беременности? 3. Обосновать диагноз новорожденного. Здоровы ли родители? 98 И.В. Терещенко Ответы на тестовые задания 1) 4 5) 4 9) 5 2) 2 6) 1 10) 1 3) 3 7) 2 11) 2 4) 5 8) 5 12) 4 Ответы на тестовые задания Ответы на вопросы ситуационных задач Ответы на вопросы ситуационных задач Задача 1. 1. Да, безусловно, о чём свидетельствуют изменения в щитовид- ной железе: выявлены множественные узловые образования. 2. Следовательно, необходимо анализировать, нет ли симпто- мов гиперфункции или гипофункции щитовидной железы. Оче- видно, у пациентки гипотиреоз, о чём свидетельствует астениче- ский симптомокомплекс, сухость и субиктеричнось кожи, специ- фический для гипотиреоза симптом «кукольного» румянца, алопе- ция, отёчность лица, конечностей (причём отёки плотные!), затор- моженность, медленная речь, сонливость днём, вялый тонус брюш- ных мышц, пупочная грыжа, нерегулярный стул. 3. Наиболее информативен контроль ТТГ. Задача 2. 1. Синдромы: ожирения; эндокринной АГ, симптомы: головные боли, лунообразное лицо, тонкие конечности, стрии, аменорея. 2. Диагностику начать с проверки уровня кортизола в крови, лучше - в моче, и с УЗИ или КТ надпочечников для выявления кор- тикостеромы. 3. Если в надпочечниках опухоли нет, сделать МРТ гипофиза для исключения аденомы гипофиза (кортикотропиномы). З 3 3. Если в надпочечниках опухоли нет, сделать МРТ гипофиза для исключения аденомы гипофиза (кортикотропиномы). Задача 3 1. Поскольку при глотании опухоль не смещается, можно счи- тать, что образование исходит не из ткани щитовидной железы. 2. Проверить ТТГ, св. Т3, св. Т4, АТ-рТТГ, общий анализ крови; сделать УЗИ щитовидной железы. 99 Пропедевтика эндокринных заболеваний Задача 4. Задача 4. 1. Необходимо проверить ТТГ, св. Т3, св. Т4, АТ-рТТГ, АТ-ТПО, АТ-ТГ. Гипотиреоз у пациентки вызван атрофической формой АИТ, о чём свидетельствует ничтожный объём щитовид- ной железы – 2,3 см3. При АИТ могут вырабатываться как блоки- рующие АТ-ТПО, АТ-ТГ, так и стимулирующие – АТ-рТТГ. Бло- кирующие АТ усугубляют гипотиреоз, могут вызывать его деком- пенсацию и увеличивать потребность в дозировке эутирокса. Из- быток АТ-рТТГ у наблюдаемой больной не вызовет тиреотоксикоз, так как тиреоидная паренхима у неё заменена фиброзной тканью, но может спровоцировать аутоиммунный процесс в орбитах. 1. Необходимо проверить ТТГ, св. Т3, св. Т4, АТ-рТТГ, АТ-ТПО, АТ-ТГ. Гипотиреоз у пациентки вызван атрофической формой АИТ, о чём свидетельствует ничтожный объём щитовид- ной железы – 2,3 см3. При АИТ могут вырабатываться как блоки- рующие АТ-ТПО, АТ-ТГ, так и стимулирующие – АТ-рТТГ. Бло- кирующие АТ усугубляют гипотиреоз, могут вызывать его деком- пенсацию и увеличивать потребность в дозировке эутирокса. Из- быток АТ-рТТГ у наблюдаемой больной не вызовет тиреотоксикоз, так как тиреоидная паренхима у неё заменена фиброзной тканью, но может спровоцировать аутоиммунный процесс в орбитах. 2. Если диагностическая версия подтвердится, то возрастной больной предстоит лечиться большими дозами глюкокортикоидов. Поэтому необходимо провести дополнительное обследование тех систем, которые могут пострадать при лечении: сердечно-сосуди- стая, углеводный обмен, электролитный баланс, костная, желудок. Задача 5. 1. Ответы на вопросы ситуационных задач Эндокринолог должен заподозрить первичный гипокорти- цизм, поскольку выражена характерная для Аддисоновой болезни пигментация кожных покровов и слизистых, при этом нет расовой и национальной окраски кожи. Артериальная гипотония и астени- ческий синдром – характерные признаки гипокортицизма. 2. Надо проверить уровень в крови кортизола (лучше – его суточ- ную экскрецию), уровень альдостерона, 17-ОН прогестерона, калия, натрия, хлоридов, альбумина и белка, креатинина, мочевины, повто- рить контроль гликемии, сделать диаскин-тест. Важно провести УЗИ или КТ надпочечников для выявления их деструкции. 3. В общем анализе крови возможна эозинофилия, общий ана- лиз мочи может быть без патологии, уровень глюкозы и холесте- рина скорее всего окажутся пониженными; на ЭКГ обнаружатся признаки гиперкалиемии. 4. Потребуется консультация фтизиатра, так как явных симпто- мов аутоиммунного генеза гипокортицизма у пациентки нет (нет витилиго). Надо исключить АИТ, поскольку аутоиммунный про- цесс в надпочечниках может сопровождаться аутоиммунным пора- жением щитовидной железы (синдром Шмидта). 100 И.В. Терещенко И.В. Терещенко Задача 6. 1. Ребёнок женского пола, поскольку хромосомный набор ХХ. Аномалия строения наружных гениталий – это псевдогермафроди- тизм, гипоспадия, очевидно, надпочечникового генеза, поскольку уровень андрогена 17 ОН-прогестерона значительно выше нормы. 1. Ребёнок женского пола, поскольку хромосомный набор ХХ. Аномалия строения наружных гениталий – это псевдогермафроди- тизм, гипоспадия, очевидно, надпочечникового генеза, поскольку уровень андрогена 17 ОН-прогестерона значительно выше нормы. 2. Допущены диагностические ошибки в определении пола плода при УЗИ-скрининге. Не было указано, что пол определить нельзя, хотя в медицинской практике это наблюдается нередко (до 10% случаев). Диагностировали мужской пол и при 2-ом, и 3-ем УЗИ. Длина ребёнка 53 см при среднем росте матери и низком – отца. Можно полагать, что костная система ребёнка формирова- лась ускоренно под влиянием избытка надпочечниковых анаболи- ков, но в заключениях УЗИ это не указано. Поэтому у беременной женщины не проверяли уровень 17 ОН-прогестерона в крови и не исключили аномалию гена CYP 21, не назначили необходимое ле- чение, не допускающее формирования псевдогермафродитизма у плода. Девочке предстоят корригирующие операции гениталий. 3. Диагноз девочки: врождённая дисфункция коры надпочечни- ков, классическая форма (адреногенитальный синдром). По- скольку ВДКН у неё имеет классическую форму, можно полагать, что оба родителя являются носителями мутантного гена CYP 21В, хотя сами здоровы. При планировании второго ребёнка необхо- димо обоих обследовать, проверить носительство мутантного гена CYP 21В. 101 Пропедевтика эндокринных заболеваний Дефиниция избранных медицинских терминов Акромегалия – опухоль гипофиза – соматотропинома, секрети- рующая в избытке СТГ. Акромегалия – опухоль гипофиза – соматотропинома, секрети- рующая в избытке СТГ. Алопеция – облысение, которое бывает гнёздным, диффузным, тотальным (бровей и др.). Алопеция – облысение, которое бывает гнёздным, диффузным, тотальным (бровей и др.). Альдостерома – опухоль клубочкового слоя надпочечника, продуцирует альдостерон. Альдостерома – опухоль клубочкового слоя надпочечника, продуцирует альдостерон. Альдостерома – опухоль клубочкового слоя надпочечника, продуцирует альдостерон. Ангулярный стоматит – мокнущие трещины в углах рта, не- редко симптом дефицита витамина В2 и/или В6. Ангулярный стоматит – мокнущие трещины в углах рта, не- редко симптом дефицита витамина В2 и/или В6. Андростерома – опухоль сетчатого слоя надпочечника, проду- цирует андрогены. Андростерома – опухоль сетчатого слоя надпочечника, проду- цирует андрогены. Андростерома – опухоль сетчатого слоя надпочечника, проду- цирует андрогены. ру р Аномальное маточное кровотечение – нарушение менстру- ального цикла. А Аномальное маточное кровотечение – нарушение менстру- ального цикла. А Анорексия – отсутствие аппетита. р у Арренобластома –злокачественная опухоль яичника, продуци- рует тестостерон. Афония – потеря звучности голоса; может говорить только шёпотом. Блефароспазм – симметричное непроизвольное сокраще- ние круговых мышц глаз, приводящее к стойкому спазматиче- скому смыканию век. Афония – потеря звучности голоса; может говорить только шёпотом. Блефароспазм – симметричное непроизвольное сокраще- ние круговых мышц глаз, приводящее к стойкому спазматиче- скому смыканию век. Булимия – повышенный аппетит. ВДКН – наследственный дефицит фермента 21-гидроксилазы в надпочечниках, вследствие чего возникает гиперсекреция надпо- чечниковых андрогенов. Булимия – повышенный аппетит. ВДКН – наследственный дефицит фермента 21-гидроксилазы в надпочечниках, вследствие чего возникает гиперсекреция надпо- чечниковых андрогенов. Гинекомастия – рост молочных желёз у мальчиков или у мужчин. Гиперандрогения – повышенная секреция андрогенов в жен- ском организме. р у у у Гиперандрогения – повышенная секреция андрогенов в жен- ском организме. Гипоспадия – врождённая аномалия строения полового члена, когда отверстие мочеиспускательного канала с головки полового члена смещено вниз и находится на теле пениса или на мошонке. Гипоспадия – врождённая аномалия строения полового члена, когда отверстие мочеиспускательного канала с головки полового члена смещено вниз и находится на теле пениса или на мошонке. ГлиНв А 1с – гемоглобин в химическом соединении с глюкозой. Глюкостерома (кортикостерома) – опухоль пучкового слоя надпочечников. Грейвса болезнь – токсический зоб, тиреотоксикоз. Диастема – расширение межзубных промежутков. ф Евнухоидизм – сниженная функция гонад вследствие эмбрио- нального поражения или вследствие поражения гонад в детском возрасте. Евнухоидизм – сниженная функция гонад вследствие эмбрио- нального поражения или вследствие поражения гонад в детском возрасте. Дефиниция избранных медицинских терминов р Инсулинома – опухоль из β-клеток поджелудочной железы с гиперсекрецией инсулина. Инциденталома – случайно выявленная опухоль эндокринной железы, может быть гипофиза, надпочечника, околощитовидной железы, тимуса и др. Инциденталома – случайно выявленная опухоль эндокринной железы, может быть гипофиза, надпочечника, околощитовидной железы, тимуса и др. 102 И.В. Терещенко Иценко-Кушинга болезнь – опухоль гипофиза, вызывающая гиперсекрецию АКТГ (кортикотропинома). р р ( р р ) Иценко-Кушинга синдром – гиперсекреция кортизола вслед- ствие кортикостеромы (глюкостеромы); может быть гиперсекре- ция кортизола другого генеза. р ру Макроглоссия – патологически большой язык. Марфана болезнь – наследственная патология соединительной ткани. Миастения – синдром повышенной мышечной утомляемости вследствие опухоли тимуса или аутоиммунного генеза. Микседема – «слизистый отёк» – крайняя клинически выражен- ная форма гипотиреоза; из-за накопления в тканях муцина и гиалу- роновой кислоты возникают тотальные отёки. НЭО – нейроэндокринные опухоли, исходящие из нейроэндо- кринных клеток ЖКТ. Могут секретировать свойственные этим тканям гормоны (соответственно различают инсулиномы, глюкаго- номы, соматостатиномы, ВИПомы, карциноидный синдром). НЭО – нейроэндокринные опухоли, исходящие из нейроэндо- кринных клеток ЖКТ. Могут секретировать свойственные этим тканям гормоны (соответственно различают инсулиномы, глюкаго- номы, соматостатиномы, ВИПомы, карциноидный синдром). Паратирома – опухоль паращитовидной железы. П б р р у р щ д Педжета болезнь – генетическое нарушение костного ремоделиро- вания, приводящее к увеличению размера и деформации костей. р р у р Педжета болезнь – генетическое нарушение костного ремоделиро- вания, приводящее к увеличению размера и деформации костей. Пиквика болезнь – крайняя степень ожирения с альвеолярной гиповентиляцией и стойкой гипоксией. Пиквика болезнь – крайняя степень ожирения с альвеолярной гиповентиляцией и стойкой гипоксией. Прогнатизм – при акромегалии из-за гиперсекреции СТГ вы- стояние нижней части лица, особенно нижней челюсти. ф Пролактинома – опухоль гипофиза из клеток, секретирующих ПРЛ. ПЮД – пубертатно-юношеский диспитуитаризм – дисфункция гипоталамо-гипофизарной системы в периоде пубертации. Полидипсия – повышенная жажда. Полифагия – чрезмерное потребление пищи, отсутствие чув- ства насыщения. д д Полифагия – чрезмерное потребление пищи, отсутствие чув- ства насыщения. СПКЯ – синдром, характеризующийся гиперандрогенией, ову- ляторной дисфункцией. СПКЯ – синдром, характеризующийся гиперандрогенией, ову- ляторной дисфункцией. р у р ф Феохромоцитома – опухоль мозгового слоя надпочечников. Цефалгия головная боль. Шиена болезнь (синдром) – послеродовый гипопитуитаризм в результате массивных кровопотерь и ишемии гипофиза. Шиена болезнь (синдром) – послеродовый гипопитуитаризм в результате массивных кровопотерь и ишемии гипофиза. Anorexia nervosa – нервная анорексия – нарушение пищевого поведения, стремление к худобе. Anorexia nervosa – нервная анорексия – нарушение пищевого поведения, стремление к худобе. Pubertas praecox – преждевременное половое созревание. Pubertas tarda – запоздалое половое созревание. Научное издание Терещенко Ирина Владимировна ПРОПЕДЕВТИКА ЭНДОКРИННЫХ ЗАБОЛЕВАНИЙ (МЕТОДЫ ИССЛЕДОВАНИЯ БОЛЬНЫХ С ЗАБОЛЕВАНИЯМИ ЭНДОКРИННОЙ СИСТЕМЫ) Монография Чебоксары, 2022 г. Редактор И.В. Терещенко Компьютерная верстка Е.В. Кузнецова Дизайн обложки Н.В. Фирсова Подписано в печать 28.12.2021 г. Дата выхода издания в свет 10.01.2022 г. Формат 60×84/16. Бумага офсетная. Печать офсетная. Гарнитура Times. Усл. печ. л. 6,045. Заказ К-921. Тираж 500 экз. Издательский дом «Среда» 428005, Чебоксары, Гражданская, 75, офис 12 +7 (8352) 655-731 info@phsreda.com https://phsreda.com Отпечатано в Студии печати «Максимум» 428005, Чебоксары, Гражданская, 75 +7 (8352) 655-047 info@maksimum21.ru www.maksimum21.ru Терещенко Ирина Владимировна Дефиниция избранных медицинских терминов Pubertas praecox – преждевременное половое созревание. Pubertas tarda – запоздалое половое созревание. Pubertas praecox преждевременное половое созре Pubertas tarda – запоздалое половое созревание. 5α-редуктаза – фермент, преобразующий тестостерон в более сильнодействующий андроген ДТС. 5α-редуктаза – фермент, преобразующий тестостерон в более сильнодействующий андроген ДТС. 103 Отпечатано в Студии печати «Максимум» 428005, Чебоксары, Гражданская, 75 +7 (8352) 655-047 info@maksimum21.ru www.maksimum21.ru ПРОПЕДЕВТИКА ЭНДОКРИННЫХ ЗАБОЛЕВАНИЙ (МЕТОДЫ ИССЛЕДОВАНИЯ БОЛЬНЫХ С ЗАБОЛЕВАНИЯМИ ЭНДОКРИННОЙ СИСТЕМЫ) Редактор И.В. Терещенко Компьютерная верстка Е.В. Кузнецова Дизайн обложки Н.В. Фирсова Издательский дом «Среда» 428005, Чебоксары, Гражданская, 75, офис 12 +7 (8352) 655-731 info@phsreda.com https://phsreda.com Отпечатано в Студии печати «Максимум» 428005, Чебоксары, Гражданская, 75 +7 (8352) 655-047 info@maksimum21.ru www.maksimum21.ru
https://openalex.org/W2899520545	https://europepmc.org/articles/pmc6230590?pdf=render	English	null	miRNA miR-21 Is Largely Dispensable for Intrathymic T-Cell Development	Frontiers in immunology	2,018	cc-by	9,928	ORIGINAL RESEARCH published: 05 November 2018 doi: 10.3389/ﬁmmu.2018.02497 miRNA miR-21 Is Largely Dispensable for Intrathymic T-Cell Development Heike Kunze-Schumacher, Samantha J. Winter, Esther Imelmann and Andreas Krueger* Institute for Molecular Medicine, Goethe University Frankfurt, Frankfurt, Germany Heike Kunze-Schumacher, Samantha J. Winter, Esther Imelmann and Andreas Krueger* Development of T cells in the thymus is tightly controlled to continually produce functional, but not autoreactive, T cells. miRNAs provide a layer of post-transcriptional gene regulation to this process, but the role of many individual miRNAs in T-cell development remains unclear. miR-21 is prominently expressed in immature thymocytes followed by a steep decline in more mature cells. We hypothesized that such a dynamic expression was indicative of a regulatory function in intrathymic T-cell development. To test this hypothesis, we analyzed T-cell development in miR-21-deﬁcient mice at steady state and under competitive conditions in mixed bone-marrow chimeras. We complemented analysis of knock-out animals by employing over-expression in vivo. Finally, we assessed miR-21 function in negative selection in vivo as well as differentiation in co-cultures. Together, these experiments revealed that miR-21 is largely dispensable for physiologic T-cell development. Given that miR-21 has been implicated in regulation of cellular stress responses, we assessed a potential role of miR-21 in endogenous regeneration of the thymus after sublethal irradiation. Again, miR-21 was completely dispensable in this process. We concluded that, despite prominent and highly dynamic expression in thymocytes, miR-21 expression was not required for physiologic T-cell development or endogenous regeneration. Edited by: Loretta Tuosto, Università degli Studi di Roma La Sapienza, Italy Edited by: Loretta Tuosto, Università degli Studi di Roma La Sapienza, Italy Reviewed by: Adrian Liston, Flanders Institute for Biotechnology, Belgium Claudio Pignata, Università degli Studi di Napoli Federico II, Italy U e s à deg S ud d o a a Sapienza, Italy Reviewed by: Adrian Liston, Flanders Institute for Biotechnology, Belgium Claudio Pignata, Università degli Studi di Napoli Federico II, Italy Correspondence: Andreas Krueger Andreas.Krueger@kgu.de Sapienza, Italy Reviewed by: Adrian Liston, Flanders Institute for Biotechnology, Belgium Claudio Pignata, Università degli Studi di Napoli Federico II, Italy Reviewed by: Adrian Liston, Flanders Institute for Biotechnology, Belgium Reviewed by: Adrian Liston, Flanders Institute for Biotechnology, Belgium Claudio Pignata, Università degli Studi di Napoli Adrian Liston, Flanders Institute for Biotechnology, Belgium g Claudio Pignata, Università degli Studi di Napoli Federico II, Italy Correspondence: Andreas Krueger Andreas.Krueger@kgu.de INTRODUCTION Specialty section: This article was submitted to T Cell Biology, a section of the journal Frontiers in Immunology Development of T cells in the thymus is a tightly controlled process involving the commitment of progenitor cells to the T lineage, generation of a diverse T-cell antigen receptor (TCR) repertoire and selection of largely non-autoreactive T cell clones. Phenotypically, the most immature thymocytes are characterized by the absence of the co-receptors CD4 and CD8 (double-negative, DN), which can be further subdivided into multiple subsets based on dynamic expression of CD117, CD25, and CD44. T-lineage commitment is completed at the CD117loCD25hiCD44+ DN2b stage upon expression of high levels of the transcription factor Bcl11b (1–4). Somatic recombination of the Trb, Trg, and Trd TCR gene loci and selection of productive rearrangements is completed at the CD44−CD25+CD28+ DN3b stage. Cells entering the αβT cell lineage acquire expression of both CD4 and CD8 (double-positive, DP), rearrange the Tra gene locus and undergo positive and negative selection. Thymocytes then mature into CD4 or CD8 single-positive (SP) cells, where negative selection continues and further maturation occurs prior to egress from the thymus. Received: 22 August 2018 Accepted: 09 October 2018 Published: 05 November 2018 Keywords: miRNA, miR-21, thymus, T cells, development, selection, regeneration Citation: Kunze-Schumacher H, Winter SJ, Imelmann E and Krueger A (2018) miRNA miR-21 Is Largely Dispensable for Intrathymic T-Cell Development. Front. Immunol. 9:2497. doi: 10.3389/ﬁmmu.2018.02497 November 2018 \| Volume 9 \| Article 2497 Frontiers in Immunology \| www.frontiersin.org miR-21 in T-Cell Development Kunze-Schumacher et al. FIGURE 1 \| Dynamic expression of miR-21 during T-cell development. Quantitative RT-PCR for miR-21 expression in sorted thymic subsets from WT mice. Expression levels of indicated cell populations were normalized to snoRNA412. Each dot represents one mouse, n = 4. T-cell development is controlled by extrinsic factors including cytokines and signals through the TCR. Furthermore, intrinsic factors such as transcriptional programs govern diﬀerent steps of intrathymic T-cell diﬀerentiation have been extensively characterized (5). In contrast, considerably less is known about post-transcriptional regulation of T-cell development, such as by miRNAs (6). Loss of all miRNAs due to deletion of key components of the miRNA processing machinery results in speciﬁc defects in T-cell development. Early loss of miRNAs results in profound thymocyte death (7). In addition, a small number of individual miRNAs have been identiﬁed to regulate distinct T-lineage developmental stages, including miR-17∼92, miR-142, and miR-181a (8–13). FIGURE 1 \| Dynamic expression of miR-21 during T-cell development. Quantitative RT-PCR for miR-21 expression in sorted thymic subsets from WT mice. Expression levels of indicated cell populations were normalized to snoRNA412. Each dot represents one mouse, n = 4. FIGURE 1 \| Dynamic expression of miR-21 during T-cell development. Quantitative RT-PCR for miR-21 expression in sorted thymic subsets from WT mice. Expression levels of indicated cell populations were normalized to snoRNA412. Each dot represents one mouse, n = 4. Functional roles of individual miRNAs can only partially explain the eﬀect of loss of all miRNAs observed in T-cell development. In addition, it is possible that some miRNAs exist that display opposing roles in this process. In order to identify miRNAs with a putative function in T-cell development we hypothesized that such miRNAs should be expressed at high levels in at least some thymocyte populations and that such miRNAs should display a pattern of strong dynamic regulation at key developmental checkpoints. miR-21 is prominently expressed in many mammalian tissues (14). In the thymus, expression levels are very high in immature DN thymocytes, followed by a steep decline toward the DP stage and modest re-expression in SP thymocytes (15–17). Citation: Expression of miR-21 is induced during T-cell activation and has been reported to support survival of memory T cells and expression of CC chemokine receptor 7 (CCR7) on naïve T cells (18, 19). In addition, it has been proposed that miR- 21 promotes PD-1-mediated tolerance by targeting PDCD4 (20). The role of miR-21 in intrathymic T-cell development remains unknown. We hypothesized that high expression levels combined with strong dynamic changes in expression of miR-21 throughout diﬀerent stages of T-cell development were indicative of a regulatory function in this process. To test this putative function, we analyzed the consequences of miR-21 deletion as well as overexpression in mice in vivo. Surprisingly, neither absence nor elevated expression of miR-21 had major eﬀects on T-cell development at steady state or in chimeric mice. Furthermore, endogenous T-cell regeneration remained unaﬀected by loss of miR-21. We conclude that despite prominent and dynamic expression, miR-21 is dispensable for steady-state and stress-induced T-cell development in mice. sorted thymocyte populations of wildtype (WT) mice starting with ETPs (CD117hiCD25−CD44+), the most immature detectable thymocyte population, toward mature SP T cells and assessed relative expression of miR-21 (Figure 1 and Figure S1). Consistent with previous reports, we found miR-21 to be diﬀerentially expressed throughout intrathymic T-cell development. Highest levels of miR-21 were observed in DN thymocytes, especially in DN2b (CD117loCD25+CD44+) and DN3b (CD25+CD44−CD28+) thymocytes. Our data revealed that lowest levels of miR-21 can be detected in post-selected DP (deﬁned as CD4+CD8+CD69+TCRβ+) and mature SP4 T cells (TCRβ+CD4+). Slightly higher expression was observable in SP8 (TCRβ+CD8+) conﬁrming previously published data. Together, our ﬁndings represent a more detailed analysis about miR-21 expression and validate dynamic expression of miR-21 during T-cell development. miR-21 Is Largely Dispensable for Steady-State T-Cell Development in the Thymus In order to test a potential role of miR-21 in intrathymic T- cell development, we ﬁrst characterized miR-21-deﬁcient mice. Absolute total thymocyte numbers were unaﬀected by miR-21 (Figure 2A). We then determined early thymocyte subsets in miR-21-suﬃcient compared to deﬁcient mice and detected a small, but statistically signiﬁcant increase in the frequency of DN2 thymocytes (Figures 2B,C). When we analyzed late T- cell development in these mice, we observed a slight decrease in frequencies of DP thymocytes (Figures 2D,E) accompanied by increased frequencies of SP T cells. Again, these changes were small. Furthermore, we revealed frequencies of γδ T cells to be similar upon loss of miR-21 (Figure 2F). We and others have shown that miRNAs are essential for the maturation of agonist-selected thymocytes (12, 21–24). To address whether miR-21 might inﬂuence the development of invariant natural killer T (iNKT) cell and T regulatory (Treg) cells, we assessed frequencies of these subsets (Figures 2G,H). We detected small but signiﬁcantly elevated frequencies of Dynamic Expression of miR-21 During T-Cell Development Previous studies determined thymic expression levels of a diverse range of diﬀerent miRNAs (15–17). For miR-21, thymic expression levels based on miRNA-sequencing were revealed to be high in DN thymocytes accompanied by an abrupt decrease toward the DP stage prior to re-expression in SP T cells. To validate global gene expression data, we November 2018 \| Volume 9 \| Article 2497 Frontiers in Immunology \| www.frontiersin.org 2 miR-21 in T-Cell Development Kunze-Schumacher et al. FIGURE 2 \| miR-21 is largely dispensable for steady-state T-cell development in the thymus. (A) Total cellularity of thymi from WT and miR-21−/−mice, n = 6 for each genotype. (B) Representative ﬂow cytometric analysis of lineage-depleted thymi from WT and miR-21−/−mice stained with antibodies against CD25 and CD44. Numbers in quadrants represent frequencies. (C) Statistical analysis of ﬂow cytometric results shown in (B), n = 5. (D) Representative ﬂow cytometric analysis of hymi from WT and miR-21−/−mice stained with antibodies against CD4 and CD8α. Numbers in quadrants represent frequencies. (E) Statistical analysis of ﬂow cytometric results shown in (D), n = 5. (F) Representative ﬂow cytometric and statistical analysis of thymi from WT and miR-21−/−mice stained with antibodies against CD4, CD8α and TCRγ δ. Numbers in right corners of plots represent frequency, n = 5. (G) Representative ﬂow cytometric and statistical analysis of thymi from WT and miR-21−/−mice stained with CD1d-tet and antibody against TCRβ. Numbers in right corners of plots represent frequency, n = 5. (H) Representative ﬂow cytometric and statistical analysis of thymi from WT and miR-21−/−mice stained with antibodies against CD4, CD25 and Foxp3. Numbers in left corners of plots epresent frequency, n = 5. FIGURE 2 \| miR-21 is largely dispensable for steady-state T-cell development in the thymus. (A) Total cellularity of thymi from WT and miR-21−/−mice, n = 6 for each genotype. (B) Representative ﬂow cytometric analysis of lineage-depleted thymi from WT and miR-21−/−mice stained with antibodies against CD25 and CD44. Numbers in quadrants represent frequencies. (C) Statistical analysis of ﬂow cytometric results shown in (B), n = 5. (D) Representative ﬂow cytometric analysis of thymi from WT and miR-21−/−mice stained with antibodies against CD4 and CD8α. Numbers in quadrants represent frequencies. (E) Statistical analysis of ﬂow cytometric results shown in (D), n = 5. (F) Representative ﬂow cytometric and statistical analysis of thymi from WT and miR-21−/−mice stained with antibodies against CD4, CD8α and TCRγ δ. Dynamic Expression of miR-21 During T-Cell Development Numbers in right corners of plots represent frequency, n = 5. (G) Representative ﬂow cytometric and statistical analysis of thymi from WT and miR-21−/−mice stained with CD1d-tet and antibody against TCRβ. Numbers in right corners of plots represent frequency, n = 5. (H) Representative ﬂow cytometric and statistical analysis of thymi from WT and miR-21−/−mice stained with antibodies against CD4, CD25 and Foxp3. Numbers in left corners of plots represent frequency, n = 5. Treg cells upon loss of miR-21. This ﬁnding is consistent with previous reports that identiﬁed miR-21 to modulate Foxp3 transcription factor levels and frequency of circulating Treg cells (25–27). In conclusion, these data suggest that loss of miR- 21 does not result in substantial defects in physiological T-cell development. Treg cells upon loss of miR-21. This ﬁnding is consistent with previous reports that identiﬁed miR-21 to modulate Foxp3 transcription factor levels and frequency of circulating Treg cells (25–27). In conclusion, these data suggest that loss of miR- 21 does not result in substantial defects in physiological T-cell development. thymocytes were skewed in miR-21−/−mice. To this end, we identiﬁed these populations based on their surface expression of CD69 and CD62L and detected no alterations in the absence of miR-21 compared to their WT counterparts (Figures 3A,B). We then asked whether miR-21 modulates selection processes. To test this, we ﬁrst analyzed surface expression of CD5 as an indicator of TCR signal strength in DP, SP4, and SP8 thymocytes of miR-21−/−mice. We detected comparable CD5 expression of DP thymocytes between WT and miR-21−/− mice, whereas SP4 and SP8 T cells of miR-21−/−mice revealed diminished expression levels (Figure 3C). We then tested a potential role of miR-21 in modulating TCR signal strength by Absence of miR-21 Does Not Affect Negative Selection Based on our previous ﬁnding that frequencies of DP thymocytes were signiﬁcantly reduced in miR-21-deﬁcient mice, we next determined whether frequencies of pre- and post-selected DP November 2018 \| Volume 9 \| Article 2497 Frontiers in Immunology \| www.frontiersin.org 3 miR-21 in T-Cell Development Kunze-Schumacher et al. GURE 3 \| Absence of miR-21 does not affect negative selection. (A) Representative ﬂow cytometric analysis of thymi from WT and miR-21−/−mice stained with ibodies against CD4, CD8α, CD69 and CD62L. Numbers in left and right corners represent frequencies of pre- and post-selection DP, respectively. (B) Statistical alysis of ﬂow cytometric results shown in (A), n = 5. (C) Statistical analysis of mean ﬂuorescence intensity of CD5 on DP, SP4 or SP8, n = 5. (D) Statistical analysis resulting Ca2+-response upon stimulation in DP, SP4 and SP8 thymocytes, n = 5. (E) Representative ﬂow cytometric analysis of thymi from WT and miR-21−/− ce, 48 h post injection with either PBS or α-CD3, stained with antibodies against CD4 and CD8α. Numbers adjacent to gates represent frequency. (F) Histograms play expression of active caspase-3 in DP thymocytes from WT and miR-21−/−mice, 48 h post injection with either PBS or α-CD3, n = 4-5 per group. (G) atistical analysis of ﬂow cytometric results shown in (E,F). Analysis of signiﬁcance were performed using one-way analysis of variance followed by Tukey’s test. icated signiﬁcant results are representative of latter test. (H) Statistical analysis of BrdU+ DP thymocytes of thymi from WT and miR-21−/−mice, 48 h post ection with either PBS or α-CD3, n = 6 for each genotype. Pooled data of two independent experiments. FIGURE 3 \| Absence of miR-21 does not affect negative selection. (A) Representative ﬂow cytometric analysis of thymi from WT and miR-21−/−mice stained with antibodies against CD4, CD8α, CD69 and CD62L. Numbers in left and right corners represent frequencies of pre- and post-selection DP, respectively. (B) Statistical analysis of ﬂow cytometric results shown in (A), n = 5. (C) Statistical analysis of mean ﬂuorescence intensity of CD5 on DP, SP4 or SP8, n = 5. (D) Statistical analysis of resulting Ca2+-response upon stimulation in DP, SP4 and SP8 thymocytes, n = 5. (E) Representative ﬂow cytometric analysis of thymi from WT and miR-21−/− mice, 48 h post injection with either PBS or α-CD3, stained with antibodies against CD4 and CD8α. Numbers adjacent to gates represent frequency. miR-21-Deﬁciency or Overexpression Do Not Alter T-Cell Development in BM Chimeras Competitive BM chimera experiments are able to reveal cell- intrinsic defects in development, which at steady state, may be masked by compensatory eﬀects (8, 30–33). Thus, we asked whether a possible cell intrinsic advantage or disadvantage of miR-21-deﬁcient BM-derived cells could occur. Lethally irradiated WT recipients were reconstituted with a 1:1 mixture of BM from WT (CD45.1) and BM cells from miR-21-deﬁcient (CD45.2) or control mice (CD45.2). After 8 weeks, we analyzed thymi of these chimeric mice and identiﬁed miR-21−/−-derived BM cells to compete in a similar manner as the respective WT controls (Figures 5A,B). Detailed analysis of frequencies of DP, SP4, and SP8 suggested no aberrant eﬀect of miR-21-deﬁcient BM cells. Accordingly, we detected comparable frequencies of these T-cell subsets in total thymus, CD45.1–or CD45.2-derived cells (Figure 5A). For DN thymocytes, we found small diﬀerences in their reconstitution levels. DN2 thymocytes of miR-21−/−origin competed in a similar manner as the provided WT control and miR-21−/−-derived DN3 thymocytes as well as DP thymocytes were detected in slightly decreased frequencies indicated by a decline in contribution. For later stages of T-cell development, this eﬀect was absent as indicated by similar reconstitution rates of miR-21−/−and WT control BM. Notably, Treg cells derived from miR-21-deﬁcient BM cells displayed a beneﬁcial reconstitution compared to their control WT counterparts. This observation can be explained by increased radioresistance of Treg cells (34–36). Therefore, host-derived Treg cells are not fully eliminated and might contribute to this skewed reconstitution. Based on our ﬁndings that loss of miR-21 does not result in aberrant T-cell development, we next aimed to elucidate whether overexpression of miR-21 controls thymopoiesis. We overexpressed miR-21 (and an empty vector control) retrovirally in lineage-depleted BM cells prior to reconstitution of WT mice. Both vectors encoded enhanced GFP (eGFP) as a reporter gene. After 8 weeks, thymi were harvested and analyzed for the development of T cells (Figures 5C,D). First, we assessed frequencies of DP, SP4, and SP8 cells in either GFP− (untransduced) or GFP+ cells (transduced) of control and miR-21-overexpressing thymi and detected no signiﬁcant alterations. In addition, we studied early thymocyte subsets and found no diﬀerences in frequencies of DN1 and DN2 upon overexpression (Figure 5D). For DN3 thymocytes, a slight increase in their frequency upon overexpression was observable when compared to their untransduced (GFP−) counterparts. However, when compared to the empty vector control, no signiﬁcant diﬀerence was detectable. Absence of miR-21 Does Not Affect Negative Selection We found similar frequencies of BrdU incorporation in DP thymocytes of WT or miR-21−/−controls treated with PBS, indicating equal rates of proliferation at steady state. Virtually no BrdU incorporation was detected in thymocytes after anti-CD3-stimulation, which is consistent with the experimental setup to monitor negative selection. Thus, we concluded that miR-21 is largely dispensable during thymic selection. miR-21-Deﬁciency or Overexpression Do Not Alter T-Cell Development in BM Chimeras Similarly, frequencies of pre- and post-selection DP thymocytes were comparable to their control counterparts upon Absence of miR-21 Does Not Affect Negative Selection (F) Histograms display expression of active caspase-3 in DP thymocytes from WT and miR-21−/−mice, 48 h post injection with either PBS or α-CD3, n = 4-5 per group. (G) Statistical analysis of ﬂow cytometric results shown in (E,F). Analysis of signiﬁcance were performed using one-way analysis of variance followed by Tukey’s test. Indicated signiﬁcant results are representative of latter test. (H) Statistical analysis of BrdU+ DP thymocytes of thymi from WT and miR-21−/−mice, 48 h post injection with either PBS or α-CD3, n = 6 for each genotype. Pooled data of two independent experiments. November 2018 \| Volume 9 \| Article 2497 Frontiers in Immunology \| www.frontiersin.org miR-21 in T-Cell Development Kunze-Schumacher et al. studying Ca2+-ﬂux upon stimulation with anti-CD3 antibody. We did not detect any signiﬁcant diﬀerences in the ability of miR-21-deﬁcient DP or SP thymocytes to respond to anti- CD3 stimulation both in terms of peak response (Figure 3D) and overall kinetics (Figure S4A). Furthermore, we evaluated whether loss of miR-21 leads to altered viability of SP thymocytes by studying ex vivo survival of these cells (Figure S5). We did not observe signiﬁcant diﬀerences in early apoptotic cells and only a mild decrease in the frequency of late apoptotic SP8 cells after 48 h in culture, indicating that survival of SP thymocytes is largely unaﬀected by loss of miR-21. To further evaluate whether a functional defect in DP thymocytes could exist that modulates negative selection, we adapted an in vivo TCR stimulation approach (28). First, we injected WT or miR- 21−/−mice with a single dose of anti-CD3 (or PBS as a control) to stimulate TCR signaling. After 48 h, we assessed frequencies of thymocytes and performed staining for activated caspase-3 to examine early apoptosis (Figures 3E-G). Thymi of control mice contained normal frequencies of DP thymocytes, whereas thymi derived from either WT or miR-21−/−anti-CD3- treated mice showed signiﬁcantly lower frequencies. Frequencies of DP thymocytes were slightly reduced in miR-21-deﬁcient mice upon treatment with anti-CD3 compared to their WT counterparts, which could be due to increased apoptosis levels in miR-21-deﬁcient mice as previously described (29). However, analysis of activated caspase-3 showed no signiﬁcant diﬀerences in apoptosis between WT and miR-21-deﬁcient DP thymocytes after treatment. In addition, we determined thymic cellularity of PBS- or anti-CD3-treated WT and miR-21−/−mice and found absolute total thymocyte numbers to be unaﬀected by miR-21 upon treatment (Figure S4B). Absence of miR-21 Does Not Affect Negative Selection Finally, we monitored proliferation in this assay by administration of a single BrdU pulse (Figure 3H). We found similar frequencies of BrdU incorporation in DP thymocytes of WT or miR-21−/−controls treated with PBS, indicating equal rates of proliferation at steady state. Virtually no BrdU incorporation was detected in thymocytes after anti-CD3-stimulation, which is consistent with the experimental setup to monitor negative selection. Thus, we concluded that miR-21 is largely dispensable during thymic selection. stem cells (HSCs), multipotent progenitors (MPPs), and common lymphoid progenitors (CLPs) upon loss of miR-21 (Figure S2), thus arguing against a pivotal role of miR-21 in the periphery. studying Ca2+-ﬂux upon stimulation with anti-CD3 antibody. We did not detect any signiﬁcant diﬀerences in the ability of miR-21-deﬁcient DP or SP thymocytes to respond to anti- CD3 stimulation both in terms of peak response (Figure 3D) and overall kinetics (Figure S4A). Furthermore, we evaluated whether loss of miR-21 leads to altered viability of SP thymocytes by studying ex vivo survival of these cells (Figure S5). We did not observe signiﬁcant diﬀerences in early apoptotic cells and only a mild decrease in the frequency of late apoptotic SP8 cells after 48 h in culture, indicating that survival of SP thymocytes is largely unaﬀected by loss of miR-21. To further evaluate whether a functional defect in DP thymocytes could exist that modulates negative selection, we adapted an in vivo TCR stimulation approach (28). First, we injected WT or miR- 21−/−mice with a single dose of anti-CD3 (or PBS as a control) to stimulate TCR signaling. After 48 h, we assessed frequencies of thymocytes and performed staining for activated caspase-3 to examine early apoptosis (Figures 3E-G). Thymi of control mice contained normal frequencies of DP thymocytes, whereas thymi derived from either WT or miR-21−/−anti-CD3- treated mice showed signiﬁcantly lower frequencies. Frequencies of DP thymocytes were slightly reduced in miR-21-deﬁcient mice upon treatment with anti-CD3 compared to their WT counterparts, which could be due to increased apoptosis levels in miR-21-deﬁcient mice as previously described (29). However, analysis of activated caspase-3 showed no signiﬁcant diﬀerences in apoptosis between WT and miR-21-deﬁcient DP thymocytes after treatment. In addition, we determined thymic cellularity of PBS- or anti-CD3-treated WT and miR-21−/−mice and found absolute total thymocyte numbers to be unaﬀected by miR-21 upon treatment (Figure S4B). Finally, we monitored proliferation in this assay by administration of a single BrdU pulse (Figure 3H). Physiological Peripheral Lymphoid Cell Frequencies in miR-21−/−mice q Next, we addressed the question whether miR-21 is important for peripheral lymphoid cell subsets. Therefore, we analyzed spleen and lymph nodes (LNs) of miR-21-deﬁcient mice and found miR-21 to be dispensable for peripheral B cells and T cell subsets (Figures 4A,B). Frequencies of agonist-selected iNKT and Treg cells in the periphery showed small diﬀerences (Figures 4C,D) that recapitulated our ﬁndings observed in the thymus. Furthermore, we analyzed the distribution of naïve, central memory, and eﬀector memory T-cell subsets in spleen (Figures 4E,F) and LN (Figures 4G,H) and detected small changes in LN of miR-21-deﬁcient mice. To further evaluate the role of miR-21, we analyzed pre-thymic progenitors in the bone marrow (BM). We detected similar frequencies of hematopoietic November 2018 \| Volume 9 \| Article 2497 Frontiers in Immunology \| www.frontiersin.org 5 miR-21 in T-Cell Development Kunze-Schumacher et al. FIGURE 4 \| Physiological peripheral lymphoid cell frequencies in miR-21−/−mice. (A) Representative ﬂow cytometric analysis and gating strategy of spleen from WT and miR-21−/−mice stained with antibodies against B220, TCRβ, CD4, and CD8α. Numbers in quadrants or adjacent to gates represent frequencies. (B) Statistical analysis of ﬂow cytometric results shown in (A), n = 4-5. (C) Representative ﬂow cytometric and statistical analysis of spleen from WT and miR-21−/−mice stained with CD1d-tet and antibody against TCRβ. Numbers in right corners of plots represent frequency, n = 4-5. (D) Representative ﬂow cytometric and statistical analysis of spleen from WT and miR-21−/−mice stained with antibodies against CD4, CD25, and Foxp3. Numbers in left corners of plots represent frequency, n = 4-5. (E) Representative ﬂow cytometric analysis of spleen from WT and miR-21−/−mice stained with antibodies against TCRβ, CD44, and CD62L to identify naïve (CD44−CD62L+), central memory (CD44+CD62L+) and effector memory (CD44+CD62L−) T-cell subsets. Numbers in or adjacent to gates represent frequency. (F) Statistical analysis of ﬂow cytometric results shown in (E), n = 4-5. (G) Representative ﬂow cytometric analysis of LNs from WT and miR-21−/−mice stained with antibodies against TCRβ, CD44, and CD62L to identify naïve (CD44−CD62L+), central memory (CD44+CD62L+) and effector memory (CD44+CD62L−) T-cell subsets Numbers in or adjacent to gates represent frequency (H) Statistical analysis of ﬂow cytometric results shown in (G) n 5 FIGURE 4 \| Physiological peripheral lymphoid cell frequencies in miR-21−/−mice. (A) Representative ﬂow cytometric analysis and gating strategy of spleen from WT and miR-21−/−mice stained with antibodies against B220, TCRβ, CD4, and CD8α. Physiological Peripheral Lymphoid Cell Frequencies in miR-21−/−mice Numbers in quadrants or adjacent to gates represent frequencies. (B) Statistical analysis of ﬂow cytometric results shown in (A), n = 4-5. (C) Representative ﬂow cytometric and statistical analysis of spleen from WT and miR-21−/−mice stained with CD1d-tet and antibody against TCRβ. Numbers in right corners of plots represent frequency, n = 4-5. (D) Representative ﬂow cytometric and statistical analysis of spleen from WT and miR-21−/−mice stained with antibodies against CD4, CD25, and Foxp3. Numbers in left corners of plots represent frequency, n = 4-5. (E) Representative ﬂow cytometric analysis of spleen from WT and miR-21−/−mice stained with antibodies against TCRβ, CD44, and CD62L to identify naïve (CD44−CD62L+), central memory (CD44+CD62L+) and effector memory (CD44+CD62L−) T-cell subsets. Numbers in or adjacent to gates represent frequency. (F) Statistical analysis of ﬂow cytometric results shown in (E), n = 4-5. (G) Representative ﬂow cytometric analysis of LNs from WT and miR-21−/−mice stained with antibodies against TCRβ, CD44, and CD62L to identify naïve (CD44−CD62L+), central memory (CD44+CD62L+) and effector memory (CD44+CD62L−) T-cell subsets. Numbers in or adjacent to gates represent frequency. (H) Statistical analysis of ﬂow cytometric results shown in (G), n = 5. overexpression (Figure 5D). Thus, we concluded that ectopic elevation of miR-21 levels does not abrogate intrathymic T-cell development. diﬀerentiation in vitro. In general, T-lineage commitment is ﬁnalized with expression of the transcription factor Bcl11b at the DN2 stage. This stage can be further subdivided, based on the expression of CD117 into DN2a and DN2b. DN2a thymocytes have not fully undergone T-lineage fate decision and still contain non-T cell options such as NK cell potential, whereas the DN2b stage reﬂects the point of no return in regards to T- lineage fate decision (37). For this reason, we sorted DN2a and DN2b cells from either WT or miR-21−/−mice and cultured those on OP9-DL1 and OP9-GFP cells for up to 15 days. We determined the frequencies of generated T, NK and B cells over T-Lineage and Alternative Lineage Fate Decisions Are Mostly Unaltered in the Absence of miR-21 In a next step, we tested whether loss of miR-21 may result in altered potential to generate lymphocytes. We utilized the OP9- DL1 and OP9-GFP system to monitor T, B, and NK lineage November 2018 \| Volume 9 \| Article 2497 Frontiers in Immunology \| www.frontiersin.org 6 miR-21 in T-Cell Development Kunze-Schumacher et al. GURE 5 \| miR-21-deﬁciency or overexpression do not alter T-cell development in BM chimeras. (A) Representative ﬂow cytometric analysis of competitive BM meras 8 weeks after transfer, CD45.1: competitor population; CD45.2: test population (WT control or miR-21−/−). Thymi were stained with antibodies against 45.1, CD45.2, CD4, and CD8α. Numbers adjacent to gates and in quadrants represent frequencies. (B) Comparative analysis of thymocyte subpopulations in 45.2 test populations (WT control or miR-21−/−) of competitive chimeras, n = 9 per group. (C) Representative ﬂow cytometric analysis of overexpression meras 8 weeks after transfer. Thymi were stained with antibodies against CD4 and CD8α. Left panels display distribution of GFP−(untransduced) thymocytes in EV mpty vector) control or OE (miR-21-overexpression) chimeras. Right panels indicate GFP+ (transduced) thymocytes. Numbers in quadrants represent frequencies. Statistical analysis of ﬂow cytometric results of DN1, DN2, DN3, DP, pre- and post-selection DP, SP4, and SP8 populations from GFP−EV, GFP−OE, GFP+ EV, P+ OE thymocyte subsets indicated as frequencies, inter alia shown in (C), n = 2-5. Analysis of signiﬁcance were performed using one-way analysis of variance owed by Tukey’s test. Indicated signiﬁcant results are representative of latter test. FIGURE 5 \| miR-21-deﬁciency or overexpression do not alter T-cell development in BM chimeras. (A) Representative ﬂow cytometric analysis of competitive BM chimeras 8 weeks after transfer, CD45.1: competitor population; CD45.2: test population (WT control or miR-21−/−). Thymi were stained with antibodies against CD45.1, CD45.2, CD4, and CD8α. Numbers adjacent to gates and in quadrants represent frequencies. (B) Comparative analysis of thymocyte subpopulations in CD45.2 test populations (WT control or miR-21−/−) of competitive chimeras, n = 9 per group. (C) Representative ﬂow cytometric analysis of overexpression chimeras 8 weeks after transfer. Thymi were stained with antibodies against CD4 and CD8α. Left panels display distribution of GFP−(untransduced) thymocytes in EV (empty vector) control or OE (miR-21-overexpression) chimeras. Right panels indicate GFP+ (transduced) thymocytes. Numbers in quadrants represent frequencies. Frontiers in Immunology \| www.frontiersin.org T-Lineage and Alternative Lineage Fate Decisions Are Mostly Unaltered in the Absence of miR-21 (D) Statistical analysis of ﬂow cytometric results of DN1, DN2, DN3, DP, pre- and post-selection DP, SP4, and SP8 populations from GFP−EV, GFP−OE, GFP+ EV, GFP+ OE thymocyte subsets indicated as frequencies, inter alia shown in (C), n = 2-5. Analysis of signiﬁcance were performed using one-way analysis of variance followed by Tukey’s test. Indicated signiﬁcant results are representative of latter test. levels upon loss of miR-21 in DN2b thymocytes (Figure 6B) indicating a preference of miR-21 in promoting alternative lineage fate decisions. However, in vitro diﬀerentiation indicated that physiological levels of miR-21 are suﬃcient to maintain adequate levels of Bcl11b. Thus, we concluded that loss of miR-21 time and observed no detectable changes compared to WT on OP9-DL1 (Figure 6A) or on OP9-GFP cells (Figure S3). Given the critical role of the transcription factor Bcl11b at this stage, we assessed its relative abundance in DN2a and DN2b of miR- 21-suﬃcient and deﬁcient mice. We identiﬁed elevated Bcl11b November 2018 \| Volume 9 \| Article 2497 Frontiers in Immunology \| www.frontiersin.org 7 miR-21 in T-Cell Development Kunze-Schumacher et al. FIGURE 6 \| T-lineage and alternative lineage fate decisions are mostly unaltered in the absence of miR-21. (A) Sorted DN2a and DN2b cells were cultured on OP9-DL1 cells for up to 15 days. Generation of T, NK, and B cells was assessed by ﬂow cytometry at indicated periods of time. Pooled data of two independent experiment. Each dot represents one mouse, n = 4-7 per group. (B) Analysis of expression of Bcl11b in sorted DN2a and b thymocytes of WT or miR-21−/−mice by quantitative RT-PCR. Expression levels were normalized to Hprt. Each dot represents one mouse, n = 4. FIGURE 6 \| T-lineage and alternative lineage fate decisions are mostly unaltered in the absence of miR-21. (A) Sorted DN2a and DN2b cells were cultured on OP9-DL1 cells for up to 15 days. Generation of T, NK, and B cells was assessed by ﬂow cytometry at indicated periods of time. Pooled data of two independent experiment. Each dot represents one mouse, n = 4-7 per group. (B) Analysis of expression of Bcl11b in sorted DN2a and b thymocytes of WT or miR-21−/−mice by quantitative RT-PCR. Expression levels were normalized to Hprt. Each dot represents one mouse, n = 4. does not result in detectable changes regarding lineage potential of DN2 thymocytes. the expression of CD25 and CD44 over time. Loss of miR-21 Does Not Affect Endogenous T-Cell Regeneration g g Having deﬁned the dispensable role of miR-21 in steady-state thymus and periphery, we next addressed whether miR-21 is contributing to stress regulated responses. We adopted an approach from Dudakov et al. (38) using sublethal total body irradiation to induce thymic stress and subsequent endogenous regeneration (Figures 7A-C). We assessed frequencies of DN and DP thymocytes every third to fourth day. As controls, we used non-irradiated mice as well as irradiated mice (both WT and miR-21−/−), which were immediately sacriﬁced upon irradiation. At day 3/4 post irradiation, we detected nearly complete absence of DP thymocytes in WT and miR-21−/− thymi, whereas frequencies of SP4 and SP8 cells were highest at this time point. This pattern was inversed at day 7/8. We observed profound changes in levels of DN thymocytes as indicated by T-Lineage and Alternative Lineage Fate Decisions Are Mostly Unaltered in the Absence of miR-21 These changes were largely dependent on the loss of DP cells. In light of this, frequencies of DN3 thymocytes reached highest levels 7 days post irradiation in WT and miR-21-deﬁcient mice, but after 14 days, frequencies declined again. We did not observe any statistically signiﬁcant diﬀerences between WT and miR- 21-deﬁcient mice. Thus, we concluded that miR-21 does not contribute to endogenous regeneration of the thymus after sublethal irradiation. Frontiers in Immunology \| www.frontiersin.org Flow Cytometry and Cell Sorting Flow Cytometry and Cell Sorting Monoclonal antibodies speciﬁc for B220 (RA3-6B2), CCR7 (4B12), CD4 (GK1.5), CD5 (53-7.3), CD8α (53-6.7), CD24 (M1/69), CD25 (PC61.5), CD28 (E18), CD44 (IM7), CD45.1 (A20), CD45.2 (104), CD62L (MEL-14), CD69 (H1.2F3), CD117 (2B8), CD127 (SB/199), CD135 (A2F10), Foxp3 (MF23), NK1.1 (PK136), TCRβ (H57-597), TCRγδ (GL3), Sca-1 (D7) were used as AmCyan, Brilliant Violet 510 (BV510), BV421, Paciﬁc Blue (PB), eFluor450, ﬂuorescein isothiocyanate (FITC), Alexa488, Alexa647, phycoerythrin (PE), peridinin chlorophyll protein-Cy5.5 (PerCP-Cy5.5), PE-Cy7, APC, APC-Cy7 and were purchased from eBioscience, BD Biosciences, or Biolegend. Allophycocyanin (APC)-conjugated CD1d/PBS-57 (αGalCer analog) loaded and unloaded tetramers were provided by the NIH Tetramer Core Facility. Cells were acquired using a BD FACSCanto II and data was analyzed using FlowJo software (Tree Star). Lin−cells were isolated from total BM by staining cell suspensions obtained from the femur and tibia with a lineage- speciﬁc antibody cocktail followed by magnetic bead depletion using the Lineage Cell Depletion Kit as per manufacturer’s instructions (Miltenyi Biotec). DN thymocytes were isolated from total thymocytes by staining cell suspensions with anti-CD4 (RL1.72) and anti-CD8 (M31), followed by lysis with Low Tox- M Rabbit Complement (Cedarlane Laboratories) and subsequent density gradient centrifugation with Lympholyte-M (Cedarlane Laboratories). Cells were sorted using a FACS Aria II cell sorter. Based on this assumption, we assessed a potential role of miR- 21 in a model of thymic regeneration. Endogenous regeneration is critical for restoration of immune competence after stress or pre-conditioning for hematopoietic stem cell transplantation (44). However, also in this model miR-21 turned out to be completely expendable. What might be the reason underlying the mild or absent phenotype in steady-state miR-21-deﬁcient mice? Notably, the most prominent functions of miR-21 have been discovered using pharmacological inhibition of miR-21 using antagonists (40, 41). In contrast, constitutive loss of miR-21 appears to be only functional in the context of malignancy (42, 43, 45). This discrepancy suggests that in the unperturbed organism, constitutive loss of miR-21 might be compensated for by a yet to be identiﬁed mechanism (46). Recently, it has been demonstrated that, in contrast to tumor cell lines, virtually no de-repression of miR-21 targets can be observed in healthy mouse liver from miR-21-deﬁcient mice or upon treatment with miR-21 inhibitors (47). This lack of target recognition by miR-21 was not due to speciﬁc characteristics of the seed region, but correlated with lack of association with polysomes, when compared to other miRNAs. DISCUSSION We conﬁrmed miRNA expression proﬁling showing that miR- 21 is prominently expressed in thymocytes and is subject to substantial changes in expression levels during T-cell development (15–17). Hence, we hypothesized that deﬁned expression levels of miR-21 were required to sustain physiologic T-cell development. We tested this hypothesis using a variety of approaches, including analysis of miR-21-deﬁcient mice November 2018 \| Volume 9 \| Article 2497 Frontiers in Immunology \| www.frontiersin.org 8 miR-21 in T-Cell Development Kunze-Schumacher et al. Loss of miR-21 does not affect endogenous T-cell regeneration. (A) Representative ﬂow cytometric analysis of thymi from WT and miR-21−/−mice antibodies against CD25 and CD44. Numbers in right bottom quadrants represent frequencies of DN3 thymocytes. Regeneration of DN thymocytes was indicated periods of time in non-irradiated mice or post sublethal irradiation. Pooled data of two independent experiments. (B) Representative ﬂow analysis of thymi from WT and miR-21−/−mice stained with antibodies against CD4 and CD8α. Numbers adjacent to right gate represent frequencies of ytes. Regeneration of DP, SP4, and SP8 thymocytes was assessed at indicated periods of time in non-irradiated mice or post sublethal irradiation. Pooled independent experiments. (C) Statistical analysis of ﬂow cytometric results shown in (A,B). Each dot represents one mouse, n = 4-5 per genotype. nalysis were performed using unpaired Mann-Whitney test. FIGURE 7 \| Loss of miR-21 does not affect endogenous T-cell regeneration. (A) Representative ﬂow cytometric analysis of thymi from WT and miR-21−/−mice stained with antibodies against CD25 and CD44. Numbers in right bottom quadrants represent frequencies of DN3 thymocytes. Regeneration of DN thymocytes was assessed at indicated periods of time in non-irradiated mice or post sublethal irradiation. Pooled data of two independent experiments. (B) Representative ﬂow cytometric analysis of thymi from WT and miR-21−/−mice stained with antibodies against CD4 and CD8α. Numbers adjacent to right gate represent frequencies of DP thymocytes. Regeneration of DP, SP4, and SP8 thymocytes was assessed at indicated periods of time in non-irradiated mice or post sublethal irradiation. Pooled data of two independent experiments. (C) Statistical analysis of ﬂow cytometric results shown in (A,B). Each dot represents one mouse, n = 4-5 per genotype. Statistical analysis were performed using unpaired Mann-Whitney test. November 2018 \| Volume 9 \| Article 2497 Frontiers in Immunology \| www.frontiersin.org 9 miR-21 in T-Cell Development Kunze-Schumacher et al. at steady state, competitive BM chimeras in the contexts of miR-21 deﬁciency and over-expression, as well as in vitro diﬀerentiation. DISCUSSION When comparing miR-21-deﬁcient and suﬃcient mice at steady state, we observed some statistically signiﬁcant diﬀerences in population sizes for DN2 cells as well as Treg cells, reduced expression levels of CD5, a surrogate marker for TCR signal strength and elevated levels of Bcl11b in DN2b cells. However, throughout the magnitude of these diﬀerences were minor and could not be validated in more functional assays, such as anti-CD3-mediated induction of negative selection or competitive BM chimeras. Together, we concluded that miR-21 is largely dispensable for intrathymic T-cell development. to explain our ﬁnding that, despite high levels of expression and dynamic changes throughout development, miR-21 does not contribute to physiologic T-cell development and thymic regeneration. Nevertheless, the underlying molecular mechanism that selectively prevents miR-21 to actively target actively translated mRNA remains unclear. So far, expression of miR-21 has almost exclusively been associated with pathology. However, ultimately, it appears unlikely that evolution should conserve an exclusively pathogenic molecule, so that the quest to identify the physiologic function of miR-21 remains ongoing. Mice miR-21 knockout mice (B6;129S6-Mir21atm1Yoli/J; termed miR- 21−/−mice throughout this article) were purchased from The Jackson Laboratory (Bar Harbor, ME). C57BL/6N mice (CD45.2) were obtained from Charles River Laboratories, Germany. B6.SJL-PtprcaPepcb/BoyJ mice (CD45.1) and C57BL/6N x PtprcaPepcb/BoyJ F1 mice (CD45.1/CD45.2 heterozygous) were bred at the ZFE, Goethe University Frankfurt. All mice were used between 4 and 20 weeks of age and maintained under speciﬁc pathogen-free conditions. g g It has been suggested that miR-21 plays a role in multiple pathological conditions. Thus, treatment of mice with miR- 21 antagonists was able to reduce pressure induced cardiac failure and similar antagomiRs were protective in autoimmune encephalomyelitis, a mouse model of multiple sclerosis (40, 41). miR-21 is better characterized as an oncogene in a variety of malignancies. Overexpression of miR-21 in mice results in the formation of pre-B-cell lymphomas, which regress once expression of miR-21 is shut oﬀ(29). Furthermore, overexpression of miR-21 promoted non-small-cell lung cancer, whereas loss of miR-21 suppressed development of tumors (42, 43). Consistent with our ﬁnding that miR-21 is essentially dispensable for steady-state T-cell development, these studies suggest that the role of miR-21 appears to be largely restricted to cellular stress or pathological settings, most prominently cancer. MATERIALS AND METHODS Despite being prominently expressed in multiple organ systems, including immune system, liver and cardiovascular system (14), information on the physiologic role of miR-21 remains scarce. miR-21 has been implicated as negative regulator of T-cell activation, survival factor for memory cells as well as downstream eﬀector of PD-1 signaling (18–20, 39). Frontiers in Immunology \| www.frontiersin.org Ethics Statement All animal experiments were performed in accordance with local and institutional guidelines and have been approved by the Regierungspräsidium Darmstadt, Abteilung Veterinärwesen. Flow Cytometry and Cell Sorting Thus, in healthy cells, miR-21 is selectively absent from translationally very active mRNA. This ﬁnding helps November 2018 \| Volume 9 \| Article 2497 Frontiers in Immunology \| www.frontiersin.org 10 miR-21 in T-Cell Development Kunze-Schumacher et al. Competitive BM Chimeras In vivo T-Cell Receptor Stimulation Mice, WT or miR-21−/−, were injected with 200 µg puriﬁed anti-mouse CD3 (17A2, Biolegend). After 48 h, thymi were harvested, stained with monoclonal antibodies against lineage markers followed by Zombie Aqua TM (Biolegend) staining to exclude dead cells. Cells were ﬁxed using formaldehyde (4%) and permeabilized prior to caspase-3 staining (Asp175, CST) followed by staining with secondary donkey anti-rabbit-Alexa647 (Poly4064, Biolegend) and ﬂow cytometric analysis. For BrdU incorporation, mice were injected i.v. (2 mg/mouse) 3 h before analysis. Staining for BrdU was performed according to the manufacturer’s instructions using a FITC anti-BrdU antibody (Biolegend, clone: 3D4) after ﬁxation, permeabilization (BD Biosciences) and DNase-treatment of the cells (Sigma-Aldrich, 30 µg). Donor BM cells from WT (CD45.1, competitor), WT (CD45.2, test) and miR-21−/−(CD45.2, test) were prepared as described above. Recipient CD45.1/CD45.2 mice were lethally irradiated (9 Gy) and reconstituted intravenously with mixtures of competitor and test cells in a 1:1 ratio 4 h after irradiation (a total of 2 × 106 cells per mouse). Mice were treated with antibiotics in the drinking water for 2 weeks and analyzed after 8 weeks. Thymus Regeneration WT or miR-21−/−mice were sublethally irradiated (5,5 Gy) and thymi were harvested at days 0, 3, 7, and 14 post irradiation. Cells were stained with monoclonal antibodies against lineage markers (CD4, CD8, CD25, CD44, and CD117) followed by viability staining and were analyzed by ﬂow cytometry. titative Real-Time PCR (qRT-PCR) Quantitative Real-Time PCR (qRT-PCR) Total RNA from diﬀerent sorted mouse T cells was prepared using the miRNeasy kit (Qiagen) as recommended by the manufacturer. Expression of miR-21 was assessed by quantitative qRT-PCR using miRNA speciﬁc looped reverse transcriptase primers and TaqMan probes for hsa-miR-21 (Applied Biosystems, Taqman miRNA Assay ID 00397). Relative Expression was calculated as % expression of snoRNA412 as house-keeping genes using the 1 cycle threshold method (Applied Biosystems, ID 001243). Retroviral Transduction For production of miR-21-expressing virus particles, HEK293T cells were transfected with pCL-Eco (co-expressing gag, pol and env derived from murine leukema virus) and MDH1-PGK- GFP_2.0-miR-21 or the empty vector control for 8 h using a calcium phosphate transfection kit (Takara). Subsequently, medium was exchanged and supernatant containing retroviral particles was harvested 24, 48, and 72 h after transfection. For transduction of lineage-depleted BM, cells were cultured overnight in minimum essential medium (Gibco) containing 20% FCS (GE Healthcare) in the presence of mouse SCF (50 ng/mL), IL-7 (25 ng/mL), Flt3-L (25 ng/mL), and IL-6 (20 ng/mL) (all obtained from Peprotech) at a density of 1 × 106 cells/mL in a 24-well plate (Sarstedt). On day 2 and 3, cells were transduced with respective viruses supplemented with polybrene (8 µg/mL; Sigma-Aldrich) using spin infection (700 g, 1.5 h, room temperature) and subsequent incubation for 4 h in a 37◦C incubator before replacing with fresh medium containing cytokines. The following day, 5 × 105 cells were injected intravenously into recipients 4 h after lethal irradiation (9 Gy). Mice were given antibiotics in the drinking water for 2 weeks and analyzed after 8 weeks. Expression of Bcl11b was determined by reverse transcription using M-MLV Reverse Transcriptase (Promega) and random hexamer Oligo(dT)12-18 primers (Progmega) according to the manufacturer’s protocol. Quantitative RT-PCR analysis of Bcl11b expression was performed using the respective Bcl11b TaqMan probe (Applied Biosystems, Mm01332818_m1). Fold diﬀerences were calculated using the 1 cycle threshold method normalized to Hprt as housekeeping gene (Applied Biosystems, Mm00446968_m1). All reactions were performed as triplicates on a Quantstudio 3 (Applied Biosystems). Cell Preparations Flt3-L, and 1 ng/mL IL-7 for OP9-DL1 T cell diﬀerentiation and 5 ng/mL IL-7 for OP9-GFP cultures (all obtained from Peprotech). After 4 days, half of the culture medium was replaced with fresh medium and at day 7 of diﬀerentiation thymocytes were harvested and separated from contaminating OP9 cells by ﬁltering the cocultured cells through a 50 µm ﬁlter (Sysmex) prior to seed onto fresh OP9 monolayers. Lineage diﬀerentiation was monitored for up to 15 days. Discrimination of dead cells were performed using 7-amino-actinomycin D according to the manufacturer’s instructions. Thymus, spleen and inguinal lymph nodes were meshed through a 70 µm cell strainer (Corning) to obtain single-cell suspensions. For spleen, red blood cells (RBCs) were depleted using Qiagen RBC Lysis Solution according to manufacturer’s instructions. Construction of miRNA Expression Vectors Construction of miRNA Expression Vectors The retroviral construct MDH1-PGK-GFP_2.0-miR-21 was generated to achieve stable overexpression of miR-21. For this reason, the 273-bp miRNA gene segment containing the miR-21 hairpin was ampliﬁed and introduced via XhoI and EcoRI in the 3’ LTR under control of the human H1 promoter of the empty vector MDH1-PGK-GFP_2.0. The empty vector was a gift from Chang-Zheng Chen (Addgene plasmid # 11375) and served as a control. For both vectors, eGFP is encoded as a reporter gene under control of the PGK promoter and served to determine transduction eﬃciency. REFERENCES 10. Li QJ, Chau J, Ebert PJ, Sylvester G, Min H, Liu G, et al. miR-181a is an intrinsic modulator of T cell sensitivity and selection. Cell (2007) 129:147–61. doi: 10.1016/j.cell.2007.03.008 1. Kueh HY, Yui MA, Ng KK, Pease SS, Zhang JA, Damle SS, et al. Asynchronous combinatorial action of four regulatory factors activates Bcl11b for T cell commitment. Nat Immunol. (2016) 17:956–65. doi: 10.1038/ni.3514 11. Ebert PJ, Jiang S, Xie J, Li QJ, Davis MM. An endogenous positively selecting peptide enhances mature T cell responses and becomes an autoantigen in the absence of microRNA miR-181a. Nat Immunol. (2009) 10:1162–9. doi: 10.1038/ni.1797 2. Li L, Leid M, Rothenberg EV. An early T cell lineage commitment checkpoint dependent on the transcription factor Bcl11b. Science (2010) 329:89–93. doi: 10.1126/science.1188989 12. Zie¸tara N, Łyszkiewicz M, Witzlau K, Naumann R, Hurwitz R, Langemeier J, et al. Critical role for miR-181a/b-1 in agonist selection of invariant natural killer T cells. Proc Natl Acad Sci USA. (2013) 110:7407–12. doi: 10.1073/pnas.1221984110 3. Li L, Zhang JA, Dose M, Kueh HY, Mosadeghi R, Gounari F, et al. A far downstream enhancer for murine Bcl11b controls its T-cell speciﬁc expression. 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Dynamic regulation of miRNA expression in ordered stages of cellular development. Genes Dev. (2007) 21:578–89. doi: 10.1101/gad.1522907 6. Dooley J, Linterman MA, Liston A. MicroRNA regulation of T-cell development. Immunol Rev. (2013) 253:53–64. doi: 10.1111/imr.12049 7. Cobb BS, Nesterova TB, Thompson E, Hertweck A, O’Connor E, Godwin J, et al. T cell lineage choice and diﬀerentiation in the absence of the RNase III enzyme Dicer. J Exp Med. (2005) 201:1367–73. doi: 10.1084/jem.20050572 16. Statistical Analysis Single-cell suspensions of thymocytes from WT and miR-21−/− mice were incubated for up to 48 h cells in minimum essential medium (Gibco) containing 20% FCS (GE Healthcare). After 0, 24 and 48 h, cells were stained with monoclonal antibodies against CD4 and CD8 followed by Annexin V detection and staining with PI according to manufacturer’s kit instructions (Biolegend). All analysis was performed using GraphPad Prism software (version 7). Data are represented as mean plus or minus SEM and P < 0.05 was considered as signiﬁcant. In all ﬁgures, ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001; ∗∗∗∗P < 0.0001; ns, not signiﬁcant. Analysis of signiﬁcance between 2 groups of mice was performed using unpaired t-tests unless otherwise speciﬁed in the ﬁgure legends. For comparison between three or more groups, one-way analysis of variance followed by Tukey’s test was used. All analysis was performed using GraphPad Prism software (version 7). Data are represented as mean plus or minus SEM and P < 0.05 was considered as signiﬁcant. In all ﬁgures, ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001; ∗∗∗∗P < 0.0001; ns, not signiﬁcant. Analysis of signiﬁcance between 2 groups of mice was performed using unpaired t-tests unless otherwise speciﬁed in the ﬁgure legends. For comparison between three or more groups, one-way analysis of variance followed by Tukey’s test was used. SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/ﬁmmu. 2018.02497/full#supplementary-material ACKNOWLEDGMENTS This work was funded by grants from the German Research Foundation (DFG) KR2320/5-1 and SFB902-B15 (to AK). We are grateful to Dr. Tobias Schmid for helpful discussions and would like to acknowledge Praveen Mathoor for assistance in cell sorting. AUTHOR CONTRIBUTIONS y y Inﬂux of Ca2+ was performed as previously described (12) with minor modiﬁcations. Brieﬂy, 5 × 106 thymocytes from CD45.1 WT mice were mixed with an equal amount of thymocytes from miR-21−/−mice and incubated for 1 h in 1 mL Ca2+- and Mg2+-free Hanks’ PBS (HBSS, pH 7.4) (Sigma-Aldrich) at room temperature. Cells were washed and loaded with Fluo-4 (3 µM) and FuraRed (6 µM) dyes (both Invitrogen) and incubated at 37◦C in Ca2+- and Mg2+-suﬃcient HBSS (Gibco). After 45 min, cells were simultaneously stained for CD45.1, CD45.2, CD4, and CD8α and stimulated with 100 µg puriﬁed anti-mouse CD3 (17A2, Biolegend) for 30 min on ice. Subsequently, cells were rested at 37◦C for 30 min. For ﬂow cytometric analysis, a baseline for 30 s was recorded prior to addition of 12 µg goat anti-rat antibody (Jackson ImmunoResearch). Data acquisition was performed for additional 210 s. Finally, 2 µg ionomycin (Sigma-Aldrich) were added as positive control. For data analysis, median peak height resulting from anti-CD3-stimulation was determined. HK-S, SW, and EI performed experiments. HK-S and AK analyzed data. HK-S and AK designed experiments. HK-S and AK wrote the manuscript. All authors reviewed and corrected the manuscript. OP9 Cocultures OP9-DL1/OP9-GFP co-culture assays were essentially performed as described previously (48). Sorted precursors (DN2a and DN2b) were plated onto subconﬂuent OP9 BM stromal cells expressing either the Notch ligand Delta-like ligand 1 (OP9-DL1) or GFP (OP9-GFP) as a control in a 24-well plate. Cocultures were performed in the presence of 10 ng/mL SCF, 5 ng/mL November 2018 \| Volume 9 \| Article 2497 Frontiers in Immunology \| www.frontiersin.org 11 Kunze-Schumacher et al. miR-21 in T-Cell Development REFERENCES Kuchen S, Resch W, Yamane A, Kuo N, Li Z, Chakraborty T, et al. Regulation of microRNA expression and abundance during lymphopoiesis. Immunity (2010) 32:828–39. doi: 10.1016/j.immuni.2010.05.009 8. Regelin M, Blume J, Pommerencke J, Vakilzadeh R, Witzlau K, Łyszkiewicz M, et al. Responsiveness of developing T cells to IL-7 signals is sustained by miR-17∼92. J Immunol. (2015) 195:4832–40. doi: 10.4049/jimmunol.14 02248 17. Kirigin FF, Lindstedt K, Sellars M, Ciofani M, Low SL, Jones L, et al. Dynamic microRNA gene transcription and processing during T cell development. 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The microRNA miR-181 is a critical cellular metabolic rheostat essential for NKT cell ontogenesis and lymphocyte development and homeostasis. Immunity (2013) 38:984–97. doi: 10.1016/j.immuni.2013.02.021 37. Rothenberg EV. The chromatin landscape and transcription factors in T cell programming. Trends Immunol. (2014) 35:195–204. doi: 10.1016/j.it.2014.03.001 38. REFERENCES Dudakov JA, Hanash AM, Jenq RR, Young LF, Ghosh A, Singer NV, et al. Interleukin-22 drives endogenous thymic regeneration in mice. Science (2012) 336:91–5. doi: 10.1126/science.1218004 22. Blume J, Zur Lage S, Witzlau K, Georgiev H, Weiss S, Łyszkiewicz M, et al. Overexpression of Vα14Jα18 TCR promotes development of iNKT cells in the absence of miR-181a/b-1. Immunol Cell Biol. (2016) 94:741–6. doi: 10.1038/icb.2016.40 39. Carissimi C, Carucci N, Colombo T, Piconese S, Azzalin G, Cipolletta E, et al. miR-21 is a negative modulator of T-cell activation. 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Blood (2010) 115:1897–905. doi: 10.1182/blood-2009-08-237784 Conﬂict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Conﬂict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. 32. Germar K, Dose M, Konstantinou T, Zhang J, Wang H, Lobry C, et al. T-cell factor 1 is a gatekeeper for T-cell speciﬁcation in response to Notch signaling. Proc Natl Acad Sci USA. (2011) 108:20060–5. doi: 10.1073/pnas.1110230108 Copyright © 2018 Kunze-Schumacher, Winter, Imelmann and Krueger. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). Frontiers in Immunology \| www.frontiersin.org November 2018 \| Volume 9 \| Article 2497 REFERENCES The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. 33. Weber BN, Chi AW, Chavez A, Yashiro-Ohtani Y, Yang Q, Shestova O, et al. A critical role for TCF-1 in T-lineage speciﬁcation and diﬀerentiation. Nature (2011) 476:63–8. doi: 10.1038/nature10279 34. Baba J, Watanabe S, Saida Y, Tanaka T, Miyabayashi T, Koshio J, et al. Depletion of radio-resistant regulatory T cells enhances antitumor November 2018 \| Volume 9 \| Article 2497 Frontiers in Immunology \| www.frontiersin.org 13
https://openalex.org/W1994086629	https://www.scielo.br/j/reben/a/pLHDpGKMhbPjftGdTfsgqDk/?lang=pt&format=pdf	Portuguese	null	Atravessando períodos nebulosos: a experiência da família da criança portadora da Síndrome de Down	Revista Brasileira de Enfermagem	2,011	cc-by	7,520	RESUMO RESUMO RESUMO RESUMO RESUMO O objetivo deste estudo foi o de compreender como é para a família ter uma criança com Síndrome de Down entre seus membros, para que, a partir dessa compreensão, as enfermeiras possam propor e realizar um cuidado com ênfase nas respostas da família aos problemas atuais e potenciais vividos. Utilizaram-se o Interacionismo Simbólico e os passos iniciais da Teoria Fundamentada nos Dados. Oito famílias compuseram o estudo, e apresentaram-se os resultados referentes a um fenômeno: “Atravessando períodos nebulosos” e suas categorias: “Deparando-se com o inimaginável”, “Moldando-se à nova realidade” e “Vivendo desilusões”. Ao longo da experiência, a família enfrenta períodos de incertezas e dúvidas, demandando novas atitudes e confronto com o desconhecido. Os profissionais da saúde, especificamente da enfermagem, têm ficado à margem do trabalho com essas famílias. Impõe-se a necessidade de orientá-las e apoiá-las, particularmente no enfrentamento de períodos de dificuldades e dúvidas, com o objetivo de minimizá-los e até evitá-los. Descritores: Descritores: Descritores: Descritores: Descritores: Síndrome de Down; Enfermagem; Família; Criança. ABSTRACT ABSTRACT ABSTRACT ABSTRACT ABSTRACT The purpose of this study was to understand how the family faces the challlenge of having a child with Down Syndrome among its members, so that, from this understanding, nurses can propose and carry out a careful emphasis on family responses to current and potential problems experienced. Symbolic Interactionism and the initial steps of Grounded Theory were used. The study comprised eight families and the results of a phenomenon were presented: “Going though cloudy periods” and its categories: “Faced with the unimaginable”, “Adapting to the new reality” and “Living with disappointments”. Throughout the experience, families face periods of uncertainty and doubt, requiring new attitudes and coping with the unknown. Health professionals, specifically in nursing, have remained outside the work done with these families. The need to guide and support such families is vital, particularly in coping with periods of difficulties and doubts, in order to minimize and possibly avoid them. p y KKKKKey wor ey wor ey wor ey wor ey words: ds: ds: ds: ds: Down syndrome; Nursing; Family; Children. Michelle Darezzo Rodrigues Nunes. Av. Caramuru, 2100, Bloco 3, Apto 932. CEP: 14030-000. Ribeirão Preto, SP. E-mail: mid13@hotmail.com AAAAAUTOR CORRESPONDENTE UTOR CORRESPONDENTE UTOR CORRESPONDENTE UTOR CORRESPONDENTE UTOR CORRESPONDENTE * Recorte de dissertação de mestrado, financiada pela FAPESP, vinculado ao Programa de Pós-Graduação da Universidade Federal de São Carlos. Revista Brasileira de Enfermagem REBEn Revista Brasileira de Enfermagem REBEn PESQUISA PESQUISA PESQUISA PESQUISA PESQUISA PESQUISA PESQUISA PESQUISA PESQUISA PESQUISA Atravessando períodos nebulosos: a e Atravessando períodos nebulosos: a e Atravessando períodos nebulosos: a e Atravessando períodos nebulosos: a e Atravessando períodos nebulosos: a experiência xperiência xperiência xperiência xperiência da família da criança portadora da Síndr da família da criança portadora da Síndr da família da criança portadora da Síndr da família da criança portadora da Síndr da família da criança portadora da Síndrome de Down* ome de Down* ome de Down* ome de Down* ome de Down* Atravesando períodos nebulosos: la experiencia de la familia del niño con Síndrome de Down Going through cloudy periods: the experience of families of children with Down Syndrome Atravessando períodos nebulosos: a e Atravessando períodos nebulosos: a e Atravessando períodos nebulosos: a e Atravessando períodos nebulosos: a e Atravessando períodos nebulosos: a experiência xperiência xperiência xperiência xperiência da família da criança portadora da Síndr da família da criança portadora da Síndr da família da criança portadora da Síndr da família da criança portadora da Síndr da família da criança portadora da Síndrome de Down* ome de Down* ome de Down* ome de Down* ome de Down* Atravesando períodos nebulosos: la experiencia de la familia del niño con Síndrome de Down Going through cloudy periods: the experience of families of children with Down Syndrome Michelle Darezzo Rodrigues Nunes Michelle Darezzo Rodrigues Nunes Michelle Darezzo Rodrigues Nunes Michelle Darezzo Rodrigues Nunes Michelle Darezzo Rodrigues NunesIIIII, Giselle Dupas , Giselle Dupas , Giselle Dupas , Giselle Dupas , Giselle DupasIIIII, Lucila Castanheira Nascimento , Lucila Castanheira Nascimento , Lucila Castanheira Nascimento , Lucila Castanheira Nascimento , Lucila Castanheira Nascimento IUniversidade Federal de São Carlos. Departamento de Enfermagem. São Carlos, SP IIUniversidade de São Paulo. Escola de Enfermagem de Ribeirão Preto, SP INTRODUÇÃO INTRODUÇÃO INTRODUÇÃO INTRODUÇÃO INTRODUÇÃO A coleta de dados se deu, no período de agosto de 2008 a julho de 2009, por meio de entrevista semiestruturada a qual foi iniciada com a questão: “Como é para vocês ter uma criança com Síndrome de Down na família?” À medida que esta questão era explorada pelos participantes, outras foram formuladas. Frases como: “Como assim?” “Me fale mais sobre isto” foram utilizadas. Em todas as entrevistas, participaram pelo menos dois membros da família, em sua maioria, a mãe e o pai; outros membros, porém, também se fizeram presentes, sendo eles avó, irmã, prima em ter- ceiro grau e prima em primeiro grau, totalizando 21 familiares. Por ainda se encontrar em fase de crescimento e desenvolvi- mento, a criança necessita de estímulos, atenção, carinho, com- preensão e proteção(3). Os primeiros anos de vida de uma criança constituem um período crítico em seu desenvolvimento cognitivo, e o papel da família nesse período é fundamental, principalmente nos casos de crianças com necessidades especiais, relacionadas à aspectos cognitivos. Seu desenvolvimento não depende só do grau em que são afetadas intelectualmente, mas também de vários out- ros fatores, sendo o ambiente familiar o principal deles(4,5). Assim, capacidades cognitivas e motoras, desde que sejam treinadas e façam parte da vida social das crianças com SD, virão a se desen- volver, pois a inteligência se constrói, dependendo do contexto familiar, social e escolar de que a criança participa(6). Foi realizada uma entrevista com cada família, com exceção de uma delas, visitada duas vezes com o objetivo de aprofundar os dados colhidos anteriormente. Todas as entrevistas foram realiza- das no domicílio dos participantes com duração média de uma hora e quarenta minutos. Todas residiam no município de São Car- los, interior de São Paulo. Cada membro entrevistado assinou o Termo de Consentimento Livre e Esclarecido, sendo deixada uma cópia para eles, após a exposição do objetivo da pesquisa e es- clarecimentos de possíveis dúvidas. Nessa mesma ocasião, foram garantidos o anonimato e o sigilo das informações coletadas, e solicitava-se autorização para gravar a entrevista. A atuação dos profissionais de saúde junto a essas famílias es- clarecendo e incentivando a precocidade de estímulos adequados é de fundamental importância(4). Outros autores destacam a ne- cessidade de um trabalho de apoio e intervenção na família, o que poderá refletir na melhora do desenvolvimento cognitivo da cri- ança portadora de SD(5). INTRODUÇÃO INTRODUÇÃO INTRODUÇÃO INTRODUÇÃO INTRODUÇÃO como referencial teórico o Interacionismo Simbólico (IS) que apre- senta a particularidade do ser humano interagir, interpretar, definir e agir no seu cotidiano, de acordo com o significado que ele atribui à situação vivida(9). Como referencial metodológico de análise, op- tou-se pela Teoria Fundamentada nos Dados (TFD) que permite a geração de teorias a partir de dados obtidos e analisados de ma- neira sistemática e concomitante, por meio de uma comparação dos dados, de um ir e vir a eles, da coleta para a análise e vice- versa(10). Por envolver seres humanos, o projeto de pesquisa foi submetido à revisão ética e ao acompanhamento do Comitê de Ética da Universidade Federal de São Carlos (UFSCar) (Processo n° CAAE-0040.0.135.000-08), tendo sido aprovado. Nas últimas décadas, o Brasil passou por transformações de- mográficas profundas que levaram ao crescimento da situação de cronicidade no perfil de morbimortalidade da população(1). Atual- mente, as condições crônicas constituem problemas de saúde que requerem gerenciamento contínuo por um período de vários anos ou décadas e abarcam uma categoria extremamente vasta de agra- vos que, aparentemente, poderiam não ter nenhuma relação entre si. No entanto, doenças transmissíveis, não transmissíveis e inca- pacidades estruturais incluem-se nessa categoria. As condições crônicas apresentam como ponto em comum ser- em persistentes e necessitarem de certo nível de cuidados perma- nentes. Independente da causa, elas exigem mudanças no estilo de vida e gerenciamento da saúde por um período de tempo(2). As- sim, a síndrome de Down (SD) pode ser considerada uma condição crônica de saúde, já que apresenta as características pontuadas acima e requer um investimento familiar e da própria pessoa acometida, para gerenciar as situações cotidianas, demandando um tempo de dedicação diária e seguimento em longo prazo. Participaram da pesquisa oito famílias de crianças com SD, as quais foram convidadas por meio de um grupo de apoio de uma cidade do interior paulista. Esse grupo foi constituído por iniciativa das próprias famílias que possuíam crianças com SD. Suas ativi- dades eram coordenadas por uma enfermeira e realizadas nas de- pendências de uma universidade localizada no município onde a pesquisa foi desenvolvida. RESUMEN RESUMEN RESUMEN RESUMEN RESUMEN El objetivo de este estudio fue lo de comprender cómo es para la familia tener un niño con Síndrome de Down entre sus membros,para que, a partir de esa comprensión, las enfermeras puedan proponer y realizar un cuidado con énfasis en las respuestas de la familia a los problemas actuales y potenciales vividos. Se utilizó el Interacionismo Simbólico y pasos iniciales de la Teoría Fundamentada en los Datos. Ocho familias compusieron el estudio y se presentaron los resultados referentes a un fenómeno: “Atravesando períodos nebulosos”, y sus categorías: “Deparándose com el inimaginable” “Moldándose a la nueva realidad” y “Viviendo desilusiones”. A lo largo de la experiéncia, la família enfrenta períodos de inseguridades y dudas, demandando nuevas actitudes y confronto com el desconocido. Los profesionales de la salud y, especificamente de la enfermería, han quedado a las orillas del trabajo con esas famílias. Se impone la necesidad de orientarlas y apoyarlas, particularmente en el enfrentamiento de períodos de dificultades y dudas, con el objetivo de minimizarlos y hasta mismo evitarlos. Descriptores: Descriptores: Descriptores: Descriptores: Descriptores: Síndrome de Down; Enfermaje; Família; Niño. * Recorte de dissertação de mestrado, financiada pela FAPESP, vinculado ao Programa de Pós-Graduação da Universidade Federal de São Carlos. 227 227 227 227 227 Rev Bras Enferm, Brasília 2011 mar Rev Bras Enferm, Brasília 2011 mar Rev Bras Enferm, Brasília 2011 mar Rev Bras Enferm, Brasília 2011 mar Rev Bras Enferm, Brasília 2011 mar-abr; 64(2): 227-33. -abr; 64(2): 227-33. -abr; 64(2): 227-33. -abr; 64(2): 227-33. -abr; 64(2): 227-33. Nunes MDR, Dupas G, Nascimento LC. Nunes MDR, Dupas G, Nascimento LC. Nunes MDR, Dupas G, Nascimento LC. Nunes MDR, Dupas G, Nascimento LC. Nunes MDR, Dupas G, Nascimento LC. INTRODUÇÃO INTRODUÇÃO INTRODUÇÃO INTRODUÇÃO INTRODUÇÃO Tratando-se de famílias com crianças em situações crônicas, as enfermeiras precisam avaliar suas experiên- cias e propor intervenções específicas para aumentar a qualidade de vida, estreitar os mecanismos de enfrentamento, manter o bem- estar e promover a saúde dos membros da família(7). Enfermeiras podem ter um importante papel na promoção de resiliência e adaptação nas famílias de crianças com SD, se elas reconhecerem e valorizarem as capacidades naturais das famílias para suportar, sobreviver e prosperar, mesmo na presença de constantes desafios associados à criança com SD(8). As entrevistas foram transcritas, na íntegra, logo após sua reali- zação, para que não perdêssemos detalhes. As famílias foram iden- tificadas por números, conforme ordem de ingresso na pesquisa. A análise dos dados foi realizada concomitantemente à coleta, procedendo-se à análise comparativa constante, seguindo os pas- sos preconizados pelo referencial, que envolvem codificação aber- ta ou substantiva (conceitualização dos dados e categorização dos dados), codificação axial ou teórica e aplicação do modelo do para- digma(11). Esta metodologia trabalha com um processo constante construção dos dados que permite ao pesquisador parar em qualquer nível de análise dos dados e expor o encontrado. Assim, este estudo foi conduzido até a codificação axial, embora esta metodologia proponha a elaboração de um modelo teórico(12). Diante de tal cenário o objetivo deste estudo foi o de com- preender como é para a família ter uma criança com SD entre seus membros, para que, a partir dessa compreensão, as enfermeiras possam propor e realizar um cuidado com ênfase nas respostas da família aos problemas atuais e potenciais vividos. Para ilustrar a apresentação dos resultados, selecionamos al- guns trechos das falas dos participantes e utilizamos a seguinte padronização: os parênteses (...) indicam recortes dentro da mes- ma fala, e as informações contidas entre colchetes [ ] referem-se a Rev Bras Enferm, Brasília 2011 mar Rev Bras Enferm, Brasília 2011 mar Rev Bras Enferm, Brasília 2011 mar Rev Bras Enferm, Brasília 2011 mar Rev Bras Enferm, Brasília 2011 mar-abr; 64(2): 227-33. -abr; 64(2): 227-33. -abr; 64(2): 227-33. -abr; 64(2): 227-33. -abr; 64(2): 227-33. MÉTODO MÉTODO MÉTODO MÉTODO MÉTODO Tratou-se de pesquisa com abordagem qualitativa, que utilizou 228 228 228 228 228 Rev Bras Enferm, Brasília 2011 mar Rev Bras Enferm, Brasília 2011 mar Rev Bras Enferm, Brasília 2011 mar Rev Bras Enferm, Brasília 2011 mar Rev Bras Enferm, Brasília 2011 mar-abr; 64(2): 227-33. -abr; 64(2): 227-33. -abr; 64(2): 227-33. -abr; 64(2): 227-33. -abr; 64(2): 227-33. Atravessando períodos nebulosos: a e Atravessando períodos nebulosos: a e Atravessando períodos nebulosos: a e Atravessando períodos nebulosos: a e Atravessando períodos nebulosos: a experiência da família da criança portadora da Síndr xperiência da família da criança portadora da Síndr xperiência da família da criança portadora da Síndr xperiência da família da criança portadora da Síndr xperiência da família da criança portadora da Síndrome de Down ome de Down ome de Down ome de Down ome de Down observações importantes que expressam comportamentos não ver- bais dos participantes ou contextualizam as falas. a mediação de profissionais podem minimizar as consequências, mostrando as possibilidades positivas, e não somente os aspectos negativos. Quanto mais adequada for a revelação do diagnóstico, menor será a situação de desamparo enfrentada pelos pais(14). Com relação a este aspecto, é importante considerar que, embora nor- malmente caiba ao médico comunicar aos pais o diagnóstico, os profissionais de enfermagem são os que permanecem 24 horas por dia nos hospitais e, por isso, os que terão maior contato com a puérpera e seu bebê. Por esta razão, esse profissional tem papel importante junto aos membros de uma família que vive tal situa- ção, devendo, pois, estar preparado para cuidar da família após a notícia, estando perto, apoiando, ouvindo, tocando, permitindo o choro e a revolta. Categoria 1: Deparando Categoria 1: Deparando Categoria 1: Deparando Categoria 1: Deparando Categoria 1: Deparando-se com o inimaginável -se com o inimaginável -se com o inimaginável -se com o inimaginável -se com o inimaginável As famílias possuem formas diferentes de receber a notícia que a criança tem SD e de encarar a situação. Algumas descobrem durante a gestação, outras após o nascimento. Tomar conheci- mento da síndrome, ainda durante a gestação, pode ser visto de duas maneiras, uma positiva, possibilitando o preparo da família para a chegada da criança, e a outra de forma pessimista, acredi- tando que seria sofrer antecipadamente. Nas famílias em que a descoberta ocorre após o nascimento, também existe outro fator que as diferencia. Algumas notam as diferenças físicas da criança, principalmente nos olhos quando elas nascem, e outras permanecem não imaginando que ela tenha uma síndrome. Até a confirmação do diagnóstico, a maioria das famílias se apega à remota possibilidade de que a presença da síndrome pos- sa ser refutável, porém após essa confirmação, a família sofre um grande golpe. É ver um sonho transformar-se, é viver algo inespe- rado, diferente do planejado e desejado. É ter de mudar seus so- nhos, seus planos de acordo com a nova situação, ver a vida alterar seu curso, sem ter nenhum controle sobre ela, não ter escolha e não poder optar. É praticamente ter que se reestruturar para uma perspectiva completamente desconhecida. Apesar dos sentimentos desencadeados na descoberta, a família aceita a ideia de ter uma criança com SD, e pais e familiares abraçam a causa de ter um filho especial, dando-lhe todo o amor que são capazes de dar(13). RESUL RESUL RESUL RESUL RESULTTTTTADOS E DISCUSSÃO ADOS E DISCUSSÃO ADOS E DISCUSSÃO ADOS E DISCUSSÃO ADOS E DISCUSSÃO O processo de coleta e análise dos dados nos permitiu identifi- car dois fenômenos significativos para as famílias participantes da pesquisa. Neste artigo, apresentamos um deles, denominado “Atravessando períodos nebulosos”. Tal fenômeno resultou da cor- relação entre as categorias: “Deparando-se com o inimaginável”, “Moldando-se à nova realidade” e “Vivendo desilusões”. Na se- quência, apresentamos as categorias e o fenômeno que as inte- gram: É preciso ter sensibilidade e ética para reconhecer que cada família tem uma história de vida e necessidades únicas. Por esta razão, é importante que o profissional que dará a notícia esteja preparado para tal, pois ele deve estar imbuído dos sentimentos que a notícia vai desencadear e ter respeito e empatia com as pessoas que vão receber o diagnóstico. Nesse contexto, faz-se necessário tomar cuidado, até mesmo com o tom de voz, com a expressão, com o uso das palavras, com o local e o momento para dar a notícia(15). Informar correta e claramente os pais sobre a SD de forma humanizada, com respeito, sem iludir, tampouco omitir como provavelmente será o desenvolvimento da criança é primor- dial para os pais. O profissional precisa ter sensibilidade para per- ceber o que eles realmente necessitam, desejam e querem saber naquele momento, sem deixar de considerar que a família pode estar vivendo momentos de angústia, de ansiedade e até mesmo de não aceitação do bebê(4). Rev Bras Enferm, Brasília 2011 mar Rev Bras Enferm, Brasília 2011 mar Rev Bras Enferm, Brasília 2011 mar Rev Bras Enferm, Brasília 2011 mar Rev Bras Enferm, Brasília 2011 mar-abr; 64(2): 227-33. -abr; 64(2): 227-33. -abr; 64(2): 227-33. -abr; 64(2): 227-33. -abr; 64(2): 227-33. Categoria 2: Moldando Categoria 2: Moldando Categoria 2: Moldando Categoria 2: Moldando Categoria 2: Moldando-se à nova realidade -se à nova realidade -se à nova realidade -se à nova realidade -se à nova realidade A ajuda e “Depois que ele foi crescendo, quando ele começou a rastejar, 229 229 229 229 229 Nunes MDR, Dupas G, Nascimento LC. Nunes MDR, Dupas G, Nascimento LC. Nunes MDR, Dupas G, Nascimento LC. Nunes MDR, Dupas G, Nascimento LC. Nunes MDR, Dupas G, Nascimento LC. que ele não gatinhava, eu tive que aprender como fazer pra estimular ele também, porque só a fisioterapia uma vez por semana [interrompeu sua fala, na intenção de dizer que essa frequência não era suficiente]. Tinha que fazer em casa tam- bém. Tive que aprender isso também. Tudo, a gente vai apren- dendo pra ir ajudando ele”. Mãe 3 ficuldades, essas famílias cultivam a esperança de que tudo irá me- lhorar e se resolver. “A médica falou: ‘sua filha tem a síndrome de Down; ela não vai andar e não vai falar’. Ela podia ter falado: ‘existem casos que as crianças não andam e não falam, mas há crianças que se desenvolvem normal. Irmã 2 Após o nascimento de uma criança com SD, a família busca adaptar-se à nova realidade e reorganizar-se para enfrentar a ex- periência de viver e conviver com a síndrome(16). Um aspecto que interfere no modo de a família lidar com o problema é o conheci- mento que ela tem sobre a síndrome. “Ele não tem noção ainda do que ele está fazendo, ele tem sete anos e pra ele é uma criancinha ainda. Fralda até menos de 2, 3 semanas [atrás] ainda tinha que usar. Não sabe ainda segurar pra ir no banheiro ou pedir pra ir no banheiro; ele sente vonta- de, ele faz”. Pai 3 A atuação da família junto à criança com SD depende do co- nhecimento que ela tem sobre a síndrome e também sobre as pos- sibilidades de tratamento(6,17). À medida que recebem explicações e informações voltadas para o que realmente querem e necessitam saber, maior compreensão sobre a SD e de como agir diante dela vai sendo construída(4). “Eu vejo assim, toda ajuda, tudo que você ouve, que falam, é tão importante, e às vezes você tá aí há muito tempo e tem gente fazendo alguma coisa que você não sabe. Quer dizer se tivesse um programa do governo, uma coisa esclarecendo, fa- lando: ‘olha vocês vão conseguir isso aqui; nós estamos ofere- cendo isso, isso e isso. Olha, o Estado fornece isso e isso pra vocês’. Categoria 2: Moldando Categoria 2: Moldando Categoria 2: Moldando Categoria 2: Moldando Categoria 2: Moldando-se à nova realidade -se à nova realidade -se à nova realidade -se à nova realidade -se à nova realidade Mas não, você tem que sair sozinha descobrindo tudo, aqui e ali, juntando tudo e tentando fazer o melhor. Então real- mente falta”. Mãe 8 A experiência dos pais aponta para o fato de que muitas per- guntas deixam de ser feitas pelo choque da informação ou por não saberem nem ao menos do que se trata a síndrome, não tendo conhecimento nem sobre o que e como perguntar. Depois do primeiro impacto, quando começam a refletir sobre as informações recebidas, surgem vários questionamentos e, normalmente, os pais não têm com quem conversar sobre o que lhes causa maior sofri- mento(4). Este é um papel do qual o profissional de saúde não pode se ausentar. Ele deve atuar com o objetivo de fortalecer a família para os enfrentamentos. As famílias dependem, nesse mo- mento, de reconhecimento do problema do filho, de ações facilita- doras para compreender as situações vividas, e quanto mais es- clarecidas estiverem, melhor será seu empenho no cuidado do fi- lho(18). A falta de sistematização das informações para essas famílias está ilustrada na fala da Mãe 8 descrita acima, a qual, por exemplo, evidencia a disponibilidade de recursos governamentais contem- plados na rede de apoio, mas que, no seu caso, esses recursos estavam subutilizados pelo seu desconhecimento. Nessa mesma família, durante nosso encontro, a Mãe8 e seu companheiro abor- daram outra situação de desilusão vivida, como a do preconceito. Eles nos disseram: Os pais também percebem que muitos dos problemas e medos que tinham e ainda têm são decorrentes de informações tardias, erradas e incompletas(15). O profissional deve estar atualizado no que diz respeito à síndrome e deve conhecer os recursos de saúde que o município possui que possam fazer parte da rede de apoio à família, de forma a ter condições de fornecer informações sufi- cientes para aquela etapa da vida da criança, sem deixar de enfati- zar as possibilidades de desenvolvimento afetivo, social e cognitivo destas crianças, quando estimuladas adequadamente, e também de responder a todas as questões apresentadas pela família(6). “Preconceito (...) vem da época dos senhores feudais até hoje (Pai 8). (...) Dói mesmo, algumas coisas doem, porque tem pessoas que tem um preconceito enraizado, aquela coisa bem difícil (...) e é lógico que nós já passamos por essas situações”. Categoria 2: Moldando Categoria 2: Moldando Categoria 2: Moldando Categoria 2: Moldando Categoria 2: Moldando-se à nova realidade -se à nova realidade -se à nova realidade -se à nova realidade -se à nova realidade Mãe 8 Na perspectiva das mães participantes do estudo, o apoio emo- cional fornecido pelos pais também foi identificado como essencial para lidar com as demandas advindas da condição da criança. Nesse sentido, a carência desse apoio foi traduzida como uma das desi- lusões vividas. No que diz respeito ao apoio e à participação dos pais, as mães relatam que a ajuda no cuidado da criança é muito restrita(21). Mães que trabalham fora exercem duplo papel, o de cuidar do filho e da casa e do trabalho fora do lar, enquanto que os pais mantêm somente as atividades profissionais fora do ambiente doméstico, deixando, assim, de auxiliar as mães nas tarefas de casa(22). Categoria 2: Moldando Categoria 2: Moldando Categoria 2: Moldando Categoria 2: Moldando Categoria 2: Moldando-se à nova realidade -se à nova realidade -se à nova realidade -se à nova realidade -se à nova realidade A nova situação vivida pela família carece de uma adequação familiar. A primeira coisa que a família busca são respostas para suas dúvidas. A maioria delas não sabe o que é a SD, e o que essa nova condição acarretará para família. A busca por informações e esclarecimentos vai de encontro à necessidade de saberem o que fazer daqui para frente, tanto no início da experiência quanto no decorrer da evolução e desenvolvimento da criança, por meio de um constante aprendizado. É ter de se ajustar ao novo. Para a família, imprime-se a necessidade de eles se mobilizarem para su- prir lacunas de conhecimento, para conseguirem lidar com a nova situação, na medida em que também aprendem, no cotidiano, as demandas que surgem, ao cuidar da criança. “A gente deseja que não seja, que nem eu esperei sair o carióti- po. Ainda quando eu fui levar o exame, eu rezava que não fosse. Nesse período, você tem a esperança que não seja. É lógico, ninguém quer”. Mãe 2 “No primeiro momento, foi, acho que como é pra todo mundo, foi um choque, né? Primeiro filho, você idealiza de uma forma e daí você fica sabendo que ela tem síndrome de Down”. Pai 4 O momento da descoberta da síndrome também tem sido foco de destaque na literatura da área. Quando o diagnóstico da SD ocorre durante a gestação, é de se supor que os pais terão uma maior oportunidade de se preparar para a aceitação da condição da criança. Contudo, devem ser consideradas as consequências dessa revelação quando a criança ainda se encontra intraútero, por exemplo, a convivência com sentimentos de angústia e do descon- hecido até o nascimento da criança, momento em que os pais poderão saber com maior precisão o grau de comprometimento da criança. “(...) Foi difícil quando veio pra gente. Quando ela veio, era uma coisa que era novidade. A gente não sabia nem o que significava síndrome de Down. Eu falei: “mas o que que é isso? Qual é essa deficiência?”Depois da pediatra [referindo-se ao momento de revelação do diagnóstico pela pediatra], a gente mais do que depressa queria esclarecimento, né? Pra ver o que era”. Pai 2 A revelação desse diagnóstico normalmente desencadeia senti- mentos de choque, decepção e revolta, entre outros(4,13). Rev Bras Enferm, Brasília 2011 mar Rev Bras Enferm, Brasília 2011 mar Rev Bras Enferm, Brasília 2011 mar Rev Bras Enferm, Brasília 2011 mar Rev Bras Enferm, Brasília 2011 mar-abr; 64(2): 227-33. -abr; 64(2): 227-33. -abr; 64(2): 227-33. -abr; 64(2): 227-33. -abr; 64(2): 227-33. Categoria 3: Vivendo desilusões Categoria 3: Vivendo desilusões Categoria 3: Vivendo desilusões Categoria 3: Vivendo desilusões Categoria 3: Vivendo desilusões Ela precisa enfrentar o nevoeiro para chegar à claridade, passar pela tensão para alcançar a serenidade durante a experiência com a SD, que permeia toda a família, não só na criança, pois todos os mem- bros são afetados por ela. A literatura aponta que as mães consideram ideal ser informa- das entre o 5º e 30º dias após o nascimento do bebê(19), portanto, nunca no momento do parto(4). Sempre que for possível, é impor- tante dar a notícia quando os cônjuges estiverem juntos, pois os pais relatam sentimentos de forte angústia e sofrimento quando a notícia é dada separadamente e, com frequência, quem recebeu primeiro a notícia fica, por um tempo, sem coragem de contar ao outro(15). A família se mobiliza para buscar uma saída, uma luz, certeza, conhecimento, segurança para agir e tranquilidade para seguir em frente. Nesta trajetória, a família, muitas vezes, se sente cansada, desanimada, triste, decepcionada e abalada. Os familiares apre- sentam-se com um desgaste físico e emocional intensos, mas esse desgaste não é capaz de impedir a família de lutar, de seguir em frente para buscar e atingir suas metas de oferecer o melhor para a criança. Além disso, depoimentos de mães relatando como souberam do diagnóstico da SD revelam com muita frequência a postura negativa de alguns profissionais nesse momento(6,15), caracterizada pelo fato de não fornecerem as explicações necessárias, deixarem as pessoas angustiadas para o enfrentamento da nova situação, não saberem transmitir amparo e segurança, nem estarem prepara- dos, ou seja, não possuírem conhecimento que lhes permita ex- plicitar informações de forma adequada(15). É necessário que os profissionais de saúde estabeleçam relações empáticas e tratem as pessoas com sensibilidade e atenção. Os períodos nebulosos são aqueles de incerteza, de dúvida, os quais algo deu errado e não se sabe o que fazer, nem por onde começar ou recomeçar. Eles ocorrem não durante toda a trajetória, mas em alguns períodos dela, demandando atitudes novas, e con- frontando-se com o desconhecido. Também não ocorrem ao mes- mo tempo, mas alternadamente, dependendo da fase em que a criança e família vivem. Quando próximo do nascimento, envolve as incertezas e decepções da descoberta, de lidar com a criança que apresenta características desconhecidas, que não se sabe o que esperar da síndrome e que não se pode prever. Categoria 3: Vivendo desilusões Categoria 3: Vivendo desilusões Categoria 3: Vivendo desilusões Categoria 3: Vivendo desilusões Categoria 3: Vivendo desilusões No decorrer da experiência, os períodos nebulosos envolvem situações de precon- ceito ou de dificuldades relacionadas ao processo de desenvolvi- mento da criança. Assim a família vive períodos de altos e baixos, momentos bons e ruins e, apesar do desgaste e esforço, encara tudo com otimismo e confiança. A família não desiste, mantém-se firme. Tratando da descoberta de alterações de saúde associadas à SD, há relatos de mães que trazem o quão angustiante é ter, além da própria experiência da síndrome, outras doenças associadas a essa condição. A esse respeito, a literatura descreve a situação de uma mãe cuja filha com SD também recebeu o diagnóstico de uma alteração cardíaca, culminando com a necessidade de realização de cirurgia. O momento cirúrgico foi descrito como sendo o dia mais difícil de sua vida, por ter de se despedir da filha sem saber se a veria novamente. Adicionalmente a esse desafio, a família enfren- tou dificuldade de desenvolvimento da criança cuja investigação levou à descoberta do autismo(20). Autores apontam que a existência de uma condição crônica gera momentos difíceis, com avanços e retrocessos nas relações entre seus membros. O cuidado da criança consome energia, tempo, retira a privacidade da família e pode provocar isolamento social e emocional(23). Essa sobrecarga dos níveis social, psicológico, finan- ceiro e dos cuidados com a criança pode estar relacionada aos sentimentos de ansiedade e incerteza quanto à sua sobrevivência, ao seu desenvolvimento, ao cuidado em longo prazo e às próprias consequências desse cuidado sobre a vida pessoal da família(24). Deste modo, a família torna-se parte essencial do cuidado de en- fermagem, já que as enfermeiras frequentemente estão envolvidas com elas durante as fases de sofrimento e, assim, têm uma opor- tunidade única de fazer diferença em sua experiência(25). O enfer- meiro precisa direcionar suas intervenções junto à família, com o objetivo de melhorar sua qualidade de vida, fortalecer seus meca- nismos de enfrentamento, manter o bem-estar e promover a saúde de seus membros(26). Outro fato relevante é o sofrimento da família por medo de a criança ficar doente e ser vítima de preconceito(4). As famílias das crianças com SD, em todos os grupos etários, notam reações neg- ativas de amigos e vizinhos, o que faz com que se sintam piores. Reações negativas de estranhos são mais frequentes nas faixas etárias de 13 a 18 anos(21). Categoria 3: Vivendo desilusões Categoria 3: Vivendo desilusões Categoria 3: Vivendo desilusões Categoria 3: Vivendo desilusões Categoria 3: Vivendo desilusões Durante a experiência da família com a criança com SD, algu- mas dificuldades são encontradas pelo caminho. Entre elas, estão: as decepções com os profissionais de saúde, principalmente no despreparo para informar o diagnóstico e fornecer informações para a família; a frustração com o desenvolvimento da criança que não ocorre como o esperado; a descoberta de algumas alterações de saúde na criança; a experiência de enfrentar situações de pre- conceito; e a carência de apoio e rede social. Essas são algumas das situações que as famílias têm de enfrentar e superar para al- cançar seus objetivos; são circunstâncias que as desestabilizam e que, muitas vezes, as levam à necessidade de uma reestruturação fami-liar, uma reorganização das atividades e que as movimentam para novas buscas por apoio e orientação. Mesmo diante das di- Com relação à maneira como os profissionais de saúde infor- mam as famílias, estudos mostram o diagnóstico ou a suspeita sendo comunicados à mãe individualmente e logo após o nasci- mento da criança, o que desencadeia tensão, angústia e estresse(4). Se o diagnóstico só é feito após o nascimento, o momento de comunicá-lo aos pais deve levar em consideração suas necessidades e também as do bebê. É importante permitir que os pais desfrutem 230 230 230 230 230 Atravessando períodos nebulosos: a e Atravessando períodos nebulosos: a e Atravessando períodos nebulosos: a e Atravessando períodos nebulosos: a e Atravessando períodos nebulosos: a experiência da família da criança portadora da Síndr xperiência da família da criança portadora da Síndr xperiência da família da criança portadora da Síndr xperiência da família da criança portadora da Síndr xperiência da família da criança portadora da Síndrome de Down ome de Down ome de Down ome de Down ome de Down os primeiros contatos com o recém-nascido e tenham a opor- tunidade de iniciar a formação do vínculo afetivo, o qual não se dá apenas pelo nascimento. Este é um processo gradual e que neces- sita de tempo, e a amamentação é um importante aliado para que a mãe e o bebê iniciem este processo. ações preconceituosas. Essas situações adversas, qualquer que sejam elas, oferecem à família a sensação de momentos onde há grande nebulosidade, dúvida, insegurança, aflição e tensão. Categoria 3: Vivendo desilusões Categoria 3: Vivendo desilusões Categoria 3: Vivendo desilusões Categoria 3: Vivendo desilusões Categoria 3: Vivendo desilusões Os pais se esforçam para diminuir e até com- bater o preconceito imposto pela sociedade(13). FFFFFenômeno: enômeno: enômeno: enômeno: enômeno: Atravessando períodos nebulosos Atravessando períodos nebulosos Atravessando períodos nebulosos Atravessando períodos nebulosos Atravessando períodos nebulosos Esse fenômeno descreve os momentos de dificuldades pelos quais a família da criança com SD precisa passar. São períodos que ninguém sabe o que tem à frente, em que tudo está encoberto, misterioso e complicado. As primeiras manifestações do fenômeno ocorrem com a gestação, seguida pelo nascimento e o momento do diagnóstico da SD. Porém, ele volta a se manifestar quando surgem novas situações em que a família não se sente segura para agir, e outras dúvidas são adicionadas a esse cenário, exigindo es- forços familiares para a superação de cada fase. Rev Bras Enferm, Brasília 2011 mar Rev Bras Enferm, Brasília 2011 mar Rev Bras Enferm, Brasília 2011 mar Rev Bras Enferm, Brasília 2011 mar Rev Bras Enferm, Brasília 2011 mar-abr; 64(2): 227-33. -abr; 64(2): 227-33. -abr; 64(2): 227-33. -abr; 64(2): 227-33. -abr; 64(2): 227-33. REFERÊNCIAS REFERÊNCIAS REFERÊNCIAS REFERÊNCIAS REFERÊNCIAS 11. Strauss A, Corbin J. Basics of qualitative research: techniques and procedures for developing grounded theory. Thousand Oaks: Sage Publications; 1990. 1. Carvalhaes MABL, Moura ECD, Monteiro CA. Prevalência de fatores de risco para doenças crônicas: inquérito populacional mediante entrevistas telefônicas em Botucatu, São Paulo, 2004. Rev Bras de Epidem [periódico na internet] 2008 Mar [citado em 2009 Jul 21]; 11(1): [10 páginas]. Disponível em: http:// www.scielosp.org/ 12. Chenitz WC, Swanson JM. From practice to Grounded Theory: qualitative research in nursing. Reading: Addison-Wesley; 1986. 13. Ramos AF, Caetano JA, Soares E, Rolim KMC. A convivência da família com o portador de Síndrome de Down à luz da Teo- ria Humanística. Rev Bras Enferm 2006; 59(3): 262-8. 2. Organização Mundial de Saúde. Cuidados inovadores para condições crônicas: componentes estruturais de ação: relatório mundial. Brasília: OMS; 2003. 2. Organização Mundial de Saúde. Cuidados inovadores para condições crônicas: componentes estruturais de ação: relatório mundial. Brasília: OMS; 2003. 14. Silva NLP, Dessen MA. Crianças com Síndrome de Down e suas interações familiares. Psicol Reflex Crit 2003; 16(3): 503-14. 3. Barbosa ECV. Humanização nas relações com a família: um desafio para a enfermagem em UTI Pediátrica. Acta Sci Health Sci 2004; 26(1): 205-12. 15. Martins, DA. Cuidando do portador de síndrome de Down e seu significante. Ciênc Cuidado Saúde 2002; 1(1): 117-22. 4. Sunelaitis RC, Arruda DC, Marcom SS. A repercussão de um diagnóstico de síndrome de Down no cotidiano familiar: pers- pectiva da mãe. Acta Paul Enferm 2007; 20(3): 264-71. 16. Motta MGC. O ser docente no tríplice mundo da criança, família e hospital: uma descrição fenomenológica das mudanças exis- tenciais [tese]. Florianópolis: Universidade Federal de Santa Catarina; 1997. 5. Voivodic MAMA, Storer MRS. O desenvolvimento cognitivo das crianças com síndrome de Down à luz das relações fami- liares. Psicol Teor Prat 2002; 4(2): 31-40. 17. Souza MJ. Família-pessoa portadora de síndrome de Down na ótica da mãe: uma contribuição para a prática de cuidar na enfermagem [tese]. Rio de Janeiro: Escola de Enfermagem Anna Nery, Universidade Federal do Rio de Janeiro;1999. 6. Iervolino AS. Estudo das percepções, sentimentos e concepções para entender o luto de familiares de portadores da síndrome de Down da cidade de Sobral - Ceará [tese]. São Paulo: Facul- dade de Saúde Pública, Universidade de São Paulo; 2005. 18. Ramalhão AB, Dupas G. CONSIDERAÇÕES FINAIS CONSIDERAÇÕES FINAIS CONSIDERAÇÕES FINAIS CONSIDERAÇÕES FINAIS CONSIDERAÇÕES FINAIS A experiência das famílias com crianças com SD aponta ne- cessidades de cuidado e momentos ímpares de atuação da equipe de saúde. O fenômeno nos leva a refletir sobre importantes es- paços de ação da enfermagem na família, no qual o enfermeiro precisa estar atento. Evidenciam-se situações de dificuldade en- frentadas pela família que vão muito além do choque da descober- As dificuldades determinam frequentes enfrentamentos para a família, manifestados pela necessidade de lidar com a postura ina- dequada dos profissionais, com a falta de apoio do marido, com a imprevisibilidade do desenvolvimento do filho, a descoberta de al- gum problema de saúde na criança ou quando passam por situ- 231 231 231 231 231 Nunes MDR, Dupas G, Nascimento LC. Nunes MDR, Dupas G, Nascimento LC. Nunes MDR, Dupas G, Nascimento LC. Nunes MDR, Dupas G, Nascimento LC. Nunes MDR, Dupas G, Nascimento LC. ta. Entre elas, estão a falta de conhecimento e a desinformação da família, o despreparo dos profissionais de saúde em fornecer infor- mações precisas sobre a síndrome, a falta de apoio social e a sub- utilização das redes de apoio pelo seu desconhecimento, a descober- ta de alterações de saúde da criança, incrementando, ainda mais, as demandas da família, a falta de parâmetros relacionados aos marcos de desenvolvimento infantil de uma criança nessa condição e, também, nas dificuldades da criança alcançá-los e, por fim, a convivência com o preconceito. Essas são circunstâncias que ge- ram momentos de preocupação, ansiedade, dúvidas, tristeza e aflição e imprimem a necessidade de o enfermeiro estar sensível e receptivo para acolher a família, coletando dados e planejando um cuidado em parceria com os envolvidos. ta. Entre elas, estão a falta de conhecimento e a desinformação da família, o despreparo dos profissionais de saúde em fornecer infor- mações precisas sobre a síndrome, a falta de apoio social e a sub- utilização das redes de apoio pelo seu desconhecimento, a descober- ta de alterações de saúde da criança, incrementando, ainda mais, as demandas da família, a falta de parâmetros relacionados aos marcos de desenvolvimento infantil de uma criança nessa condição e, também, nas dificuldades da criança alcançá-los e, por fim, a convivência com o preconceito. CONSIDERAÇÕES FINAIS CONSIDERAÇÕES FINAIS CONSIDERAÇÕES FINAIS CONSIDERAÇÕES FINAIS CONSIDERAÇÕES FINAIS Essas são circunstâncias que ge- ram momentos de preocupação, ansiedade, dúvidas, tristeza e aflição e imprimem a necessidade de o enfermeiro estar sensível e receptivo para acolher a família, coletando dados e planejando um cuidado em parceria com os envolvidos. sionais da saúde e, particularmente, aqueles da enfermagem, têm ficado à margem do trabalho com essas famílias. Assim, impõe-se a necessidade de orientá-las de forma acurada; de oferecer acesso às redes de apoio disponíveis; de acionar o serviço social e outros afins, quando necessário; de apoiar e de estimular espaços de in- teração entre o casal, com o objetivo de auxiliar na distribuição dos papéis e demandas da família e agindo, em especial, no en- frentamento de períodos de dificuldades e dúvidas, com o objetivo de minimizá-los e até evitá-los. A finalização desta pesquisa permitiu, ainda, a identificação de lacunas do conhecimento nessa área no âmbito da enfermagem brasileira. Impõe-se a necessidade de maior aproximação dessa população e sugere-se que pesquisas futuras possam ser desen- volvidas, abordando questões identificadas neste estudo. Mere- cem ser explorados: a comunicação entre a família e a equipe de saúde; a interação dos membros da família, especificamente do casal; as estratégias de organização da família e redistribuição de papéis por ocasião do nascimento da criança com SD; o segui- mento da criança com SD na família; a família no manejo de situ- ações de preconceito, e a organização econômica da família no cotidiano do cuidar da criança com SD. A enfermagem deve proporcionar outros cenários de encontro com essas famílias, além do hospitalar, já que as necessidades da mesma extrapolam o momento do diagnóstico. É preciso buscá- los nos retornos aos atendimentos regulares a que a criança é sub- metida ou por meio de busca ativa na comunidade. Os “períodos nebulosos” enfrentados pela família mostram essas oportunidades de ação da enfermagem e a necessidade de criação de espaços de contatos privilegiados para intervenções junto às famílias. Os profis- Rev Bras Enferm, Brasília 2011 mar Rev Bras Enferm, Brasília 2011 mar Rev Bras Enferm, Brasília 2011 mar Rev Bras Enferm, Brasília 2011 mar Rev Bras Enferm, Brasília 2011 mar-abr; 64(2): 227-33. -abr; 64(2): 227-33. -abr; 64(2): 227-33. -abr; 64(2): 227-33. -abr; 64(2): 227-33. 24. Shapiro J, Blacher J, Lopez SR. Maternal reactions to children with mental retardation. In Burack JA, Hodapp RM, Zigler E, organizadores. Handbook of mental retardation and development. child with a disability. J Child Health Care 2003; 7(4): 231-47. 23. Elsen I, Marcon SS, Silva MRS, organizadores. O viver em família e sua interface com a saúde e a doença. 2a ed. Maringá: Eduem; 2004. 24. Shapiro J, Blacher J, Lopez SR. Maternal reactions to children with mental retardation. In Burack JA, Hodapp RM, Zigler E, organizadores. Handbook of mental retardation and development. REFERÊNCIAS REFERÊNCIAS REFERÊNCIAS REFERÊNCIAS REFERÊNCIAS Vivendo a ambivalência: o significado da visita para os pais de neonatos internados em Unidade de Tratamento Intensivo. Acta Paul Enferm 2003; 16(3): 41-50. 7. Paula ES, Nascimento LC, Rocha SMM. Roles assessment in families of children with chronic renal failure on peritoneal dial- ysis. Inter J Nurs Pract 2008; 14(3): 215-20. 19. Mustacchi Z. Síndrome de Down. In: Mustacchi Z, Peres S. Genética baseada em evidências: síndromes e heranças [CD ROM]. São Paulo: Centro Israelita de Assistência ao Menor; 2000. 8. Van Riper M. Families of children with Down syndrome: re- sponding to a “change in plans” with resilience. J Pediatr Nurs 2007; 22(2): 116-28. 20. Phillips L. Nia’s history continued. Learn Disabilty Pract 2007; 10(5): 28-30. 9. Charon JM. . . . . Symbolic interacionism: an introduction, an inter- pretacion, an integration. 3rd ed. Englewood Cliffs: Prentice Hall; 1989. 21. Sari HY, Baser G, Turan JM. Experiences of mothers of children with Down syndrome. Paediatr Nurs 2006; 18(4): 29-32. 10. Glaser BG, Strauss AL. The discovery of grounded theory: strategies for qualitative research. New York: Aldine de Gruyter; 1967. 22. Pelchat D, Lefebvre H, Perreault M. Differences and similari- ties between mothers’ and fathers’ experiences of parenting a 232 232 232 232 232 Rev Bras Enferm, Brasília 2011 mar Rev Bras Enferm, Brasília 2011 mar Rev Bras Enferm, Brasília 2011 mar Rev Bras Enferm, Brasília 2011 mar Rev Bras Enferm, Brasília 2011 mar-abr; 64(2): 227-33. -abr; 64(2): 227-33. -abr; 64(2): 227-33. -abr; 64(2): 227-33. -abr; 64(2): 227-33. Atravessando períodos nebulosos: a e Atravessando períodos nebulosos: a e Atravessando períodos nebulosos: a e Atravessando períodos nebulosos: a e Atravessando períodos nebulosos: a experiência da família da criança portadora da Síndr xperiência da família da criança portadora da Síndr xperiência da família da criança portadora da Síndr xperiência da família da criança portadora da Síndr xperiência da família da criança portadora da Síndrome de Down ome de Down ome de Down ome de Down ome de Down Atravessando períodos nebulosos: a e Atravessando períodos nebulosos: a e Atravessando períodos nebulosos: a e Atravessando períodos nebulosos: a e Atravessando períodos nebulosos: a experiência da família da criança portadora da Síndr xperiência da família da criança portadora da Síndr xperiência da família da criança portadora da Síndr xperiência da família da criança portadora da Síndr xperiência da família da criança portadora da Síndrome de Down ome de Down ome de Down ome de Down ome de Down child with a disability. J Child Health Care 2003; 7(4): 231-47. Cambridge: Cambridge University Press; 1998. p. 606-36 25. Bousso RS, Angelo M. Buscando preservar a integridade da unidade familiar: a família vivendo a experiência de ter um filho na UTI. Rev Esc Enferm USP 2001; 35(2): 172-9. 23. Elsen I, Marcon SS, Silva MRS, organizadores. O viver em família e sua interface com a saúde e a doença. 2a ed. Maringá: Eduem; 2004. 24. Shapiro J, Blacher J, Lopez SR. Maternal reactions to children with mental retardation. In Burack JA, Hodapp RM, Zigler E, organizadores. Handbook of mental retardation and development. 26. Paula ES, Nascimento LC, Rocha SMM. Religião e espiritua- lidade: experiência de famílias de crianças com Insuficiência Renal Crônica. Rev Bras Enferm 2009;62(1): 100-6. 233 233 233 233 233
https://openalex.org/W3038037949	https://www.mdpi.com/2077-0383/9/7/2046/pdf?version=1593928924	English	null	High Prevalence of Sarcopenia in Older Trauma Patients: A Pilot Study	Journal of clinical medicine	2,020	cc-by	6,182	Received: 8 June 2020; Accepted: 27 June 2020; Published: 29 June 2020 Abstract: Sarcopenia is related to adverse outcomes in various populations. However, little is known about the prevalence of sarcopenia in polytrauma patients. Identifying the number of patients at risk of adverse outcome will increase awareness to prevent further loss of muscle mass. We utilized data from a regional prospective trauma registry of all polytrauma patients presented between 2015 and 2019 at a single level-I trauma center. Subjects were screened for availability of computed tomography (CT)-abdomen and height in order to calculate skeletal mass index, which was used to estimate sarcopenia. Additional parameters regarding clinical outcome were assessed. Univariate analysis was performed to identify parameters related adverse outcome and, if identiﬁed, entered in a multivariate regression analysis. Prevalence of sarcopenia was 33.5% in the total population but was even higher in older age groups (range 60–79 years), reaching 82 % in patients over 80 years old. Sarcopenia was related to 30-day or in-hospital mortality (p = 0.032), as well as age (p < 0.0001), injury severity score (p = 0.026), and Charlson comorbidity index (p = 0.001). Log rank analysis identiﬁed sarcopenia as an independent predictor of 30-day mortality (p = 0.032). In conclusion, we observed a high prevalence of sarcopenia among polytrauma patients, further increasing in older patients. In addition, sarcopenia was identiﬁed as a predictor for 30-day mortality, underlining the clinical signiﬁcance of identiﬁcation of low muscle mass on a CT scan that is already routinely obtained in most trauma patients. Keywords: sarcopenia; polytrauma; prevalence; mortality; skeletal mass index; muscle mass J. Clin. Med. 2020, 9, 2046; doi:10.3390/jcm9072046 High Prevalence of Sarcopenia in Older Trauma Patients: A Pilot Study Robert C. Stassen, Kostan W. Reisinger, Moaath Al-Ali, Martijn Poeze, Jan A. Ten Bosch and Taco J. Blokhuis * Department of Traumatology, Maastricht University Medical Centre+, 6229HX Maastricht, The Netherlands; robert.stassen@mumc.nl (R.C.S.); k.reisinger@zuyderland.nl (K.W.R.); moaath.alali@mumc.nl (M.A.-A.); martijn.poeze@mumc.nl (M.P.); jan.ten.bosch@mumc.nl (J.A.T.B.) * Correspondence: taco.blokhuis@mumc.nl Department of Traumatology, Maastricht University Medical Centre+, 6229HX Maastricht, The Netherlands; robert.stassen@mumc.nl (R.C.S.); k.reisinger@zuyderland.nl (K.W.R.); moaath.alali@mumc.nl (M.A.-A.); martijn.poeze@mumc.nl (M.P.); jan.ten.bosch@mumc.nl (J.A.T.B.) * Correspondence: taco.blokhuis@mumc.nl Journal of Clinical Medicine Journal of Clinical Medicine Journal of Clinical Medicine Journal of Clinical Medicine www.mdpi.com/journal/jcm 2. Patients and Methods Approval for this study was acknowledged by the local ethics committee of the Maastricht University Medical Centre (MUMC+, Maastricht, the Netherlands). Requirement for informed consent was waived because of the retrospective nature of this study. Skeletal mass Index = Skeletal muscle mass (SMM)/height2 To estimate prevalence of sarcopenia in the study population, sarcopenia was deﬁned according to cutoﬀvalues for SMI as described by Prado et al. [18]. These values were determined at 52.4 cm2/m2 and 38.5 cm2/m2 for males and females, respectively. Two independent investigators measured the L3 muscle area of all patients and these data were used to calculate the interobserver agreement. Intra-observer agreement was assessed by repeating 50 L3-measurements 6 months after initial analysis. 1. Introduction Sarcopenia, a disease characterized by progressive loss of skeletal muscle mass (SMM), muscle strength, and physical performance, is a common condition among senior adults [1]. It is associated with a wide range of adverse outcomes and leads to a decreased quality of life and increased mortality [1–4]. The clinical relevance of sarcopenia has been extensively described in various disorders [5–8]. In addition, the predictive value of skeletal muscle mass measurements for complications has been demonstrated in various areas of surgery, such as general, vascular, colorectal, and liver transplant surgery [7,9,10]. With worldwide increased life expectancy and associated increased incidence in geriatric polytrauma patients, prevalence of sarcopenia in trauma patients is expected to increase as well [11,12]. To date, prevalence of sarcopenia in polytrauma patients is largely unknown. Evidence on adverse outcome related to sarcopenia in polytrauma patients is scarce and quality of available studies is limited [13–15]. One limiting factor is the heterogeneity in polytrauma patients, including all age groups, diﬀerent trauma mechanisms and a variety in pre-existing medical conditions. In addition, use of diﬀerent deﬁnitions for sarcopenia hampers the comparison of studies in trauma patients [1,16,17]. www.mdpi.com/journal/jcm www.mdpi.com/journal/jcm J. Clin. Med. 2020, 9, 2046 2 of 10 Nevertheless, on arrival of a trauma patient, a total-body computed tomography (CT) scan is obtained almost routinely, and CT-based measurement of skeletal muscle mass is the gold standard for quantiﬁcation of SMM. Therefore, the abdominal CT scan oﬀers the opportunity to identify patients with low muscle mass directly. Nevertheless, on arrival of a trauma patient, a total-body computed tomography (CT) scan is obtained almost routinely, and CT-based measurement of skeletal muscle mass is the gold standard for quantiﬁcation of SMM. Therefore, the abdominal CT scan oﬀers the opportunity to identify patients with low muscle mass directly. The aim of this pilot study was to investigate the prevalence of low muscle mass in older polytrauma patients, as well as to explore the relation between low muscle mass and mortality, complications, and inﬂammatory response. We hypothesize that a low skeletal muscle mass on abdominal CT, as an indicator of sarcopenia, is common in polytrauma patients of increased age and that it could aggravate their clinical outcome by increasing the risk of complications. In addition, we hypothesize that low muscle mass is a predictor of 30-day or in hospital mortality and that it induces an increased early inﬂammatory response during hospitalization. Skeletal mass Index = Skeletal muscle mass (SMM)/height2 (1) (1) 2.1. Patients and Determination of Muscle Mass Data from polytrauma patients (injury severity score (ISS) ≥16) who were admitted to a single level-I trauma center between January 2015 and December 2019 were assessed for study eligibility. Patient records were retrospectively checked for presence of an abdominal CT scan and patient height. Only when both abdominal CT scan on admission and height were available, patient data were included for analysis. Measurement of SMM was performed using abdominal CT images by two independent observers using OsiriX Lite 11.0.2 open software on transverse slides of abdominal CT scan at the level of third lumbar (L3) vertebra as described before [18,19]. All measurements were performed in a semi-automated fashion by setting tissue of interest threshold at −30 to +110 Hounsﬁeld Units (HU) for skeletal muscle [20]. Automatically generated areas of interest were corrected manually. Total muscle mass area was automatically calculated and displayed in square centimeters. y p y q Skeletal mass index (SMI), a derivative of the skeletal muscle mass (SMM) [21], was then calculated using the following equation: 3.1. Patients Data from 846 polytrauma patients were assessed for eligibility. In 428 patients, no abdominal CT-scan was available. Further exclusion was due to missing data regarding patient height (n = 179) and death within 24 hours due to brain injury (n = 1). Therefore, data from 239 polytrauma patients were included in analysis of prevalence and from 238 for complications, excluding the single patient who died of severe brain injury within 24 hours. Patient demographics are listed in Table 1. One-hundred ﬁfty nine of 239 (66.5%) patients were male, and 80 of 239 (33.5%) were female. Average age was 49 years (range 6–89, SD 21.45), with a non-signiﬁcant distribution among gender (48 (± 20) and 53 (± 24) years for males and females, respectively, p = 0.078). For the total population, the following means were observed: ISS 26.7 (± 9.9), body mass index (BMI) 25.0 kg/m2 (± 4.3), Charlson Comorbidity Index 1.7 (± 2.1). Patients were hospitalized for an average of 19 days (± 17). Mean L3 SMI for males and females was 57.4 cm2/m2 (± 10.24) and 42.7 cm2/m2 (± 7.82), respectively. Table 1. Patient characteristics. No. 2.2. Clinical Outcome Complications were retrieved from patient data by two observers. For this purpose, complications (pneumonia, urinary tract infection, delirium and mortality (both in hospital and 30-day)) and duration of intensive care unit (ICU) admission and hospitalization were all scored. Diagnosis of pneumonia was based on chest radiographs and antibiotic treatment [22]. Urinary tract infection was deﬁned as positive urinary culture and initiation of antibiotic therapy [23]. Delirium was diagnosed by a geriatrician in patients with altered mental status and if they received medical treatment [24]. In addition, inﬂammatory variables (leukocytes and C-reactive protein (CRP) on admission, after 24 and 48 h) were evaluated. Data from patients with severe head trauma who deceased within 24 h were included in the analysis of prevalence of sarcopenia. However, their data were excluded from analysis of complications. J. Clin. Med. 2020, 9, 2046 3 of 10 2.3. Statistical Analysis Frequencies are presented as absolute numbers and percentages. Continuous data is presented as mean (± standard error of the mean). Normal distribution was tested using Kolmogorov–Smirnov test. Diﬀerences between groups were analyzed using Pearson χ2 test for dichotomous variables. Conﬁdence intervals were calculated using logistic regression analysis. First, univariate analysis was performed to select parameters directly related to adverse outcome. Dependent variables that were identiﬁed in univariate analysis were subsequently entered into a multivariate logistic regression analysis. The inﬂuence of sarcopenia on 30-day mortality was determined using a log rank test. The interobserver agreement (R.S., M.A.) of L3 muscle index assessment of sarcopenia was analyzed by the Pearson correlation index. Two-tailed P values less than 0.05 were considered signiﬁcant. All statistical analyses were performed using SPSS (version 25.0; SPSS Inc. Chicago, IL, USA). 3.1. Patients of Patients (%) Mean (SD) Sarcopenic Non-Sarcopenic Signiﬁcance Gender Male 159 (66.5%) 51 108 Female 80 (33.5%) 30 50 Age Male 48 (± 20) 52 (± 21.9) 46 (± 18.7) p = 0.046 female 53 (± 24) 66 (± 22.7) 46 (± 21.0) p < 0.0001 >80 20 (8.3%) Male 84 (± 3) 5 1 Female 83 (± 2) 12 2 BMI <18.5 7 (2.9%) 4 (1.7%) 3 (5.1%) p = 0.174 18.5–24.9 120 (50.5%) 54 (22.7%) 66 (27.7%) p < 0.0001 25–29.9 88 (36.9%) 20 (8.4%) 68 (28.6%) p =0.006 >30 23 (9.7%) 2 (0.8%) 21 (8.8%) p = 0.005 Length of hospital stay (days) 19 (± 17) 17.6 (± 15.9) 20.1 (± 17.3) p = 0.29 Length of stay ICU (days) 6 (± 8) 5.4 (± 7.5) 6.2 (± 8.6) p = 0.48 Injury severity score 26.7 (± 9.9) 25.9 (± 9.3) 27.1 (± 10.2) p = 0.34 Charlson comorbidity index 0–1 145 (60.9%) 37 108 >2 94 (39.9%) 44 50 SMI Male <52.4 52 (21.8%) Female <38.5 30 (11.8%) Complications Pneumonia 43 (18.1%) 17 (20.9%) 26 (16.4%) Urinary tract infection 11 (5.5%) 2 (2.5%) 9 (5.6%) Delirium 51 (21.0%) 22 (27.2%) 29 (18.4%) Mortality within 1 month 18 (7.5%) 10 (12.3%) 8 (5.1%) p = 0.032 Mortality within 1 year 8 (3.4%) 4 (4.9%) 4 (2.5%) Mortality after 1 year 5 (2.1%) 3 (3.7%) 2 (1.3%) Number of patients requiring emergency (<24 h) surgery 108 (45.4%) 33 (41.3%) 75 (47.5%) Number of patients requiring ICU admission 181 (76.1%) 127 (80.4%) 54 (67.5%) BMI: body mass index; SD: standard deviation; ICU: intensive care unit; SMI: skeletal mass index. Table 1. Patient characteristics. J. Clin. Med. 2020, 9, 2046 p ICU admis 4 of 10 3.2. Prevalence 3.2. Prevale Prevalence of sarcopenia using the criteria as deﬁned by Prado et al. was 80 out of 239 patients (33.5%), of whom 52 (65%) were male and 28 out of 80 (35%) were female (see Table 2). Mean SMI in the sarcopenic group was 42.8 (± 6.9) while this was 57.5 (± 10.5) for the non-sarcopenic group. SMI in males was higher compared to females (57.4 cm2/m2 (± 10.2) vs 42.7 cm2/m2 (± 7.8), respectively). In the older cohort (> 80 years), prevalence increased to 85%, with an even distribution between males and females (83.3% and 85.7%, respectively). Patients deﬁned as sarcopenic were older than non-sarcopenic patients (57 years (± 22.9) and 46 years (± 19.5), respectively, p < 0.0001). ISS was comparable in sarcopenic (ISS = 25.9) and non-sarcopenic (ISS = 27.2) patients. The relation between age, ISS, and SMI is represented in Figure 1. Prevalence of sarcopenia using the criteria as defined by Prado et al. was 80 out of 239 patie (33.5%), of whom 52 (65%) were male and 28 out of 80 (35%) were female (see Table 2). Mean SM the sarcopenic group was 42.8 (± 6.9) while this was 57.5 (± 10.5) for the non-sarcopenic group. S in males was higher compared to females (57.4 cm²/m² (± 10.2) vs 42.7 cm²/m² (± 7.8), respective In the older cohort (> 80 years), prevalence increased to 85%, with an even distribution between ma and females (83.3% and 85.7%, respectively). Patients defined as sarcopenic were older than n sarcopenic patients (57 years (± 22.9) and 46 years (± 19.5), respectively, p < 0.0001). ISS w comparable in sarcopenic (ISS = 25.9) and non-sarcopenic (ISS = 27.2) patients. The relation betw age, ISS, and SMI is represented in Figure 1. Table 2. Sarcopenia prevalence in polytrauma population. General Population (%) Age 60–79 (%) Age ≥80 (%) Group Females 11.8 (n = 28/239) 13.6 (n = 9/66) 60 (n = 12/20) Males 21.8 (n = 52/239) 24.2 (n = 16/66) 25 (n = 5/20) Table 2. Sarcopenia prevalence in polytrauma population. General population (%) Age 60–79 (%) Age ≥ 80 (% Group Females 11.8 (n = 28/239) 13.6 (n = 9/66) 60 (n = 12/20 Males 21.8 (n = 52/239) 24.2 (n = 16/66) 25 (n = 5/20 Figure 1. Survival curves for sarcopenia and no sarcopenia. Table 2. Sarcopenia prevalence in polytrauma population. Table 2. 3.2. Prevalence 3.2. Prevale Sarcopenia prevalence in polytrauma popula Figure 1. Survival curves for sarcopenia and no sarcopenia. 3.4. Complications and Inﬂammatory Response The incidence of complications was 68 (45.3%) in non-sarcopenic patients and 41 (52.6%) in sarcopenic patients (p = 0.28). No signiﬁcant diﬀerences were observed between sarcopenic and non-sarcopenic cohorts regarding prevalence of pneumonia (p = 0.25), urinary tract infection (p = 0.34), delirium (p = 0.085) and ICU (p = 0.48) or hospital length of stay (p = 0.29). The inﬂammatory response in sarcopenic patients showed a signiﬁcant increase in leukocyte levels at 48 hours compared to non-sarcopenic patients (11.95 (± 3.64) vs 10.08 (± 3.04), respectively, p = 0.002). The other time points (admission and 24 hours after admission) showed no diﬀerence for leukocyte levels (p = 0.18 and p = 0.45, respectively). 3.5. Interobserver Agreement of CT Based Muscle Measurement by Osirix Interobserver agreement analysis of all measurements showed a strong and signiﬁcant correlation (R2 = 0.99; p < 0.0001). The interclass correlation coeﬃcient (ICC) of sarcopenia assessment by CT image analysis using Osirix was 0.99 (p < 0.0001) with a Cronbach alpha of 0.99. The interobserver coeﬃcient of variation (CV) was 10.1%. Intraobserver agreement analysis of 50 L3-measurements showed a signiﬁcant correlation (0.863; p < 0.0001) in the repeated measurements. 3.3. In Hospital Mortality 3.3. In Hospital Mortality 3.3. In Hospital Mortality Eighteen (7.6%) patients died within one month or during hospital admission. Log rank analy identified sarcopenia as an independent predictor of 30-day mortality (p = 0.032, Figure 1). Eighteen (7.6%) patients died within one month or during hospital admission. Log rank analysis identiﬁed sarcopenia as an independent predictor of 30-day mortality (p = 0.032, Figure 1). In univariate analysis, sarcopenia (p = 0.045) was identiﬁed as a predictor for 30-day or in-hospital mortality. In addition, age (p = 0.005), Charlson Comorbidity Index (p = 0.001), ISS (p = 0.026), surgical procedures (p = 0.038), and hospital length of stay (p = 0.004) were signiﬁcant predictors of mortality within one month. In the binary logistic regression analysis age (OR, 1.08; 95% CI 1.01–1.165; p = 0.018), ISS (OR, 1.19; 95% CI 1.04–1.20; p = 0.003) and hospital length of stay (OR, 0.83; 95% CI 0.74–0.92; p < 0.0001) remained as independent predictors of mortality within one month or during hospital admission. Logistic regression results are summarized in Table 3. 5 of 10 J. Clin. Med. 2020, 9, 2046 Table 3. Logistic regression analysis of mortality within one month. Table 3. Logistic regression analysis of mortality within one month. Univariate Analysis Multivariate Analysis Mortality Odds Ratio p-Value Odds Ratio p-Value Gender Male 9/150 1 Female 9/79 2.14 (0.82–5.6) 0.115 Age 1.05 (1.02–1.08) 0.005 BMI 0.93 (0.82-1.04) 0.21 Sarcopenia No 8/157 1 Yes 10/81 2.62 (0.99–6.93) 0.45 Charlson Comobidity Index 1.46 (1.20–1.77) 0.001 Injury Severity Score 1.05 (1.00–1.09) 0.026 1.19 (1.00–1.41) 0.05 Surgery during hospitalization No 1 Yes 0.36 (0.14–0.98) 0.38 Inﬂammatory parameters Plasma CRP at hospitalization 0.97 (0.87–1.07) 0.55 Plasma Leukocyte at hospitalization 1.01 (0.98–1.03) 0.59 Plasma CRP after 24 h 0.99 (0.99–1.01) 0.85 Plasma Leukocytes after 24 h 1.08 (0.95–1.24) 0.25 Plasma CRP after 48 h 0.99 (0.99–1.00) 0.19 Plasma leukocytes after 48 h 1.13 (0.94–1.36) 0.20 1.77 (1.06–2.96) 0.029 Complications Pneumonia No 14/195 1 Yes 4/43 1.33 (0.41–4.25) 0.63 Urinary tract infection No 18/227 Yes 0/11 0.33 Delirium No 11/187 1 Yes 7/51 2.55 (0.93–6.94) 0.06 ICU length of stay 0.99 (0.93–1.06) 0.84 Hospital length of stay 0.89 (0.82–9.63) 0.004 0.67 (0.50–0.89) 0.006 CRP: C-reactive protein. 4. Discussion The clinical signiﬁcance of sarcopenia, deﬁned in this study as a decreased skeletal muscle mass (SMM), is becoming evident in many ﬁelds of medicine. The depletion of muscle mass is a risk factor for infection during hospitalization in both non-cancer patients and cancer patients [9,25,26]. Furthermore, it has been revealed that sarcopenia in cancer patients is associated with treatment J. Clin. Med. 2020, 9, 2046 6 of 10 toxicity, poor functional status, increased length of hospital stay, prolonged rehabilitation care, and increased mortality (Huaiying 2019) [9,18,27–29]. The prevalence of sarcopenia in community-dwelling populations is up to 29%, ranging from 12% to 60% in patients with colorectal cancer [30,31]. However, despite the direct clinical relevance, the prevalence of sarcopenia in polytrauma patients is unclear. This study identiﬁed a prevalence of 34% in the overall polytrauma study group, increasing to more than 80% in polytrauma patients aged over 80 years. Furthermore, our data indicate a relationship between sarcopenia and 30-day or in-hospital mortality, regardless of gender, underlining the clinical signiﬁcance of sarcopenia in polytrauma patients. Our data are in line with other studies that have described the prevalence of sarcopenia in various populations [32–34]. In general populations, von Haehling and colleagues describe a prevalence of 5–13% in people aged 60–70 years, increasing to 11–50% in those aged 80 or above. These numbers are comparable to the prevalence of sarcopenia in populations suﬀering from obstructive pulmonary disease (COPD), renal failure and cancer [5,35,36]. The present study shows a higher prevalence of sarcopenia in polytrauma patients compared to other studies using the same age groups. Although the reason for this higher prevalence is beyond the scope of the current analysis, it raises questions on the causal relationship between sarcopenia and accidents. Sarcopenia is associated with an increased risk of falling [37], and in a part of the current population, a fall was registered as mechanism of trauma. Another aspect that has to be taken into account in interpretation of the high prevalence in our population is the applied deﬁnition of sarcopenia. In the present study, the criteria for skeletal muscle mass as described by Prado were used. Other deﬁnitions of sarcopenia use functional tests in addition to the quantiﬁcation of muscle mass, such as the EWGSOP-II deﬁnition that uses hand-grip strength and gait speed [1]. However, in the polytrauma population these parameters are not readily obtainable. 4. Discussion This limitation is mostly due to the extremity injuries frequently present in polytrauma patients, but also admission to the ICU and (prolonged) ventilation makes it impossible to obtain these data. Obtaining data during the admission is not straight forward, as loss of muscle mass and muscle function occurs within days during bedrest [38]. In contrast, a total-body CT scan is performed almost routinely upon arrival of a polytrauma patient, providing detailed imaging of the abdomen. The quantiﬁcation of skeletal muscle mass is therefore readily available, and our study indicates that this parameter is directly related to survival. The relation between sarcopenia and mortality, as indicated by many publications, appears in the univariate analysis of our data as well. In addition, a clear prediction of 30-day mortality was found. The identiﬁcation of sarcopenia in these analyses is in line with available literature, showing increased mortality within one year of diagnosing sarcopenia [39,40]. A recent study by Leeper et al. indicates sarcopenia to be a strong predictor of 6-month post-discharge mortality in elderly trauma patients [41]. However, in the multivariate analysis in our study, we were unable to maintain sarcopenia as an independent risk factor for 30-day or in-hospital mortality. This is likely due to the small sample size in our study, as well as the stronger correlation between mortality and other factors such as age and trauma severity. Notwithstanding this eﬀect, in the study by Leeper et al., an association is described between mortality and a variety of factors including ICU and hospital length of stay and injury scoring systems like Injury Severity Score and Abbreviated Injury Scale. Our data are in accordance with their ﬁndings. Obtaining an abdominal CT scan is becoming a more and more widespread routine in trauma patients [42]. Our data showed the same trend, as the percentage of patients excluded for missing CT data decreased per year. Surprisingly, data from routinely obtained trauma CT scans are only seldom used for muscle mass measurement in clinical practice. We believe that the addition of skeletal mass measurement analysis in trauma patients has a direct beneﬁcial eﬀect, since treatment of low muscle mass can be initiated immediately, and further loss of muscle mass could be prevented. One consideration in using only CT derived data is, however, the mentioned cutoﬀvalues for the L3 index, which are used to estimate sarcopenia. 4. Discussion As these values are based on obese patients with cancer, caution is advised when translating these values to other populations. Ideally, cutoﬀvalues for sarcopenia 7 of 10 J. Clin. Med. 2020, 9, 2046 should be established within each speciﬁc patient population and BMI category. A much larger sample size would be required to undertake cutoﬀvalues analyses by gender and by Body Mass Index (BMI). It is important to realize that there has been mounting evidence on the clinical importance of BMI in the ﬁeld of traumatology. An observational prospective study of Childs et al. shows an increased risk of infections, acute renal failure, length of ICU stay, hospital length of stay, and duration of mechanical ventilation in polytrauma patients with a BMI > 30 [43]. In addition, our analyses suggest diﬀerences in prevalence of sarcopenia between diﬀerent BMI ranges, with an increasing portion of sarcopenic trauma patients in higher BMI ranges. However, it is important to acknowledge that BMI does not distinguish between muscle and fat tissues. Increase in routinely obtained CT-scanning gives the opportunity to assess CT-based anthropometric parameters of fat in addition to muscle mass. A recent study of Poros et al. showed that CT-based assessment of abdominal fat is suitable in revealing pathologic body composition in trauma patients [44]. Abdominal fat measurements might add valuable information in relation to complications and mortality in trauma patients in future studies. p y p The current study was a pilot study, where a retrospective analysis of collected data was performed as a ﬁrst step to elucidate the clinical impact of sarcopenia in trauma patients. This study has therefore limitations that have to be kept in mind in interpretation of the results. One of the limitations of this study is its retrospective nature. Because only data from patients with an abdominal CT were included in our analysis, there is a potential inclusion bias. Still, all cases were retrieved from a regional prospective trauma registry, thereby limiting the eﬀect of potential selection bias. Another limitation is the heterogeneity of the study population. Traumatic events occur in any age group with diﬀerent grades of severity. In order to limit the eﬀect of heterogeneity, only patients with an ISS ≥16 were included, and age-speciﬁc analysis was obtained regarding the prevalence of sarcopenia and its complications. 4. Discussion Finally, only CT data were analyzed, and other markers of frailty such as functional and nutritional status were not included in our analysis. In future studies, functional tests should, if possible, be included in the criteria for sarcopenia. Moreover, the cut-oﬀvalues for skeletal mass index on CT measurements may require diﬀerent cut-oﬀ points based upon reported outcome in this speciﬁc population. 5. Conclusions The prevalence of sarcopenia in elderly polytrauma patients is high, reaching 85% in people over 80 years old. Our analysis also shows that sarcopenia is an independent predictor for 30-day mortality. Since abdominal CT scans are now almost routinely obtained in trauma patients, we advocate the measurement of skeletal mass measurement to detect decreased muscle mass early. Early identiﬁcation of people at risk for sarcopenia will lead to early diagnosis and therapy, and more awareness will help to prevent additional loss of muscle mass during the admission after trauma. Author Contributions: Conceptualization, R.C.S. and T.J.B.; data curation, M.A.-A.; formal analysis, R.C.S., M.P., and T.J.B.; methodology, K.W.R. and J.A.T.B.; supervision, K.W.R., M.P., J.A.T.B., and T.J.B.; writing—original draft, R.C.S., K.W.R., and M.A.-A.; writing—review and editing, M.P., T.J.B. and J.A.T.B. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Acknowledgments: We acknowledge support by the Open Access Publication Funds of the Göttingen Unive Acknowledgments: We acknowledge support by the Open Access Publication Funds of the Göttingen University. Conﬂicts of Interest: The authors declare no conﬂicts of interest. Acknowledgments: We acknowledge support by the Open Access Publication Funds of the Göttingen University. Conﬂicts of Interest: The authors declare no conﬂicts of interest. Conﬂicts of Interest: The authors declare no conﬂicts of interest. Conﬂicts of Interest: The authors declare no conﬂicts of interest. 2. Cruz-Jentoft, A.J.; Baeyens, J.P.; Bauer, J.M.; Boirie, Y.; Cederholm, T.; Landi, F.; Martin, F.C.; Michel, J.-P.; Rolland, Y.; Schneider, S.M.; et al. Sarcopenia: European consensus on deﬁnition and diagnosis: Report 1. Cruz-Jentoft, A.J.; Bahat, G.; Bauer, J.; Boirie, Y.; Bruyère, O.; Cederholm, T.; Cooper, C.; Landi, F.; Rolland, Y.; Sayer, A.A.; et al. 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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/). © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
https://openalex.org/W3116323164	https://revistaseletronicas.pucrs.br/index.php/civitas/article/download/30279/26373	English	null	An advocacy coalition analysis of the game RapeLay	Civitas - Revista de Ciências Sociais	2,020	cc-by	6,915	1 International Christian University (ICU), Tokyo, Japan. 2 The views expressed herein are those of the author. Japanese names follow the Japanese standard naming starting with the family name followed by the given name. Non-East-Asian names follow the traditional Western standard of given name followed by the family name. Dilton Rocha Ferraz Ribeiro1 orcid org/0000 0003 3243 2 orcid.org/0000-0003-3243-2062 diltonferraz@gmail.com Keywords: Virtual pornography. Lolicon. Japan. Pop culture. Received: 25 Jul. 2018 Accepted: 24 Oct. 2019 Published: 23 Dec. 2020 Resumo: Utilizando o modelo de coalizões de defesa busca-se analisar o complexo sistema relacionado a fantasias de estupro envolvendo imagens fic tícias de menores em jogos de videogame e computador. O foco desse artigo é especialmente no jogo chamado “RapeLay”. Há duas coalizões opostas sobre esse tema: a “coalizão de proteção infantil” e a “coalizão pro status quo”. Cada grupo busca ou mudar políticas públicas ou a manutenção da atual modelo ja ponês. Esse artigo analisa como essas coalisões são formadas e se são eficazes. Palavras-chave: Pornografia virtual. Lolicon. Japão. Cultura pop. Resumen: Utilizando el modelo de coalición de defensa, se busca analizar ese complejo sistema relacionado con las fantasías de violación que involucran imágenes ficticias de menores en videojuegos y juegos de computadora. El en foque de este artículo está especialmente en el juego llamado “RapeLay”. Hay dos coaliciones opuestas sobre este tema: la “coalición de protección infantil” y la “coalición de status quo”. Cada grupo busca o cambia de las políticas públicas o el mantenimiento del modelo japonés actual. Este artículo analiza cómo se forman estas coaliciones y si son efectivas. Palabras clave: Pornografía virtual. Lolicon. Japón. Cultura pop. Revista de Ciências Sociais Programa de Pós-Graduação em Ciências Sociais Civitas 20 (3): 454-463, set.-dez. 2020 e-ISSN: 1984-7289 ISSN-L: 1519-6089 Revista de Ciências Sociais Programa de Pós-Graduação em Ciências Sociais Civitas 20 (3): 454-463, set.-dez. 2020 e-ISSN: 1984-7289 ISSN-L: 1519-6089 1 International Christian University (ICU), Tokyo, Japan. An advocacy coalition analysis of the game RapeLay: the regulation of sexual violence and virtual pornography in Japan Uma análise do jogo RapeLa com base no modelo de coali ões de defesa a An advocacy coalition analysis of the game RapeLay: the regulation of sexual violence and virtual pornography in Japan Uma análise do jogo RapeLay com base no modelo de coalizões de defesa: a viabilidade da regulamentação da pornografia virtual no Japão Un análisis del juego RapeLay basado en el modelo de coaliciones de defensa: la viabilidad de la reglamentación de la pornografía virtual en Japón Abstract: The Advocacy Coalition Framework provides an efficient lens to analyze a complex subsystem concerning the controversial issue of fictitious depictions of children and rape fantasies in games, especially a game released in Japan called RapeLay. There are two opposing coalitions in Japan: the “children protection coalition” and the “pro status quo coalition”. Each group seeks to influence people to either change or to maintain the current non-criminalization of these products. This article analyzes how these coalitions are bound together and whether they are efficient. Received: 25 Jul. 2018 Accepted: 24 Oct. 2019 Published: 23 Dec. 2020 Introduction2 Since the 1980s, Japanese popular culture has continued to grow as a relevant global commodity and an increasing aspect of Japan’s soft power (Kawasaki 2007; Heng 2014). This world centered on fantasy and on cuteness has a wide variety of genres including pornography and violence. This pornographic subgenre of pop culture can be heavily focused on sexual violence and fictitious depictions of children in animation, comics Dilton Rocha Ferraz Ribeiro An advocacy coalition analysis of the game RapeLay 455 Dilton Rocha Ferraz Ribeiro An advocacy coalition analysis of the game RapeLay 455 and video games. As Japanese pop culture can be centered on a notion of cuteness, these fictitious characters are often drawn in ways to look cute and young. Debates about the desirability and existence of these materials have been polarizing with individuals placing them as expressions of free speech and sexuality, while others call for their criminalization and prohibition3 (Takeuchi 2015). this article reflect the author’s opinion but rather on how people seek to change certain policies. This paper is divided in four parts. The first briefly comments on the advocacy coalition framework. The second section seeks to give some general background on Japanese pop culture. The third part focuses on the arguments put forward by both coalitions acting in this subsystem and whether they are successful. The conclusion is that there are two large and opposite coalitions: one seeking to change Japan’s legislation and another one seeking to maintain the status quo. Both coalitions are stable and their members share deep policy core beliefs. Moreover, they have achieved different levels of success. The academic literature on these issues focuses on: analyzing cute culture (Kinsella 1998; Okano 2014), the relationship between cuteness and gender (Kinsella 1995; Allison 2006), media theory (Lamarre 2013); a psychological or anthropological analysis of otaku (Allison 2006; Hinton 2014), an economic analysis of otaku culture (Kam 2013), a philosophical study of otaku (Saito 2013; Azuma 2009), and legal and sociological issues concerning otaku and virtual pornography (Galbraith 2011; Shibata 2008; Kato 2008; Mathews 2011). This paper seeks to contribute to this literature with an analysis of the forces that drive or attempt to influence changes in the way Japan regulates animation, games and comics, especially concerning fictitious depictions of children. Introduction2 This research adds to the body of work by taking into account that individuals get together with others who share the same beliefs to influence local or global policies. In other words, individuals who appreciate Japan’s pop culture can act together to maintain the current system and those who dislike such materials also join forces to change policies to prohibit certain behaviors. In order to understand how these forces act and whether their proposals are successful, this research is based on the advocacy coalition framework. This methodology seeks to understand the actors within this subsystem, whether they share core policy beliefs, how they act, which arguments they support and how effective they are in bringing about policy change. The advantage of this analysis is that it is possible to visualize whether and how actors seek to change or maintain policies and whether or the degree to which they are successful. However, 3 Galbraith, Patrick W. 2011. “Lolicon: the reality of ‘virtual child pornography’ in Japan”. Image & Narrative 12 (1): 83-114. Accessed March 7, 2017. http://www.imageandnarrative.be/index.php/imagenarrative/article/viewFile/127/98. The advocacy coalition framework This paper relies on the Advocacy Coalition Framework to analyze the prohibition or the acceptability of sexually violent games in Japan focusing on the game RapeLay. Developed by Paul Sabatier, the Advocacy Coalition Framework is grounded on the view that interest groups gather in policy communities that are a part of a policy domain (Birkland 2011). This coalition presupposes a variety of actors that forms groups and coalitions of groups based on shared beliefs (Birkland 2011). Although members of a group might diverge over small questions, these are limited in scope, as the shared beliefs are what keep the group together (Vicente 2015). The coalition groups then compete seeking to translate their beliefs into public policies mobilizing political resources and through policy-oriented learning (Vicente 2015). The assumption part of the Advocacy Coalition Framework is that policy actors have three belief systems (Weible and Sabatier 2017). The first system, deep core beliefs, concerns “fundamental normative values and ontological axioms” (Weible and Sabatier 2017, 140). The second system, policy core belief, may, normatively, “reflect basic orientation and value priorities of the policy subsystem” and, empirically, “include overall assessments of the seriousness of the problem, Civitas 20 (3): 454-463, set.-dez. 2020 456 Otaku became a worldwide term commonly used to refer to avid fans of Japanese animation (anime), Japanese comics (manga), games, idols and Japanese popular culture in general (Azuma 2009). Not coincidentally, the term surfaced concomitantly with the boom of kawaii culture in the late 1970s and 1980s (Kinsella 1998). The current meaning of this word can be traced back to when Nakamori, in his column entitled “Otaku Research” (otaku no kenkyū), criticizes people who he grouped under an invented category of “otaku”4 (Galbraith 2015). Although his general statement was gender neutral, according to Galbraith (2015, 26), however, Nakamori particularly criticized “men sexually attracted to fictional girl characters”. Nakamori calls this sexual attraction a two-dimensional complex arguing that otaku cannot talk to women and focus on idol singers who do not display femininity. basic causes of the problem, and preferred solutions” (Weible and Sabatier 2017, 140-141). Finally, secondary beliefs are “a subset of the policy subsystem or the specific instrumental means for achieving the desired outcomes outlined in the policy core beliefs” (Weible and Sabatier 2017, 141). The advocacy coalition framework Outside of the core beliefs classifications, but nevertheless important to this framework is the notion of “devil shift”, which happens when “actors exaggerate the power and maliciousness of their opponents” (Weible and Sabatier 2017, 141). In the words of Weible and Sabatier (2017, 148), advocacy coalitions “are defined by actors who share policy core beliefs and who coordinate their actions […] to influence a policy subsystem”. The policy subsystem “is a primary unit of analysis” and “defined by a policy topic, territorial scope, and the actors or indirectly influencing policy system affairs” (Weible and Sabatier 2017, 139). The game RapeLay is analyzed taking into account different actors sharing core policy beliefs seeking to influence the regulation of sexually oriented games featuring fictitious children. Accordingly, it is necessary to briefly explain some terms central to the world of Japanese pop culture related to this discussion. Bishōjo or cute girl characters surfaced in late 1970s concomitantly with kawaii culture and soon became an increasing feature of many anime and manga (Galbraith 2015). The growth the number of people who produced works focusing on bishōjo characters and people buying these products led to writings on a “lolicon boom” (Galbraith 2015, 2011). Lolicon is a contraction for Lolita Complex, which can be used to refer to the desire for cute girls. In Japan, there is a word, moé, which relates specifically with the desire for such characters and works that might or intend to produce such effects and avoids imprecisions that the expression lolicon can encompass (Lamarre 2013). 4 Morikawa, Kaichirō. 2012. Otaku. Working words: new approaches to japanese studies. Center for Japanese Studies: UC Berkley. Acces sed November 9, 2017. http://eprints.cdlib.org/uc/item/5zb9r8cr. RapeLay Simulation games can also appeal to fans of moé characters using violence and sex. RapeLay (reipurei) provides an example of such games. RapeLay simulates rape, stalking and violence. The player incorporates an individual whose purpose in the game is to violate women. It was produced in 2006 and released only in the Japanese market. The title is formed with the amalgamation of the words “rape” and “play”. In the game, the player controls a guy who gropes and rapes three women of the same family. The game starts with the player using the control of the mouse to grope and undress one of these three women on a crowded platform or train (Galbraith 2017). It follows with the player forcing sexual intercourse with any of these three women. The game includes an “internal ejaculation counter” that informs the player when one of his victims are about to get pregnant” (Galbraith 2017, 1). In most bishōjo video games, girls are repre sented by anime style cartoons rather than by pictures of real girls. This allows the game developers to circumvent Japanese child por nography laws, which do not prohibit the simu lated representation of characters who appear to be under 18 years old [...] Nevertheless, it is common to find a disclaimer on the video game covers stating that all the characters are older than 18 (Gutiérrez 2014, 60). As the current interpretation of Japanese laws does not prohibit depictions of fictitious children in sexual media, including video game, inside and outside of Japan two main coalitions concerning the representation of children in media took shape. One, which I call “children protection coalition”, seeks to change Japan’s legislation and impose a crime on the production, sell, distribution and possession of depictions of children in pornographic animation, comics and video games. The other opposite coalition, which I call the “status quo coalition”, seeks to maintain the current legal framework excluding depictions of children as a crime. One of the main platforms of policy core beliefs happens in the academic and feminist contexts where advocates of both coalitions put forward arguments in favour or against any policy change in this subsystem. The player, and consequently, the violator, is a man. All his three victims are women, members of the same family. The game’s story seems to be equally straight forward and chilling. Kawaii and otaku Kawaii, or cute essentially means childlike. Kinsella (1995, 220) adds that kawaii culture celebrates “sweet, adorable, innocent, pure, simple, genuine, gentle, vulnerable, weak, and inexperienced social behaviour and physical appearances”. It is a style that denies maturity replacing it for innocence sometimes coupled with an effort to act stupid, dependent and weak (Kinsella 1995). Although of old origin, kawaii remerged from the 1970s and paved the way to a nationwide cuteness phenomenon that encompassed clothes, a variety of accessories, television shows, animations, comics and music (Okano 2014; Cheok and Fernando 2012). The kawaii culture is fundamentally centered on consumerism (Lamarre 2013). The fervent consumers of kawaii products are usually labelled otaku. Arguably, simulation games are an example of moé. These games can be explained as computer or video games focusing on romance or relationships, which might or not include erotic elements (Jones 2005). Although there are many subgenres of simulation games, it is possible to highlight three major subdivisions. First, BL ou boys’ love refers to games targeted towards a female market and focuses on romantic or erotic relationships between male characters (Taylor 2007). Otome games are the ones that the player is a female and the purpose of the game is to interact Dilton Rocha Ferraz Ribeiro An advocacy coalition analysis of the game RapeLay 457 Dilton Rocha Ferraz Ribeiro An advocacy coalition analysis of the game RapeLay 457 1000 to 2000 copies (Kagami, 2010). The list of gender-based violent games produced in the West did not stop in 1982. A number of games such as Custer’s Revenge, Slaves of the Red Mansion, Slap the Whore, Slap the Booty, and Sim Girl include scenes of rape and violence against women (Gutiérrez 2014). Perhaps, what is different and draws attention to games such as RapeLay is that the violence and sexuality is directed to virtual characters that look young and childlike. Gutiérrez (2014), for example, affirms that: with attractive male characters. The most popular are bishōjo games, that is, the ones that the player is a male who has to interact with different cute – kawaii – girl characters (Jones 2005). RapeLay The story focuses on a male who decides to take revenge on a girl who had previously reported him for an attempted rape. The male-player then decides to sexually abuse this adolescent girl, her 12-year- old sister, and her mother (Gutiérrez 2014). In order to advance in the game, the male-player has assure the characters do not get pregnant or get abortions to then turn them into sex slaves (Gutiérrez 2014). If not, one of the characters might stab the protagonist to death and the game ends. Commenting on RapeLay, Nakasatomi (2009) affirms the game depicts misogynistic attitudes and naturalizes violence against women and girls. In his view, games like RapeLay strengthens rape myths and instils the notion that women and underage girls can be sexually violated without legal consequences from these actions (Nakasatomi, 2009). In other words, RapeLay and Sexually violent video games are not only produced and sold in Japan. Arguably, the first raped-themed video game, Mystique Company, was produced in the United States in 1982 (Gutiérrez, 2014). The game sold 80,000 copies. For comparative purposes, RapeLay sold around Civitas 20 (3): 454-463, set.-dez. 2020 458 is focused. The model of control, dependence, submission and “adoration” that they reproduce is an explicit and “normalised” form of gen der-based violence which tends to construct a deeply sexist and patriarchal stereotype in this type of video game (Gutierrez 2014, 61). similar games would not only push individuals with rape tendencies to commit this crime but also lead people who are not rape-prone to accept rape myths and view the degradation of women and underage girls as a normal activity of male dominance. On a legal standpoint, the Act on Regulation and Punishment of Acts Relating to Child Prostitution and Child Pornography, and the Protection of Children is applicable to any image of a child depicting sexual intercourse or a conduct similar to sexual intercourse, or any image that the child is naked or partially naked, or any image that depicts a child touching another person genital organ.5 Nakasatomi (2009) thus argues that the lack of a criminalization of virtual depictions of child pornography leads to an endless availability of materials featuring the exploitation, abuse and torture of children in the virtual world. 5 Act on regulation and punishment of acts relating to child prostitution and child pornography, and the protection of children. Acces sed March 23, 2018. http://www.japaneselawtranslation.go.jp/law/detail/?ft=2&re=02&dn=1&yo=children&x=12&y=15&ia=03&ky=&page=4. 6 Omori, Sawa. 2014. Manga and anime: Japan still treating children as sexual objects. Al Jazeera. Agust 11, 2014. Accessed October 14, 2017. http://www.aljazeera.com/indepth/opinion/2014/08/manga-animejapan-still-treatin-201484145420634173.html. 7 Equality Now. 2009. Women’s action 33.1 Japan: rape-simulator games and the normalisation of sexual violence. Accessed November 9, 2017. http://www2.ohchr.org/english/bodies/cedaw/docs/ngos/Equality_Now_June2009_Japan_CEDAW44.pdf. 8 Women’s Action. 2009. Women’s Action 33.1 Japan. Accessed October 23, 2019. http://www2.ohchr.org/english/bodies/cedaw/ docs/ngos/Equality_Now_June2009_Japan_CEDAW44.pdf. 9 Equality Now. 2009.Women’s action 33.1 Japan: rape-simulator games and the normalisation of sexual violence. Accessed November 9, 2017. http://www2.ohchr.org/english/bodies/cedaw/docs/ngos/Equality_Now_June2009_Japan_CEDAW44.pdf. 10 Equality Now. 2009. Women’s action 33.1 Japan: rape-simulator games and the normalisation of sexual violence. Accessed November 9, 2017. http://www2.ohchr.org/english/bodies/cedaw/docs/ngos/Equality_Now_June2009_Japan_CEDAW44.pdf. 11 European Women’s Lobby (EWL). Rape is not a game. Accessed October 23, 2019. http://www.womenlobby.org/Rape-is-not-a-game. 12 Lah, Kyung. 2010. ‘Rapelay’ video game goes viral amid outrage. CNN, March 31, 2010. Accessed November 9, 2017. http://edition.cnn. com/2010/WORLD/asiapcf/03/30/japan.video.game.rape/index.htm. 13 Pidd, Helen. 2009. Amazon pulls ‘rape’ computer game. The Guardian, February 12, 2009. Accessed November 9, 2017. https://www. theguardian.com/technology/2009/feb/13/amazon-pulls-rape-computer-game. RapeLay Some members of the Komeito party, however, did not share the same core policy belief and joined the coalition as they sought the same policy outcome. These politicians, consequently, are not on a stable alliance as this is just a join of forces due to convenience. The United Nations, although not addressing RapeLay specifically, issued reports condemning Japan’s policy on the virtual representation of children in adult media. The Report of the United Nations’ Special Rapporteur on child trafficking and child prostitution from the United Nations15 states that virtual child abuse breaches international human rights norms, which prohibit any pornographic representation of a child, regardless of whether the child is real or virtual. This Report followed the reasoning that virtual depictions of children constitute harm and foster social tolerance towards exploitative acts and might contribute to the formation of a subculture of child abuse. The Rapporteur recommended Japan to further amend its legislation to be in tune with international human rights law. Some of the actors opposite “status quo coalition” also advocate their policy core belief on academic and feminist perspectives. Patrick Galbraith (2017, 9), for example, argues that “the RapeLay controversy demonstrates a confluence of anti-pornography advocacy and conservative agendas in Japan in the 2000s”. He stresses that a number of Japanese feminists support the production and commercialization of games such as RapeLay on the grounds of freedom of expression and the threat of government interference on people’s private lives deciding on what would constitute “good sex”, that is, heterosexual and reproductive sexual intercourse (Galbraith 2017, 9). Galbraith (2017) mentions feminists such as Fujimoto Yukari who asserts that manga and media in general are safe spaces to think about sex in an environment where girls are both producers and consumers of products containing depictions of sexual violence since the 1970s. In this view, rape fantasies do not reflect a desire to rape or be raped, as sexual fantasies in general are not always about representations of objects of desire in reality. There is a separation of desires for sexual violence and for fictional underage characters, that is, the image itself and desires for real children (Galbraith 2006). This coalition has, however, one outlier: some members of the Komeito Party. RapeLay The reasoning is that if the virtual depictions look like children and portray them in a sexual way, the material – in this case a video game – should be under the regulation of child pornography. Concluding that although there are no real children, virtual pornography can distort the user’s sexual desires, Nakasatomi calls for an amendment of Article 2.3 of the Child Prostitution and Child Pornography Act to include virtual depictions of children in pornography as part of child pornography. Connecting virtual child pornography with the abuse of women in general, Gutiérrez (2014) summarizes Nakasatomi view’s concerning RapeLay when he affirms that: The desire to change the current policy towards the virtual pornography in order to protect children is followed by other academics such as Shibata (2008), Takeuchi (2015), Matthews (2011).6 Moreover, some human rights organizations and certain media outlets share this policy core belief. The game RapeLay also draw attention the New York-based non-governmental organization Equality Now,7which started a campaign called “Women’s Action 33.1 Japan: Rape-simulator games and the normalisation of sexual violence”.8 This human rights organization called the producer of RapeLay to “withdraw immediately from sale […] all games, including RapeLay, which involve rape, stalking or other forms of sexual violence or which otherwise denigrate women”.9 Moreover, the organization launched a letter writing campaign to the Japanese Government calling them to “ban the sale of computer games such as RapeLay, which normalize and promote sexual violence against women and girls”.10 In a similar condemnation, the European Women’s Lobby (EWL) affirmed RapeLay “promotes a hostile attitude towards women, girls and male sexuality, and enforces and promotes harmful and discriminatory gender stereotypes”.11 The media was also involved in this debate on the virtual depiction of children in adult games. Western media such as CNN12, The Guardian,13 and the It could thus be said that gender-based vio lence is represented at the core of the sexual relationships on which this type of video game Dilton Rocha Ferraz Ribeiro An advocacy coalition analysis of the game RapeLay 459 Dilton Rocha Ferraz Ribeiro An advocacy coalition analysis of the game RapeLay 459 Belfast Telegraph14 wrote pieces on the shocking nature of RapeLay, which stressed the game’s issue with women’s right and child pornography in tone with the “children protection coalition”. prohibition of the production of other rape-based games. 14 Fennelly, Gary. 2009. Exclusive: Amazon selling rape simulation game Rapelay. The Belfast Telegraph. February 12, 2009. Accessed November 9, 2017. https://www.belfasttelegraph.co.uk/business/technology/exclusive-amazon-selling-rape-simulation-game-rapelay-28533501.html. 15 ONU. 2016. Report of the special rapporteur on the sale of children, child prostitution and child pornography on her visit to Japan – addendum – comments by the government of Japan on the report of the special rapporteur regarding her mission to Japan. Human Rights Council Thirty-first session A/HRC/31/58/Ass. 3, p. 2. March 7 2016. Accessed March 23, 2018. http://www.ohchr.org/EN/Issues/ Children/Pages/CountryVisits.aspx. 16 Komeito. 2019. Accessed October 23, 2019. https://www.komei.or.jp/ 17 Ando, Ritsuko. 2014. Japan bans child porn, but makes exceptions for manga comics. The Huffington Post, June 18, 2014. Accessed October 15, 2017. https://www.huffingtonpost.com/2014/06/18/japan-bans-child-porn_n_5506114.html. 9 3 9 p jp 17 Ando, Ritsuko. 2014. Japan bans child porn, but makes exceptions for manga comics. The Huffington Post, June 18, 2014. Accessed October 15, 2017. https://www.huffingtonpost.com/2014/06/18/japan-bans-child-porn_n_5506114.html. RapeLay Komeito is a relatively small social conservative and nationalist- leaning party that is part of the ruling governmental coalition.16 Toyama Kiyohiko is one of the proponents for criminalizing fictitious depictions of children in violent and sexual media.17 Accordingly, the coalition against virtual depictions of children in pornographic materials in Japan is formed by some academics, some human rights organizations, the United Nations, some politicians (especially of the Komeito Party) and, to some extent, the media. They share the policy core belief that these depictions are harmful to children and foster a culture of child abuse. As a policy outcome, they hope to amend Japan’s legislation to criminalize virtual child pornography. On this specific case, the “children protection coalition” sought to remove the production and sell of RapeLay and to assure the Fujimoto’s perspective is in tune with Saitō Tamaki’s (2011) theory. For Saitō (2011) the distinction between reality and non-reality is blurred and otaku’s love for fictional characters represents multilayers of forms of love that are different from one another. Although there’s no intrinsic Civitas 20 (3): 454-463, set.-dez. 2020 460 (jidō poruno kisei hōan ni mukete no iken-sho, 2013;19 hontōni mamorubeki mono, 2013).20 The current Government of Japan with its ruling Liberal Democratic Party is itself part of this “pro status quo coalition” for not taking any effective steps to criminalize virtual child pornography and prohibit sexual violent games such as RapeLay. Moreover, the Government submitted a response to the United Nations report on child trafficking and child prostitution asserting that this report is inaccurate, non-objective, contains insufficient information and lacks understanding of Japan and its culture.21 The document stressed that the Special Rapporteur’s arguments regarding virtual child pornography represent her personal opinion on the issue and no domestic law and binding international law point to the understanding that that virtual child pornography is in fact child pornography. Consequently, instead of promising to amend its legislation, the Government took a clear position against criminalizing virtual child pornography. difference between loving a fictional character and an actual human, the former represents a completely different being. Saitō (2011) asserts the test to know a real otaku is whether they genuinely feel sexually drawn to fictional images. Consequently, the sexual attraction for fictional characters and humans are different forms of sexuality. 18 Kōeki Shadanhōjin Nihon Mangakka Kyōkai. Hontōni mamorubekimono 2013. Accessed November 9, 2017. https://www.nihonman gakakyokai.or.jp/?tbl=information&id=3260. 19 Kōeki Shadanhōjin Nihon Mangakka Kyōkai, Jidō poruno kisei hōan ni mukete no iken-sho 2013. Accessed November 9, 2017. https:// www.nihonmangakakyokai.or.jp/?tbl=information&id=3136. 20 Kōeki Shadanhōjin Nihon Mangakka Kyōkai, Hontōni mamorubeki mono 2013. Accessed November 9, 2017. https://www.nihonman gakakyokai.or.jp/?tbl=information&id=3260. 21 ONU. 2016. Report of the special rapporteur on the sale of children, child prostitution and child pornography on her visit to Japan – ad dendum – comments by the government of Japan on the report of the special rapporteur regarding her mission to Japan. Human Rights Council thirty-first session A/HRC/31/58/Ass. 3, p. 2. [hereinafter Government Comments U.N.]. Accessed March 23, 2018. http://www. ohchr.org/EN/Issues/Children/Pages/CountryVisits.aspx. 22 Ken Akamatsu. n.d. Twitter. Accessed March 23, 2018. https://twitter.com/KenAkamatsu. RapeLay Fujimoto (Galbraith 2017) is analysing this issue in a similar lenses to assert that desires for fictional underage characters and sexual fantasies do not reflect on real life individuals and desires. Moreover, not only those are separate things but the regulation of these issues open a window to allow government interference in people’s lives by determining what would constitute accepted forms of sexuality coupled with a disregard to freedom of expression. Shigematsu (1999, 138) affirms that fictional characters should not be “blamed as the cause of the sexualisation of actual young girls or their molestation.” She adds that “[h]ow individuals consume, appropriate, and transform rorikon manga (or other media) and how they act thereafter cannot be controlled or determined in advance”. Fans of pop culture are also part of the “pro status quo coalition”. Among artists, Akamatsu Ken is one of the most vocal opponents online to the criminalization of virtual pornography.22 Moreover, a number of websites offer platforms to discuss and coordinate movements in support of the non-criminalization of virtual child pornography and others offer translations and download links of games such as RapeLay. Indeed, a simple Google search leads to a number of websites offering free downloads of RapeLay. McLelland (2015, 15) shares the same view affirming that it is “the two-dimensionality of these characters, and lack of reference to any physically existing person that many manga fans find so attractive”. Moreover, artists also lobbied against any amendment of Japan’s legislation usually on the grounds of freedom of expression. The Japan Cartoonists Association18 argued that the criminalization of virtual depictions of children would impact the creative industry, would implement a fear of persecution leading to the destruction of artwork (“book burnings”) and would hinder ideas and harm freedom of expression causing “unnecessary adverse effects” The Advocacy Coalition Framework and the RapeLay case The RapeLay controversy concerns the broader policy subsystem regarding virtual depictions of minors in sexual and violent situations. There Dilton Rocha Ferraz Ribeiro An advocacy coalition analysis of the game RapeLay 461 Dilton Rocha Ferraz Ribeiro An advocacy coalition analysis of the game RapeLay 461 are two internationally based coalitions looking to achieve opposite objectives. As policy goals, one coalition seeks to prohibit games such as RapeLay and, more broadly, change Japan’s legislation to criminalize virtual depictions of children in pornographic manga, animation and games. Academics such as Nakasatomi, Western media outlets such as CNN and The Guardian, non-governmental organizations such as Equality Now and Japan’s Lighthouse Project,23 and branches of the United Nations form this coalition. Moreover, social conservative politicians from the Liberal Democratic Party and especially from the Komeito Party also share these policy objectives. This coalition is a “children protection coalition” because their main policy core belief is that Japan needs to ban games such as RapeLay and virtual child pornography in order to protect children from exploitation and to avoid promoting a culture of child abuse. in Japan concerning virtual child pornography. This coalition is formed by academics such as Galbraith, feminists such as Fujimoto and Shigematsu, the Japan Cartoonists Association, artists such as Akamatsu Ken and arguably most members of the current Government of Japan. The “pro status quo coalition” based their views on the arguments that a number of sexual and violent manga, anime and games are produced by women themselves (agency) and reflect desires, which do not belong to the realm of reality. Accordingly, to this coalition, sexual desires towards virtual characters are different to and separate from any real human being. Moreover, they advocate freedom of speech arguing that change on the legislation would severally impact the media industry in Japan and would place restrictions on the creative drive of artists. However, this coalition arguably has one outlier, that is, one actor that does not necessarily share these core beliefs: the Government. The ruling political party does not justify its position on feminist views rooted on agency or on freedom of expression. It simply points out the lack of studies and proof that virtual child pornography is prejudicial to children and is inertial on amending Japan’s criminal laws. 23 Fujiwara, Shihoko. 2014. Japan outlaws possession of child porn, but anime and manga still legal. Interview by Carol Off. CBC Radio, June 06, 2014. Accessed November 9, 2017. http://www.cbc.ca/radio/asithappens/friday-moncton-shooting-rcmp-friend-ireland-s-magdale ne-laundries-squirrel-cpr-and-more-1.2903540/japan-outlaws-possession-of-child-porn-but-anime-and-manga-still-legal-1.2903543. The Advocacy Coalition Framework and the RapeLay case Consequently, the core policy belief share by all actors on this coalition is that there is no evidence proving that virtual child pornography is damaging and Japan should not ban virtual pornography. Most actors of the “children protection coalition” place their arguments within a feminist framework of the protection of women against exploitation of an uneven society. However, social conservative politicians do not share feminist views. On the contrary, they usually sponsor “traditional” heterosexual forms of union leading to matrimony and procreation. Indeed, one of the leading politicians against games such as RapeLay and virtual child pornography is the anti- feminist and anti-sex education Yamatani Eriko (Nakasatomi, 2009). Due to this divergence, this coalition is more centered on the core belief that children must be protected and sexual violence has no place in media forms. There is thus the question of the effectiveness of these coalitions. The pro status quo coalition is stable and effective for three main reasons. First, this coalition does not need to get support to change policies. On the contrary, they mainly want the non-application of laws that, in their view, might hinder free expression. Secondly, artists and fans can be quite vocal in their support for manga, animation and games on the Internet and on real life. Third, the Government seems, at least up to now, to be supporting this coalition, which hinders any possibility of change through the Parliament. The only non-stable member The opposite coalition seeks, as policy goals, to make sure Japan preserves its current content-free laws that do not criminalize virtual depictions of children in any situation. In a more specific analysis, they also sought to preserve the production and commercialization of games such as RapeLay. This opposition is, therefore, a “pro status quo coalition”, that is, it seeks to preserve the status quo Civitas 20 (3): 454-463, set.-dez. 2020 462 that is, non-real representations of individuals, especially children. In the context of Japanese pop-culture, there are two opposing coalitions: the “children protection coalition” and the “pro status quo coalition”. They act in a complex set of arrangements that include academic papers, books, websites, social networks and political debates. Each group seeks to influence people to either change policies towards the production and commercialization of virtual child pornography, or to maintain the current non-criminalization of these products. of this coalition is the current Government. The Advocacy Coalition Framework and the RapeLay case This is due to the fact that there is no evidence that most members of the ruling party supports agency-based feminism and the non-limitation of freedom of expression. Accordingly, although the “pro status quo coalition” is arguably stable and effective on its policy goals, they might face a starker opposition in case the majority of the Parliament decides to shift core beliefs and support a change in the current legislation. However, this does not mean that the “children protection coalition” is not effective. This coalition is stable and managed to lobby for the amendment of the Child Prostitution and Child Pornography Act to criminalize the possession of child pornography. Moreover, after strong opposition from this coalition on the production and commercialization of games such as RapeLay, the self-regulatory agency Ethics Organization of Computer Software banned the production and commercialization of games such as RapeLay in Japan (Nakasatomi 2009).This is a surprising victory for the “children protection coalition” as they were able to accomplish one of their policy goals despite strong lobbying from the Japan Cartoonists Association and artists such as Akamatsu Ken. Moreover, the Ethics Organization of Computer Software, formed by game producers, moved past the interests of their member organizations and acquiesced with a policy goal from the “children protection coalition”. Most members of both groups arguably share the same core beliefs, as both groups have been stable for more than a decade. Moreover, they act and react to each other. RapeLay is an example of this action-reaction system. The game was produced and commercialized within its niche. After the game’s sale reached the stoplight on the website “Amazon”, there was a chain action from the “children protection coalition”, which lead to a reaction from the “pro status quo coalition”. In this case, both coalitions were, to some extent, victorious. The “children protection coalition” managed to remove the game from Japanese stores and the “pro status quo coalition” succeeded in keeping Japanese laws intact. However, even though the “children protection coalition” was victorious in its claims concerning the game, this is limited to a punctual aspect, as they remain unable to succeed in changing the legal framework that allows for the fictitious depictions of children. In other words, the “pro status quo coalition” has been, at least so far, more effective and successful. Conclusion The Advocacy Coalition Framework provides efficient lenses to analyze the complex subsystem concerning rape fantasies and fictitious depictions of children in adult media. 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https://openalex.org/W4229450206	https://www.nature.com/articles/s41598-022-11657-z.pdf	English	null	Association between bronchopulmonary dysplasia and early respiratory morbidity in children with respiratory distress syndrome: a case–control study using nationwide data	Scientific reports	2,022	cc-by	6,420	Association between bronchopulmonary dysplasia and early respiratory morbidity in children with respiratory distress syndrome: a case–control study using nationwide data OPEN Jeong Eun Shin1,2, Haerin Jang1,2, Jung Ho Han1, Joonsik Park1, Soo Yeon Kim1, Yoon Hee Kim1, Ho Seon Eun1, Soon Min Lee1, Kook In Park1, Myung Hyun Sohn1, Min Soo Park1,3 & Kyung Won Kim1,3* Bronchopulmonary dysplasia (BPD) can cause respiratory morbidity beyond the neonatal period. We aimed to analyze the association of BPD on childhood lower respiratory illness (LRI) and asthma among patients diagnosed with respiratory distress syndrome (RDS). This case–control study analyzed data between 2002 and 2015 from a nationwide database. We included 55,066 children with RDS. Two-year LRI and asthma at ages 3 and 5 were assessed. Readmission for LRIs within 2 years of birth occurred in 53.9% and 37.9% of the BPD (n = 9470) and non-BPD (n = 45,596) cases, respectively. In the BPD group, the median number of hospitalizations, mechanical ventilation and oxygen use rates were significantly higher, while the hospitalization duration was significantly longer (P < 0.001 for all). The relative risk of BPD was 1.42 (1.39–1.45) on total readmission and 6.53 (5.96–7.15) on intensive care unit readmission. Asthma prevalence was significantly higher in BPD group (57.6% vs. 48.9% at age 3 and 44.3% vs. 38.2% at age 5, P < 0.001). In children with RDS, BPD could affect repetitive and worse LRI as an independent risk factor for respiratory morbidity during the first 2 years of life. BPD may also be a crucial risk factor for asthma in preschoolers. Respiratory distress syndrome (RDS) requiring surfactant replacement occurs in approximately 1% of total live births globally. RDS has been steadily increasing in Korea and in western countries1,2. Following the introduc- tion of pulmonary surfactant administration in the 1990s, very low birth weight (VLBW) and extremely low birth weight (ELBW) infant survival rates in Korea increased by 1.6 and 2.7 times, respectively, over 20 years3.h g ( ) y p y y The prevalence of bronchopulmonary dysplasia (BPD), a chronic lung disease caused by RDS, has increased because of improved preterm infant survival4. Despite advances in perinatal care, approximately 20–30% of VLBW infants develop BPD1,5. Children with BPD have higher rehospitalization rates in early childhood, mostly because of respiratory illness, longer hospital days, and higher wheezing or asthma rates than preterm or ELBW infants without BPD6. In Asia, few studies have investigated respiratory morbidity of BPD patients in preterms7. No study has evaluated the impact of BPD in children born with RDS worldwide. www.nature.com/scientificreports www.nature.com/scientificreports Methods D We analyzed the number of admissions and total days admitted for both general admission and ICU care. Prescription and treatment records were used to analyze whether respiratory support was necessary. Mechanical ventilation and non-invasive ventilation were evaluated, along with oxygen prescrip- tion records. Treatment codes used to identify each treatment are listed in Supplementary Table S5. y pp y Mortality was analyzed in all patients who died before age 3. Cause of death data was collected from the Statistics Korea database, which is based on death certificate forms that specify the primary cause of death using ICD-10 codes. We classified the cause of death into four categories: death due to respiratory diseases; death due to non-respiratory diseases; death due to external causes, including all accidents; and death due to miscellaneous causes. ICD-10 codes used for classification are organized in Supplementary Table S6. Asthma prevalence and morbidity assessment. Each patient was evaluated for asthma at ages 3 and 5. Asthma patients were defined as children who visited a hospital at least twice yearly with an asthma diagnosis using code J45 (asthma) or J46 (status asthmaticus), including all subsections, and prescribed with asthma medi- cation at the same visit9. Asthma medications were determined based on medical charge codes for prescribed drugs (Supplementary Table S7) including oral/intravenous corticosteroids, inhaled corticosteroids, inhaled cor- ticosteroids/long-acting beta 2 agonist combination inhalers, leukotriene receptor antagonists, short-acting beta 2 agonists for inhalation, and aminophylline/theophylline. For asthma at age 3, medical records 24–36 months after birth were retrieved, and the number of doctor visits with asthma diagnosis and asthma medication pre- scriptions were counted. For asthma at age 5, medical records 48–60 months after birth were retrieved to define asthma. Patients who died before reaching 24 or 48 months of age were excluded. Among asthma patients, we also examined those who were hospitalized within this period because of asthma. The number of admissions and total days admitted were used to evaluate asthma severity. Statistical analysis. Data were analyzed using R version 3.3 and SAS version 9.4 (SAS Institute Inc., Cary, NC). Descriptive statistics were used to summarize the entire study population. Results are presented as frequen- cies or medians and interquartile ranges, as appropriate. Groups were compared using Welch’s two-sample t test for continuous variables and Pearson’s Chi-square test with Yates’ continuity correction for categorical variables. The Mann–Whitney U test was used for non-normal distributions. Methods D Data source and study population. All diagnosed diseases are recorded in the NHIS using codes based on the 10th Revision of the International Classification for Disease (ICD-10). From the NHIS database, we extracted data on children born between 2002 and 2015 who fulfilled either of the following criteria within 2 years of birth: diagnosed with RDS using ICD-10 code P22.0 or received exempted health insurance fees because of RDS with code V142. Patients were excluded from the study if death occurred during the initial neo- natal ICU (NICU) hospitalization or if major congenital anomalies were present. Major congenital anomalies were defined as malformation or deformation of the nervous, circulatory, respiratory, digestive, or musculoskel- etal systems; chromosomal abnormality; inborn errors of metabolism; or hydrops fetalis. Patients with external minor congenital anomalies, anomalies limited to the limbs, or isolated urogenital anomalies were included. ICD-10 codes used for the exclusion criteria are presented in Supplementary Table S1. Because of the nature of the data, medical records are only registered after birth registration. Hence, medical records of infants who received health care before birth registration are recorded under their birth mothers. As there were a few cases where birth registration was performed after discharge from the NICU, we excluded patients whose first medical record was registered in an outpatient clinic. Characteristics of RDS patients. Demographic data, diagnosed illnesses, prescribed therapies, and admission history were collected. BPD was defined as a diagnosis with ICD-10 code P27.1 within 2 years of birth. VLBW infants born after 2011 could be identified using diagnosis codes that specify birth weight < 1500 g (Supplementary Table S2). The date of the first medical record was used as the date of birth for each patient; age was calculated using this date. Comorbidity was evaluated in medical records within 2 years of birth. Pat- ent ductus arteriosus ligation, intraventricular hemorrhage, pulmonary hypertension, necrotizing enterocolitis, retinopathy of prematurity, and neonatal sepsis were confirmed using their corresponding ICD-10 codes (Sup- plementary Table S3). Lower respiratory morbidity and mortality assessment. We evaluated medical records of patients hospitalized within 2 years of birth with a diagnosed LRI. Supplementary Table S4 contains the ICD-10 codes for bronchitis, bronchiolitis, pneumonia, croup, laryngitis, and tracheitis. The first admissions recorded for each patient were most likely NICU admissions immediately after birth; thus, we only included records after dis- charge from the first admission. www.nature.com/scientificreports/ emergency care, and pharmaceutical services. At each doctor visit, institutions submit electronic health care utilization information to the NHIS for reimbursement, which is integrated into the NHIS database8. The NHIS provides access to the database for research, allowing nationwide large-scale and longitudinal analyses in Korea. We aimed to determine the influence of BPD on childhood lower respiratory illness (LRI) and asthma among a population who received pulmonary surfactant for neonatal RDS. Association between bronchopulmonary dysplasia and early respiratory morbidity in children with respiratory distress syndrome: a case–control study using nationwide data OPEN To our knowledge, this is the first large-scale nationwide study analyzing BPD as a risk factor for early respiratory morbidities and pediatric asthma and the first to investigate infants with RDS, not preterm or ELBW infants.hh i g p The National Health Insurance Service (NHIS) provides health insurance to most Koreans (99.01%). The NHIS covers many medical services, including medical visits, admissions, intensive care unit (ICU) care, 1Department of Pediatrics, Yonsei University College of Medicine, 50‑1 Yonsei‑ro, Seodaemun‑gu, Seoul 03722, Korea. 2These authors contributed equally: Jeong Eun Shin and Haerin Jang. 3These authors jointly supervised this work: Min Soo Park and Kyung Won Kim. email: kwkim@yuhs.ac Scientific Reports \| (2022) 12:7578 \| https://doi.org/10.1038/s41598-022-11657-z www.nature.com/scientificreports/ Methods D Patients readmitted within 2 years of birth because of lower respiratory illness. Data are expressed as n (%), median (range), or median (interquartile range). The Mann–Whitney U test was used for non-normal distributions in continuous variables. BPD bronchopulmonary dysplasia, NIV non-invasive ventilator, ICU intensive care unit. presentation of any identifying data, the requirement for informed consent was waived by the same IRB com- mittee. All methods were carried out in accordance with Korean government’s guideline for health and medical data utilization. Methods D P < 0.05 was considered statistically signifi- cant. Ethical approval. This study protocol was reviewed and approved by the Institutional Review Board of Severance Hospital (approval No. 4-2019-0383). As this was an observational study without intervention and the https://doi.org/10.1038/s41598-022-11657-z Scientific Reports \| (2022) 12:7578 \| www.nature.com/scientificreports/ BPD (n = 9470) Non-BPD (n = 45,596) P-value Male 5176 (54.7) 26,321 (57.7) < 0.001 Perinatal comorbidity PDA, treated surgically 184 (1.9) 91 (0.2) < 0.001 Intraventricular hemorrhage 528 (5.6) 287 (0.6) < 0.001 Pulmonary hypertension 824 (8.7) 1266 (2.8) < 0.001 Necrotizing enterocolitis 1018 (10.7) 941 (2.1) < 0.001 Retinopathy of prematurity 7460 (78.8) 17,274 (37.9) < 0.001 Neonatal sepsis 4849 (51.2) 11,261 (24.7) < 0.001 Readmission for LRI within 2 years of birth 5102 (53.9) 17,273 (37.9) < 0.001 Death within 2 years of birth 264 (2.8) 387 (0.8) < 0.001 Table 1. Patient characteristics. Data are expressed as n (%). LRI lower respiratory illness, BPD bronchopulmonary dysplasia, PDA patent ductus arteriosus. P-value was calculated using Welch’s two-sample t test for continuous variables and Pearson’s Chi-square test with Yates’ continuity correction for categorical variables. p ( ) p y bronchopulmonary dysplasia, PDA patent ductus arteriosus. P-value was calculated using Welch’s two-sample t test for continuous variables and Pearson’s Chi-square test with Yates’ continuity correction for categorical variables. Table 2. Patients readmitted within 2 years of birth because of lower respiratory illness. Data are expressed as n (%), median (range), or median (interquartile range). The Mann–Whitney U test was used for non-normal distributions in continuous variables. BPD bronchopulmonary dysplasia, NIV non-invasive ventilator, ICU intensive care unit. BPD (n = 5102) Non-BPD (n = 17,273) P-value Number of readmissions 2 (1–40) 1 (1–29) < 0.001 Hospital stay, days 13 (6–30) 8 (5–15) < 0.001 Ventilator use 760 (14.9) 459 (2.7) < 0.001 NIV use 201 (3.9) 82 (0.5) < 0.001 Oxygen use 2094 (41.0) 2505 (14.5) < 0.001 ICU admission 1030 (20.1) 760 (4.3) < 0.001 Number of ICU admissions* 1 (1–6) 1 (1–8) < 0.001 ICU hospital stay, days 34 (18–61) 14 (9–25) < 0.001 Table 2. Patients readmitted within 2 years of birth because of lower respiratory illness. Data are expressed as n (%), median (range), or median (interquartile range). The Mann–Whitney U test was used for non-normal distributions in continuous variables. BPD bronchopulmonary dysplasia, NIV non-invasive ventilator, ICU intensive care unit. Table 2. Table 1. Patient characteristics. Data are expressed as n (%). LRI lower respiratory illness, BPD Figure 1. Trend in the relative risk of bronchopulmonary dysplasia on total readmission (A) and ICU admission (B) because of respiratory diseases. ICU intensive care unit. Table 3. Asthma patients categorized per BPD status. Data are expressed as n (%), median (interquartile range), or median (range). a Between 36 and 48 months of age. b Between 60 and 72 months of age. The percentage of patients with asthma at ages 3 and 5 was calculated using the total number of patients alive at ages 3 and 5. BPD bronchopulmonary dysplasia. BPD (n = 5760) Non-BPD (n = 27,369) P-value Asthma at age 3 (n = 16,467) 3217 (57.6) 13,250 (48.9) < 0.001 Number of admitted patientsa 772 (24.0) 2289 (17.3) < 0.001 Number of admissions per person* 1 (1–13) 1 (1–12) 0.157 Hospital days per person 7 (5–12) 6 (5–10) 0.003 Asthma at age 5 (n = 12,812) 2468 (44.3) 10,344 (38.2) < 0.001 Admissions due to asthmab 369 (15.0) 1070 (10.3) 0.439 Number of admissions per person* 1 (1–12) 1 (1–11) < 0.001 Hospital days per person 6 (4–10) 6 (5–9) 0.777 Table 3. Asthma patients categorized per BPD status. Data are expressed as n (%), median (interquartile range), or median (range). a Between 36 and 48 months of age. b Between 60 and 72 months of age. The percentage of patients with asthma at ages 3 and 5 was calculated using the total number of patients alive at ages 3 and 5. BPD bronchopulmonary dysplasia. Table 3. Asthma patients categorized per BPD status. Data are expressed as n (%), median (interquartile range), or median (range). a Between 36 and 48 months of age. b Between 60 and 72 months of age. The percentage of patients with asthma at ages 3 and 5 was calculated using the total number of patients alive at ages 3 and 5. BPD bronchopulmonary dysplasia. Respiratory morbidities in VLBW infants. Of the total population, 33,437 patients (60.7%) were included in a subgroup analysis based on birth weight of < 1500 g or > 1500 g. BPD was diagnosed in 58.7% and 7.2% of the < 1500 g and > 1500 g groups, respectively. Results BPD (n = 5760) Non-BPD (n = 27,369) P-val Asthma at age 3 (n = 16,467) 3217 (57.6) 13,250 (48.9) < 0.00 Number of admitted patientsa 772 (24.0) 2289 (17.3) < 0.00 Number of admissions per person* 1 (1–13) 1 (1–12) 0.15 Hospital days per person 7 (5–12) 6 (5–10) 0.00 Asthma at age 5 (n = 12,812) 2468 (44.3) 10,344 (38.2) < 0.00 d d h b ( ) ( ) Results Respiratory morbidities/mortality within 2 years in BPD patients. From 2002 to 2015, 69,245 patients (0.95% of total live births in Korea) were diagnosed with RDS (P22.0). After applying the exclusion criteria, 55,066 patients (79.5%) were finally included in the analysis (Supplementary Fig. S1), 9470 (17.2%) of whom were diagnosed with BPD. Perinatal co-morbidities including surgically treated patent ductus arteriosus, intraventricular hemorrhage, pulmonary hypertension, necrotizing enterocolitis, retinopathy of prematurity, and neonatal sepsis were significantly higher in the BPD group than the non-BPD group (P < 0.001, Table 1). Rehospitalization for LRI within 2 years of birth occurred in 53.9% of BPD patients and 37.9% of non-BPD patients (P < 0.001, Table 1). p Among readmitted patients, the median number of readmissions within 2 years of birth was twice as high in the BPD group than the non-BPD group (2 (1–40) vs. 1 (1–29), P < 0.001, Table 2). Patients in the BPD group showed a significantly longer hospital stay and higher rates of mechanical ventilator, non-invasive ventilator, and oxygen use than those in the non-BPD group (14.9% vs. 2.7%, 3.9% vs. 0.5%, and 41.0% vs. 14.5%, respectively, P < 0.001, Table 2). Furthermore, compared with those in the non-BPD group, patients in the BPD group were 4.7 times more likely to require ICU care and stayed in the ICU 2.4 times longer (P < 0.001, Table 2).hi y q y g The relative risk (RR) of BPD on readmission because of LRI significantly and steadily increased from 2002 to 2015 (1.42, 95% confidence interval [CI], 1.39–1.45, Fig. 1A). The RR of BPD on ICU admission was much higher than that on total readmission (6.53, 95% CI, 5.96–7.15, Fig. 1B). The RRs of both total readmission and ICU admission showed an increasing trend between 2005 and 2006; the RR of ICU admission peaked in 2010 (8.60, 95% CI, 6.32–11.70, Fig. 1).hi g Of the 55,066 patients, 651 (1.18%) died within 2 years. The mortality rate was significantly higher in the BPD group than the non-BPD group (2.8% vs. 0.8%, P < 0.001, Table 1). Respiratory morbidity caused 42.4% and 25.3% of deaths in the BPD and non-BPD groups, respectively (P < 0.001, Supplementary Table S8). https://doi.org/10.1038/s41598-022-11657-z Scientific Reports \| (2022) 12:7578 \| cientificreports/ www.nature.com/scientificreports/ scientificreports/ Figure 1. Trend in the relative risk of bronchopulmonary dysplasia on total readmission (A) and ICU admission (B) because of respiratory diseases. ICU intensive care unit. Discussionh This study analyzed the association between BPD and childhood respiratory morbidities in Korean children with RDS born between 2002 and 2015. In the study population, 17.2% of children were diagnosed with BPD; 58.7% of the < 1500 g and 7.2% of the > 1500 g birth weight subgroups had BPD. The incidence and severity of LRIs requiring readmission within 2 years of birth were significantly higher in the BPD group than the non-BPD group. BPD increased the frequency of readmission, hospitalization stay, ICU admission rate and duration, and overall ventilator and oxygen use in admitted patients. The RR of BPD on rehospitalization slightly decreased after adjusting for birth weight; however, BPD was still significantly associated with rehospitalization in both birth weight groups. Pediatric asthma prevalence and severity were also higher in the BPD group than the non- BPD group at ages 3 and age 5, approximately 3 times higher than in the general population.h g p g g , pp y g g p p The high incidence of early life LRIs in BPD patients means they are vulnerable to respiratory infections and potential further decline of lung function because of LRI-related causes. BPD is characterized by disrupted alveolar and vascular growth due to pulmonary inflammation, oxidative stress, and mechanical trauma to the fragile immature lungs10. Most alveolarization occurs within the first 2 years of life, and pulmonary function follows a predictable progression from birth through childhood until full maturation at age 22; therefore, any adverse exposure before or during development has the potential to change the trajectory of lung development and cause a loss of lung function with age10–13. Also, even late preterm children had decreased childhood lung function compared with those delivered at term14.h p The RR of BPD on readmission decreased from 2002 to 2005 but rapidly increased during 2005–2006, which was further emphasized when looking at ICU admission (Fig. 1). This result can be partially explained by the increase in preterm infant survival. In VLBW infants, the survival rate of infants born in Korea between 2005 and 2009 was 85.3%, up 14.5% from that of infants born between 2000 and 2004 (74.5%)15. When comparing 2000 and 2010, preterm births increased both in absolute number (23,914 vs. 27,304) and in the proportion of total births (3.8% vs 5.8%), although the total number of births decreased by 35%16. Compared with the non-BPD group, the BPD group had a significantly higher readmission frequency; more days in the hospital; and higher mechanical ventilator, non-invasive ventilator, and oxygen use on readmission within 2 years in both the < 1500 g and > 1500 g birth weight subgroups. The number of ICU admissions was not higher in patients with BPD in the > 1500 g group (Supplementary Table S9). pp y Patients with BPD showed similar RRs for readmission because of LRI in both the < 1500 g (1.23, 95% CI 1.17–1.29, Supplementary Fig. S2) and > 1500 g groups (1.28, 95% CI 1.22–1.34). All patients with RDS born between 2011 and 2015 had an RR for readmission of 1.36 (Supplementary Fig. S2). Asthma prevalence and asthma‑related morbidities in BPD. Patients born between 2002 and 2012 were included in the analysis. Of 33,129 children, asthma prevalence at ages 3 and 5 was 49.7% and 38.7%, respectively. The proportion of patients diagnosed with asthma was significantly higher in the BPD group than the non-BPD group at both ages 3 (57.6% vs. 48.9%, P < 0.001) and 5 (44.3% vs. 38.2%, P < 0.001). Total asthma prevalence decreased at age 5 (Table 3). The number of hospital admissions for asthma and the duration of hos- https://doi.org/10.1038/s41598-022-11657-z Scientific Reports \| (2022) 12:7578 \| www.nature.com/scientificreports/ Figure 2. Relative risk of bronchopulmonary dysplasia on asthma diagnosis at ages 3 (A) and 5 (B). Figure 2. Relative risk of bronchopulmonary dysplasia on asthma diagnosis at ages 3 (A) and 5 (B). Figure 2. Relative risk of bronchopulmonary dysplasia on asthma diagnosis at ages 3 (A) and 5 (B). igure 2. Relative risk of bronchopulmonary dysplasia on asthma diagnosis at ages 3 (A) and 5 (B). pitalization among admitted patients were higher in the BPD group than in the non-BPD group (Table 3). The RR of BPD on asthma diagnosis was 1.06–1.37 at age 3 and 1.05–1.23 at age 5 (Fig. 2). pitalization among admitted patients were higher in the BPD group than in the non-BPD group (Table 3). The RR of BPD on asthma diagnosis was 1.06–1.37 at age 3 and 1.05–1.23 at age 5 (Fig. 2). www.nature.com/scientificreports/ Long-term follow-up is necessary to investigate whether prematurity of BPD itself is a predominant risk factor for respira- tory morbidities or if BPD is only a major synergistic factor. y y j y g Previous studies analyzed BPD as a risk factor for respiratory morbidity or asthma in VLBW or extremely preterm infants. Our study focused on a nationwide population with RDS requiring surfactant replacement. As the diagnostic code in claims data depends on input by each physician, the accuracy of diagnostic code input is critical. The diagnostic code for “RDS” must be accompanied by a pulmonary surfactant prescription and registered with the rare incurable disease code V142, which provides financial benefits for RDS patients from the national health insurance. Extraction of patient data using code V142 is more accurate and reliable than the diagnostic code related to ‘prematurity’ which has not been regulated by obligation for prescription or redemp- tions. We were able to compare the asthma prevalence in the RDS group with that in the general population using the same operational diagnosis9. However, as this is large-scale real-world data, the possibility of prescribing asthma medications for preventive purposes cannot be excluded in some BPD patients.h This study has some limitations, including those inherent in any retrospective study using claims data. Although a large number of participants in our study allowed us to investigate patients with RDS extensively, we could not obtain all perinatal and environmental variables because of insufficient detailed medical records. Therefore, we were unable to distinguish the extent of immaturity and BPD on respiratory morbidity. BPD diag- nosis in our study population was based on the input of a diagnostic code by physicians and was presumed to be defined as “oxygen use for 28 days”. Subgroup analysis among VLBW infants was only possible in infants born after 2011 when the Korean Classification of Disease was updated. Infants with transient tachypnea of newborn (TTN) or pneumonia may have been incorrectly diagnosed as RDS and included in our study population. How- ever, previous national multicenter studies reported that 93.5% and 87.9% of total RDS patients were < 37 weeks and < 2500 g respectively, whereas patients with TTN or pneumonia constituted only minimal portion in preterm or low birth weight infants33,34. www.nature.com/scientificreports/ investigation using a large-scale longitudinal cohort study containing detailed medical records is necessary to clarify the predominant risk factors for this high prevalence of asthma in the RDS population. y g Interestingly, the prevalence of asthma was not affected by BPD when we limited the population to only < 1500 g birth weight infants (data not shown); however, LRI-related readmission was influenced by BPD in the < 1500 g birth weight subgroup. This finding is partially consistent with the results of Jackson et al.20 who suggested asthma diagnosis in children born before 28 weeks gestation is predominantly associated with post- NICU risk factors, especially low socioeconomic status, but not with neonatal complications, including BPD. Kim et al.21 reported that small for gestational age infants were associated with poor pulmonary function showing obstructive patterns within BPD patients within the first year of life. Therefore, the influence of BPD on asthma may be diminished when the population is limited to extremely preterm or VLBW infants because birth weight and prematurity are relatively predominant risk factors for asthma. Moreover, because asthma development is affected by multiple factors including genetic, prenatal, prematurity, birth weight, and postnatal environmental factors22,23, the impact of BPD on asthma may be less than that on LRIs. We found asthma-related hospitalization and the influence of BPD on asthma decreased at age 5 than at age 3, suggesting the influence of BPD decreases as children age. However, asthma development in BPD patients can result from LRI-related hospitalizations and their severity during the first 2 years of life. Previous studies have shown asthma is closely related to early life respiratory syncytial virus infection, and viral prophylaxis reduced childhood asthma prevalence23–25. p y y y p p y p BPD diagnosis in early life can cause an early decline in adult lung function. Several longitudinal cohort stud- ies revealed a decreased forced expiratory volume and decreased forced expiratory volume/forced vital capacity ratio at school age in preterm-born children26,27. Decreased lung function in childhood has also been suggested to be a risk factor for adult chronic obstructive pulmonary disorder28–30. BPD patients may persistently present with obstructive lung disease into adulthood31,32. However, long-term outcomes of survivors with BPD who were born before the current era of perinatal care may not be generalizable to more recent survivors. www.nature.com/scientificreports/ Therefore, we suggest that the overlapping or misdiagnosis of TTN or pneumonia with RDS in our cohort has minimal effects on the conclusion we draw from the study. Conclusion By investigating the annual trends of decreased BPD mortality for over 10 years, we were able to explore the association between BPD and respiratory morbidity from a macroscopic viewpoint. In addition, we revealed the real burden on readmitted patients by focusing on the severity of the patients. y g y Our study demonstrated that children with BPD required rehospitalization because of respiratory morbidi- ties twice as frequently as children without BPD. The BPD population received approximately five times more intensive care, assessed by ventilator use and ICU admission rate than the non-BPD population. The prevalence of pediatric asthma at ages 3 and 5 were three times higher in BPD children than in the general population. Regular follow-up and care for patients with BPD are crucial, and a follow-up plan may be necessary to manage their long-term health. Received: 18 October 2021; Accepted: 19 April 2022 Discussionh The proportion of BPD in the RDS population increased to 23.1% in 2007, compared with 17.0% in 200216. Two epidemics of mycoplasma pneumonia occurred, in 2006 and from 2010 to 201117, which might have affected BPD patients more severely. Rehospitalization rates in children with BPD during the first 2 years of life were similar to those reported in the United States18,19. A recent study by Sol et al.9 showed that the average prevalence of asthma in all Korean children born between 2002 and 2015 peaked at 18.4% at age 3 and decreased to 14.7% at age 5. Therefore, we can infer that the prevalence of asthma is approximately 3 times higher in BPD patients than in the general population. Moreover, non-BPD patients with RDS were diagnosed with asthma 2.5 times more than the general population. Further Scientific Reports \| (2022) 12:7578 \| https://doi.org/10.1038/s41598-022-11657-z www.nature.com/scientificreports/ Acknowledgementsh g This study was supported by a Severance Hospital Research fund for Clinical excellence (SHRC) (C-2020–0014) nd a grant (22213MFDS486) from Ministry of Food and Drug Safety in 2022. References References 1. Stoll, B. J. et al. Neonatal outcomes of extremely preterm infants from the NICHD Neonatal Research Network. Pediatrics 126 443–456 (2010). 1. Stoll, B. J. et al. Neonatal outcomes of extremely preterm infants from the NICHD Neonatal Research Network. Pediatrics 126, 443–456 (2010). 2. Shin, J. E. et al. 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Surfactant therapy for neonatal respiratory distress syndrome: A review of Korean experiences over 17 years. J. Korean Med. Sci. 24, 1110–1118 (2009). y J , ( ) 34. Choi, J. S., Bae, C. W., Jang, Y. S., Lee, N. H. & Hong, W. R. Policy issues on neonatal intensive care for low birth weight infants. Research Monograph of Korea institute for health and social affairs. 2012-47-9 (2012). y ( ) 4. Choi, J. S., Bae, C. W., Jang, Y. S., Lee, N. H. & Hong, W. R. Author contributions J.E.S. and H.J. designed the study, collected data, carried out the initial analysis, interpretation, and drafted the manuscript. J.H.H., J.P., and S.Y.K. contributed to administrative, technical support. Y.H.K., H.S.E., and S.M.L. reviewed and revised the manuscript. K.I.P. and M.H.S. contributed to the conceptualization and supervision of the study. M.S.P. and K.W.K. conceptualized and designed the study, critically reviewed the manuscript for important intellectual contents, obtained funding, coordinated and supervised the study. All authors approved the final manuscript as submitted and agree to be accountable for all aspects of the work. https://doi.org/10.1038/s41598-022-11657-z Scientific Reports \| (2022) 12:7578 \| www.nature.com/scientificreports/ Competing interests The authors declare no competing interests. Additional information Supplementary Information The online version contains supplementary material available at https://doi.org/ 10.1038/s41598-022-11657-z. Correspondence and requests for materials should be addressed to K.W.K. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2022, corrected publication 2022 Competing interests The authors declare no competing interests. Additional information Supplementary Information The online version contains supplementary material available at https://doi.org/ 10.1038/s41598-022-11657-z. Correspondence and requests for materials should be addressed to K.W.K. 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. © The Author(s) 2022, corrected publication 2022 Author contributions Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2022, corrected publication 2022 https://doi.org/10.1038/s41598-022-11657-z Scientific Reports \| (2022) 12:7578 \|
https://openalex.org/W2044824735	https://bmcgenomdata.biomedcentral.com/counter/pdf/10.1186/1471-2156-14-27	English	null	Genomic deletion of CNGB3 is identical by descent in multiple canine breeds and causes achromatopsia	BMC genomic data	2,013	cc-by	10,062	RESEARCH ARTICLE Open Access Yeh et al. BMC Genetics 2013, 14:27 http://www.biomedcentral.com/1471-2156/14/27 Yeh et al. BMC Genetics 2013, 14:27 http://www.biomedcentral.com/1471-2156/14/27 Genomic deletion of CNGB3 is identical by descent in multiple canine breeds and causes achromatopsia Connie Y Yeh1,2†, Orly Goldstein3†, Anna V Kukekova3,4, Debbie Holley5, Amy M Knollinger6, Heather J Huson7, Susan E Pearce-Kelling8, Gregory M Acland3 and András M Komáromy1,2* * Correspondence: komaromy@cvm.msu.edu †Equal contributors 1Department of Clinical Studies, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA 2Department of Small Animal Clinical Sciences, College of Veterinary Medicine, Michigan State University, East Lansing, MI 48824, USA Full list of author information is available at the end of the article Determining the deletion breakpoints of the CNGB3 mutation in the Alaskan malamute (AM) breed and establishing a diagnostic test The exact size of the genomic deletion on CFA29 that includes the CNGB3 gene in cd-affected AMs has not yet been defined. BAC clone analysis and FISH hybridization suggested that the deletion should be between 150 kb and 1 Mb [13]. To identify the breakpoints of the deletion, we sought to genotype an AM-derived cd-affected colony dog on a fine scale. AM-colony dogs are cd-affected dogs from a pedigree segregating the disease originated by cross- breeding a purebred, cd-affected AM to a beagle-mix dog (details about the colony have been described by Sidjanin and colleagues [13]). Primers designed to amplify genomic regions within the neighboring genes successfully amplified fragments from an affected dog within the genes solute carrier family 7 (cationic amino acid transporter, y + system) member 13 (SLC7A13), WW domain containing E3 ubi- quitin protein ligase 1 (WWP1), cyclic nucleotide binding domain containing 1 (CNBD1), and matrix metallopeptidase 16 (MMP16) (Figure 1; primer pairs 1, 2, 41–43, and 44–46 in Table S1A in Additional file 1). Primers, evenly dis- tributed between WWP1 and CNBD1 genes, amplified the proximal (primer pairs 3–9) and distal (primer pairs 20–40) regions and yet failed to amplify the region CFA29: 35,699,379-36,103,729 (primer pairs 10–19) in the cd- affected dog (Table S1A in Additional file 1). Achromatopsia is naturally occurring in two canine breeds, where the condition is also referred to as cone degeneration (cd). In the Alaskan malamute (AM), it is caused by a genomic deletion of the entire CNGB3 gene, while in the German shorthaired pointer it results from a missense mutation in exon 6 [13]. Both of these genetic defects are functional null mutations, and the phenotypic manifestations resemble those observed in human patients [14]. The genomic deletion in cd-affected AMs that includes the CNGB3 gene was not fully characterized. The length of the deletion and the inclusion of other adjacent genes were not identified, which resulted in an inability to identify carrier dogs [13]. The clinical signs of canine achromatopsia, predominantly day-blindness, typically manifest by 8–12 weeks of age when retinal development is completed in dogs [15-18]. Cones develop normally but once they are no longer functional, their inner and outer segments gradually deteriorate, followed by a slow loss of cones throughout the animal’s lifetime [16,19]. The loss of cone function can be confirmed by electroretinography [14,15]. However, affected dogs remain ophthalmoscopically normal. Background breeds. In the present study, we report in-depth the identical phenotype and genotype of AM-achromatopsia in a new, unrelated canine breed, the miniature Australian shepherd (MAS). We also found the same cd-allele in a Siberian husky and Alaskan sled dogs in a heterozygous state. We identified the breakpoints and determined the length of the genomic CNGB3-deletion to be 404,820 bp, involving two other genes. All the cd-alleles found in the different breeds were shown to be IBD, strongly suggesting an affected founder effect. Congenital achromatopsia, also called rod monochromacy and day-blindness, is a rare autosomal recessive condition that results in complete loss of cone photoreceptor function, while the rod photoreceptors remain intact [1]. The disease is characterized by decreased visual acuity, photophobia, nystagmus, and complete colorblindness [1]. Thus far, mutations in five genes have been identified to cause achromatopsia; they encode key components of the cone phototransduction cascade: the alpha (PDE6C) and gamma (PDE6H) subunits of cone cyclic guanosine monophosphate (cGMP) phosphodiesterase [2,3], the alpha subunit of cone transducin (GNAT2) [4,5], as well as the alpha (CNGA3) and beta (CNGB3) subunits of cone cyclic- nucleotide gated channel [6-8]. In the majority of patients, achromatopsia is a channelopathy and caused by mutations in either the CNGA3 or CNGB3 gene, with CNGB3 being affected most commonly [9-12]. Abstract BMC Genetics 2013, 14:27 http://www.biomedcentral.com/1471-2156/14/27 Page 2 of 12 Abstract Background: Achromatopsia is an autosomal recessive disease characterized by the loss of cone photoreceptor function that results in day-blindness, total colorblindness, and decreased central visual acuity. The most common causes for the disease are mutations in the CNGB3 gene, coding for the beta subunit of the cyclic nucleotide-gated channels in cones. CNGB3-achromatopsia, or cone degeneration (cd), is also known to occur in two canine breeds, the Alaskan malamute (AM) and the German shorthaired pointer. Results: Here we report an in-depth characterization of the achromatopsia phenotype in a new canine breed, the miniature Australian shepherd (MAS). Genotyping revealed that the dog was homozygous for a complete genomic deletion of the CNGB3 gene, as has been previously observed in the AM. Identical breakpoints on chromosome 29 were identified in both the affected AM and MAS with a resulting deletion of 404,820 bp. Pooled DNA samples of unrelated purebred Australian shepherd, MAS, Siberian husky, Samoyed and Alaskan sled dogs were screened for the presence of the affected allele; one Siberian husky and three Alaskan sled dogs were identified as carriers. The affected chromosomes from the AM, MAS, and Siberian husky were genotyped for 147 SNPs in a 3.93 Mb interval within the cd locus. An identical shared affected haplotype, 0.5 Mb long, was observed in all three breeds and defined the minimal linkage disequilibrium (LD) across breeds. This supports the idea that the mutated allele was identical by descent (IBD). Conclusion: We report the occurrence of CNGB3-achromatopsia in a new canine breed, the MAS. The CNGB3-deletion allele previously described in the AM was also observed in a homozygous state in the affected MAS, as well as in a heterozygous carrier state in a Siberian husky and Alaskan sled dogs. All affected alleles were shown to be IBD, strongly suggesting an affected founder effect. Since the MAS is not known to be genetically related to the AM, other breeds may potentially carry the same cd-allele and be affected by achromatopsia. Keywords: Achromatopsia, Alaskan malamute, Alaskan sled dog, Australian shepherd, Cone degeneration, CNGB3, Day-blindness, Identical by descent, Siberian husky © 2013 Yeh 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. Yeh et al. BMC Genetics 2013, 14:27 http://www.biomedcentral.com/1471-2156/14/27 Yeh et al. Determining the deletion breakpoints of the CNGB3 mutation in the Alaskan malamute (AM) breed and establishing a diagnostic test Once the breakpoints of the deletion were established, a test was designed to genotype normal, carrier and affected dogs by using primers flanking the deletion, which would only amplify the affected allele, combined with primers within the deleted sequence, which would only amplify the normal allele (Figure 2A; see Methods for details). A subset of 15 colony dogs segregating the disease was genotyped (Figure 2B). Two affected dogs (dogs 1 and 4) were positive for the mutated allele PCR (Figure 2B-B1) and negative for the wildtype allele PCR (Figure 2B-B2). One normal dog known to not carry the mutated allele (dog 15) was negative for the mutated allele PCR and positive for the wildtype allele PCR. Three obligated carriers (dogs 2, 3, 5 and 6) were positive to both PCR reactions. Dogs 7–14 were all phenotypically normal, and the genetic test identified dogs 7, 10 and 13 as carriers, and the rest as homozygous wildtype. These primers were also used together in a multiplex PCR (Figure 2-B3). We extended our screening to include samples from ten purebred AMs we had in our laboratory. Among these ten animals were one dog diagnosed with day-blindness at 7 months of age and its parents, one dog diagnosed with day-blindness, one dog suspected of having progressive retinal atrophy (PRA), and five dogs not known to exhibit day-blindness. The parents of the affected dog were genotyped as carriers of the cd- mutation and their offspring as homozygous affected. The rest of the dogs were all genotyped as homozygous normal, including one day-blind AM (data not shown). A complete ophthalmic examination, including slit lamp biomicroscopy, indirect ophthalmoscopy, applanation tonometry, and streak retinoscopy, was performed and found to be within normal limits (Figure 3A). Systemically, the dog appeared to be healthy as assessed by complete physical examination, complete blood count, and chemistry panel. As a mean for preliminary assessment of retinal dys- function, chromatic pupillary light reflexes were evaluated qualitatively by use of diffuse red (630 nm – stimulation of long- and medium- wavelength absorbing cones) and blue (480 nm – stimulation of melanopsin-containing intrin- sically photosensitive retinal ganglion cells) lights of high intensity (200 kcd/m2) [22-24]. The severely reduced pupillary reflexes to the red light with normal pupillary reflexes to the blue light were suggestive of lost cone but normal inner retinal function (Figure 3B). Determining the deletion breakpoints of the CNGB3 mutation in the Alaskan malamute (AM) breed and establishing a diagnostic test Fine mapping identified a 404,820 bp deletion at position 35,699,378-36,104,197 that included the distal end of CPNE3-exon 1 and continued to include the rest of the gene, the complete CNGB3 gene, and the first four exons of CNBD1, to end in the middle of intron 4. Numbered black lines represent exons. Figure is not drawn to scale. presentation of the cd locus and the CNGB3 deletion. The initial screening confirmed the presence of four genes within 7A13, WWP1, CNBD1, and MMP16. Fine mapping identified a 404,820 bp deletion at position 35,699,378-36,104,197 that d of CPNE3-exon 1 and continued to include the rest of the gene, the complete CNGB3 gene, and the first four exons of middle of intron 4. Numbered black lines represent exons. Figure is not drawn to scale. Figure 1 Schematic presentation of the cd locus and the CNGB3 deletion. The initial screening confirmed the presence of four genes within the disease locus: SLC7A13, WWP1, CNBD1, and MMP16. Fine mapping identified a 404,820 bp deletion at position 35,699,378-36,104,197 that included the distal end of CPNE3-exon 1 and continued to include the rest of the gene, the complete CNGB3 gene, and the first four exons of CNBD1, to end in the middle of intron 4. Numbered black lines represent exons. Figure is not drawn to scale. 404,820 bp deletion (position 35,699,378-36,104,197). The deletion starts in the first coding exon of CPNE3 (Copine III) and includes the rest of the gene, the complete CNGB3 gene, and exons 1–4 of the CNBD1 gene (Figure 1). 12 months of age for visual deficits in bright daylight. The authors were made aware of other day-blind dogs related to the MAS in this study but were unable to obtain more information or blood samples from the breeder. Visual deficits were first noted at 3-months of age. This dog collided with objects in bright daylight outside but his vision greatly improved under dim light conditions, such as at night. The dog was unable to locate a ball while playing fetch in daylight unless he heard it bounce nearby or smelled it (Movie in Additional file 2). No other health problems were reported by the owner. Determining the deletion breakpoints of the CNGB3 mutation in the Alaskan malamute (AM) breed and establishing a diagnostic test To refine the breakpoints of the deletion, a forward primer from the proximal end of the interval (“left end 7 F” in pair 9, Table S1A in Additional file 1) was paired with two reverse primers from the distal end (“right set 4 1R” in pair 19 and “right set 4 1.5R” in pair 20, Table S1A in Additional file 1). Both of these primer pair combinations are more than 406 kb apart in a normal dog (CFA29: 35,698,350-36,104,507 and CFA29: 35,698,350-36,105,809) and are expected to fail in a PCR reaction. Results confirm this hypothesis: both primer pairs failed to amplify DNA from a normal dog. However, in the cd-affected AM-colony dog the first primer pair (left_end_7F/right_set4_1R) amplified a 1,336 bp fragment, and the second primer pair (left_end_7F/right_set4_1.5R) amplified a fragment larger than 2,600 bp. Sequencing the shorter fragment revealed a The main formation of dog breeds took place in the last 200 years. Some breeds have evolved from dogs with unknown ancestry and have been maintained as closed lines, while others were formed by cross-breeding of existing breeds. The relationship among dog breeds at the molecular level has been established [20,21]. It is not surprising that closely related breeds often share diseases caused by the same identical by descent (IBD) allele. It is less expected that a disease could be caused by an IBD mutation in not closely related breeds. Identification of common disease mutations among breeds that are not closely related indicates that large scale genetic screening of unrelated breeds can provide unexpected information about disease-causing alleles segregating in individual Yeh et al. BMC Genetics 2013, 14:27 http://www.biomedcentral.com/1471-2156/14/27 Yeh et al. BMC Genetics 2013, 14:27 Page 3 of 12 http://www.biomedcentral.com/1471-2156/14/27 ............. 1 2 17 18 404,820 bp deletion (position 35,699,378-36,104,197) CFA29 SLC7A13 CNGB3 Position 34,654,160-34,662,401 1 2 16 17 ............... CPNE3 WWP1 Position CPNE3 Position 35,699,270-35,731,014 CNGB3 Position 35,752,880-35,896,148 CNBD1 Position 36,034,290-36,380,294 MMP16 Position 36,921,131-37,055,570 35,507,176-35,623,260 ............. 1 2 3 4 5 CNBD1 10 11 Figure 1 Schematic presentation of the cd locus and the CNGB3 deletion. The initial screening confirmed the presence of four genes within the disease locus: SLC7A13, WWP1, CNBD1, and MMP16. Determining the deletion breakpoints of the CNGB3 mutation in the Alaskan malamute (AM) breed and establishing a diagnostic test When using both primer pairs in a multiplex reaction, only one band of 289 bp long is observed in affected dogs (B3, dogs number 1 and 4), two bands are observed in carrier dogs (B3, dogs 2, 3, 5, 6, 7, 10, and 13) and only one band of 242 bp long is observed in the normal dogs (B3, dogs 8, 9, 11, 12, 14, and 15). Figure 2 cd-PCR based test. A. A schematic presentation of the test: primers flanking the deletion (D_A_3F/D_A_3R), combined with primers within the deleted sequences (CNGB3_4F/CNGB3_4R) were designed to amplify an affected and a normal chromosome, respectively. Primers flanking the deletion would not amplify a normal chromosome since they are more than 404 kb apart, but would give a product of 289 bp from an affected chromosome. The primer pair within the deletion would only amplify a normal chromosome and result in a 242 bp fragment. Dashed line represents the deleted region. B. cd-PCR based test results are observed in a colony pedigree genotyped for the deletion. A PCR product of 289 bp molecular weight is observed when using primers flanking the deletion (D-A-3 F/3R) in affected dogs (B1, dogs number 1 and 4), and in carrier dogs (B1, dogs number 2, 3, 5, 6, 7, 10, and 13). A PCR product is observed when using primers within the deleted sequence (CNGB3_4F/4R) in normal dogs (B2, dogs number 8, 9, 11, 12, 14, and 15) and in carrier dogs (B2, dogs number 2, 3, 5, 6, 7, 10, and 13). When using both primer pairs in a multiplex reaction, only one band of 289 bp long is observed in affected dogs (B3, dogs number 1 and 4), two bands are observed in carrier dogs (B3, dogs 2, 3, 5, 6, 7, 10, and 13) and only one band of 242 bp long is observed in the normal dogs (B3, dogs 8, 9, 11, 12, 14, and 15). Sequencing of the positive product revealed that the animal was homozygous for the complete genomic deletion of the CNGB3 gene with the same breakpoints as in the cd- affected AMs (Figure 4). the AM and MAS, showing identical breakpoints of the deletion (Figure 4). No new mutated alleles were found in the MAS group, and none were found in the Samoyed group or the Australian shepherds. Determining the deletion breakpoints of the CNGB3 mutation in the Alaskan malamute (AM) breed and establishing a diagnostic test Electroreti- nography revealed an absence of cone-mediated function, whereas the rods continue to function normally (Figure 3C). Combined with the other clinical findings these functional abnormalities were most suggestive of achromatopsia. Genomic DNA was extracted from a whole blood sample to determine if the dog was affected by a mutation in the CNGB3 gene using the previously described multiplex PCR test. The PCR results confirmed a deletion within the same locus: amplification was positive while using the primers specific to the mutated allele (D_A_3F/D_A_3R in Figure 2A), and negative while using the primers specific to the wildtype allele (CNGB3_4F/CNGB3_4R in Figure 2A). Phenotype and genotype characterization of CNGB3-deletion-achromatopsia in a miniature Australian shepherd (MAS) One affected, male-neutered MAS from a family of day- blind dogs was initially presented at 5 months and then Yeh et al. BMC Genetics 2013, 14:27 Yeh et al. BMC Genetics 2013, 14:27 http://www.biomedcentral.com/1471-2156/14/27 Page 4 of 12 http://www.biomedcentral.com/1471-2156/14/27 Figure 2 cd-PCR based test. A. A schematic presentation of the test: primers flanking the deletion (D_A_3F/D_A_3R), combined with primers within the deleted sequences (CNGB3_4F/CNGB3_4R) were designed to amplify an affected and a normal chromosome, respectively. Primers flanking the deletion would not amplify a normal chromosome since they are more than 404 kb apart, but would give a product of 289 bp from an affected chromosome. The primer pair within the deletion would only amplify a normal chromosome and result in a 242 bp fragment. Dashed line represents the deleted region. B. cd-PCR based test results are observed in a colony pedigree genotyped for the deletion. A PCR product of 289 bp molecular weight is observed when using primers flanking the deletion (D-A-3 F/3R) in affected dogs (B1, dogs number 1 and 4), and in carrier dogs (B1, dogs number 2, 3, 5, 6, 7, 10, and 13). A PCR product is observed when using primers within the deleted sequence (CNGB3_4F/4R) in normal dogs (B2, dogs number 8, 9, 11, 12, 14, and 15) and in carrier dogs (B2, dogs number 2, 3, 5, 6, 7, 10, and 13). Determining the deletion breakpoints of the CNGB3 mutation in the Alaskan malamute (AM) breed and establishing a diagnostic test Allele frequencies in each breed sample were estimated as 2% in the MAS, 0.877% in the Siberian huskies, and were undetectable in the Samoyeds and Australian shepherds. A larger number of sled dogs will be required to get a better estimation of the mutated allele in that group of dogs. Screening for the mutated allele in other canine breeds g The presence of the CNGB3-deletion mutation in a MAS, a breed not known to be closely related to the AM [20], suggested that other breeds, more closely related to the AM, might carry the affected allele. We also wanted to expand our screening in the MAS population in an attempt to evaluate the frequency of the affected allele. For that, we collected DNA samples from dogs of five different breeds and unrelated to each other for at least three generations (no parents or grandparents in common): MAS (49 dogs), Australian shepherd (118 dogs), Siberian husky (57 dogs), Samoyed (60 dogs), and Alaskan sled dogs (38 dogs). The screening was done on pooled samples (see Methods for details). One Siberian husky and three Alaskan sled dogs were identified as carrying the cd- affected allele. All three sled dogs were from a subgroup of distance runners and did share a common ancestor four and five generations back. The Siberian husky and the three sled dogs’ mutated alleles were sequenced and aligned to Establishing identical by descent (IBD) in the canine breeds affected by CNGB3-deletion-achromatopsia and determining the minimal linkage disequilibrium (LD) Establishing identical by descent (IBD) in the canine breeds affected by CNGB3-deletion-achromatopsia and determining the minimal linkage disequilibrium (LD) To further support the idea that the affected alleles observed in the three different breeds are IBD, we sought to genotype the DNA flanking the deletion, and determine if all affected alleles share the same genetic variations. Seven primer pairs were used to amplify a region up to 5.5 kb upstream and up to 13.7 kb downstream of the mutation (Table S1B in Additional file 1). The genotypes were compared between a purebred, cd-affected AM and three AMs not affected with CNGB3-deletion- achromatopsia (one of the three unaffected AMs was an obligated carrier, the parent of the affected dog). We also Yeh et al. BMC Genetics 2013, 14:27 http://www.biomedcentral.com/1471-2156/14/27 Page 5 of 12 Figure 3 Ocular fundus, chromatic pupillary light reflexes, and electroretinogram of a day-blind MAS. A. Compiled image of the normal ocular fundus of the day-blind MAS. B. Chromatic pupillary light reflexes. i. Day-blind MAS: Normal pupillary constriction was observed with the blue light stimulus (480 nm, 200 kcd/m2), whereas no pupil constriction was observed with the red light stimulus (630 nm, 200 kcd/m2) of the same light intensity. ii. Normal canine: Complete pupillary constrictions were elicited with both bright red and blue light. C. Electroretinograms. Compared to a normal, age-matched, mixed-bred colony dog (ii) with normal cone-mediated ERG responses (ii, black arrows), the recordings of the day-blind MAS (i) revealed normal rod- but absent cone-mediated retinal function. ure 3 Ocular fundus, chromatic pupillary light reflexes, and electroretinogram of a day-blind MAS. A. Compiled image of the normal ar fundus of the day blind MAS B Chromatic pupillary light reflexes i Day blind MAS: Normal pupillary constriction was observed with the Ocular fundus, chromatic pupillary light reflexes, and electroretinogram of a day-blind MAS. A. Compiled imag Figure 3 Ocular fundus, chromatic pupillary light reflexes, and electroretinogram of a day-blind MAS. A. Compiled image of the normal ocular fundus of the day-blind MAS. B. Chromatic pupillary light reflexes. i. Day-blind MAS: Normal pupillary constriction was observed with the blue light stimulus (480 nm, 200 kcd/m2), whereas no pupil constriction was observed with the red light stimulus (630 nm, 200 kcd/m2) of the same light intensity. ii. Normal canine: Complete pupillary constrictions were elicited with both bright red and blue light. C. Electroretinograms. Establishing identical by descent (IBD) in the canine breeds affected by CNGB3-deletion-achromatopsia and determining the minimal linkage disequilibrium (LD) Compared to a normal, age-matched, mixed-bred colony dog (ii) with normal cone-mediated ERG responses (ii, black arrows), the recordings of the day-blind MAS (i) revealed normal rod- but absent cone-mediated retinal function. mutation were added (Table S1C in Additional file 1). One hundred and thirteen additional polymorphisms were identified, to total a 147 polymorphism haplotype in a 3.93 Mb interval (Table S3 in Additional file 1). The fine- mapping was carried out on five dogs: one carrier AM and its affected offspring, one affected AM-colony dog, one affected MAS, and one carrier Siberian husky. A normal boxer was also genotyped as a control reference. These five dogs represented eight affected chromosomes from three different breeds. The genotypes showed that the genotyped a cd-affected AM-colony dog, a purebred affected and unaffected MAS, and a normal boxer. Haplotype analysis identified a shared haplotype be- tween the cd-affected AM and the cd-affected MAS, composed of 34 polymorphisms in a ~20-kb segment (Table S2 in Additional file 1). This haplotype was not observed in the normal MAS or the AMs that did not carry the affected allele. To further determine how far the deletion-associated haplotype expands, 32 more amplicons surrounding the Page 6 of 12 Yeh et al. BMC Genetics 2013, 14:27 http://www.biomedcentral.com/1471-2156/14/27 Figure 4 Alignment of the cd-mutation in four breeds. Alignment of the sequences obtained from cd-affected alleles of four different breeds: AM, MAS, Siberian husky, and Alaskan sled dog. All four breeds had the same 404,820 bp deletion with identical breakpoints between the first coding exon of CPNE3 and intron 4 of CNBD1. Figure 4 Alignment of the cd-mutation in four breeds. Alignment of the sequences obtained from cd-affected alleles of four different breeds: AM, MAS, Siberian husky, and Alaskan sled dog. All four breeds had the same 404,820 bp deletion with identical breakpoints between the first coding exon of CPNE3 and intron 4 of CNBD1. purebred cd-affected AM and its carrier parent have a different haplotype from the AM-colony dog at the proximal end (32,670,916-34,655,058), suggesting a shared haplotype of 1.0–1.5 Mb (Table S3 in Additional file 1, and Figure 5A and B). The cd deletion allele from the purebred AMs and the cd-affected MAS shared the same 1.0–1.5 Mb haplotype (Figure 5B and C). Discussion Seddon and colleagues [27] found genetic heterogeneity of day-blindness in AMs in Australia. CNGB3-achromatopsia is the most common form of achromatopsia in humans [9,12]. In dogs, two different CNGB3 mutations were identified that result in the achromatopsia phenotype [13]. One is the D262N missense mutation in German shorthaired pointers, while the other is a genomic deletion of CNGB3 found in the AM [13]. Both of these mutations lead to the same disease phenotype with complete loss of cone function [14]. In our study, we found that affected AMs have a 404,820 bp deletion containing three known genes, CPNE3, CNGB3, and CNBD1. Since dogs with either the D262N missense mutation or the genomic deletion of CNGB3 show very similar disease phenotype, the loss of the CPNE3 and CNBD1 genes does not appear to have phenotypic con- sequences. CPNE3 is part of a family of proteins known as the copines. They all share a similar structure and interact with a range of cell signaling and cytoskeletal proteins in response to increases in intracellular calcium [25,26]. They are known to be responsible for calcium- dependent phospholipid binding [25,26]. CNBD1 has not been fully characterized. Future studies comparing the two types of CNGB3 mutations may shed light on the function and importance of CPNE3 and CNBD1 by identifying subtle phenotypic differences. We also report the occurrence of achromatopsia in a MAS based on the identical classical clinical phenotype and genomic CNGB3 deletion that was reported in the AM. Pooled-sample screening identified other arctic breeds closely related to the AM carrying the affected allele: one Siberian husky and three Alaskan sled dogs. We are not aware of any reports about day-blindness in these arctic breeds; however, the occurrence of achromatopsia would not be surprising based on our findings. The presence of the same large genomic CNGB3 deletion in several related and unrelated canine breeds suggests that these mutated alleles are IBD. To further confirm this idea, we expanded the locus genotyping to a 3.93 Mb interval. One hundred and forty seven SNPs were identified and confirmed an identical shared affected haplotype across the three breeds, a segment of 0.5–1.04 Mb in size. Comparing haplotypes of large segments of DNA between unrelated dogs gives the opportunity to observe historical recombin- ation, instead of meiosis recombination within a specific pedigree. Establishing identical by descent (IBD) in the canine breeds affected by CNGB3-deletion-achromatopsia and determining the minimal linkage disequilibrium (LD) The affected chromosome carried by the Siberian husky had the same proximal haplotype as the MAS but the distal end was dif- ferent than both the AM and the MAS starting at position 36,106,350 (Figure 5D). This reduces the shared haplotype across all breeds to 0.5 Mb. The two SNPs defining the minimal LD across all three breeds were 34,655,058 and 36,106,350 (1.04 Mb of sequenced interval on the affected chromosome). Within that interval, a 0.5-Mb segment at the proximal end was not genotyped (between 34,655,058 and 35,192,980; Figure 5, dashed frame). Therefore the shared affected haplotype across all breeds is a minimum of 0.5 Mb and a maximum of 1.04 Mb (Figure 5). The shared haplotype is composed of 53 polymorphisms (Table S3 in Additional file 1, red box). This shared haplotype supports the idea that the mutated alleles in the AM, Siberian husky and the MAS are IBD. Within the 0.5 Mb-interval, one microsatellite was identified, showing different alleles between the affected chromosomes (Table S3 in Additional file 1, polymorphisms number 77). This allelic difference most likely represents a mutation rather a recombination event, due to high mutation rate of microsatellites, and has not been included in the haplotype analysis. Yeh et al. BMC Genetics 2013, 14:27 http://www.biomedcentral.com/1471-2156/14/27 Page 7 of 12 Page 7 of 12 http://www.biomedcentral.com/1471-2156/14/27 A. B. C. D. Figure 5 Schematic presentation of the shared haplotype across five dogs from three different breeds. Alignment of the affected haplotype observed in five dogs carrying the cd-affected chromosome. Single nucleotide polymorphisms (SNPs) are represented by letters, and their location is denoted in the header. The minimal shared haplotype is boxed in red (0.5 Mb) and the maximum shared haplotype is marked with the dashed extended box (1.04 Mb). A. AM-derived colony dog affected with cd. B. Affected haplotype observed in a homozygous state in a purebred AM affected with cd and in a heterozygous state in its parent. C. MAS affected with cd. D. Siberian husky carrying the cd-affected chromosome. Only the affected chromosome is presented in the diagram. Distances from the mutation are represented in the bottom scale and are drawn to scale. A. B. C. D. Figure 5 Schematic presentation of the shared haplotype across five dogs from three different breeds. Establishing identical by descent (IBD) in the canine breeds affected by CNGB3-deletion-achromatopsia and determining the minimal linkage disequilibrium (LD) Alignme h l t b d i fi d i th d ff t d h Si l l tid l hi (SNP ) Figure 5 Schematic presentation of the shared haplotype across five dogs from three different breeds. Alignment of the affected haplotype observed in five dogs carrying the cd-affected chromosome. Single nucleotide polymorphisms (SNPs) are represented by letters, and their location is denoted in the header. The minimal shared haplotype is boxed in red (0.5 Mb) and the maximum shared haplotype is marked with the dashed extended box (1.04 Mb). A. AM-derived colony dog affected with cd. B. Affected haplotype observed in a homozygous state in a purebred AM affected with cd and in a heterozygous state in its parent. C. MAS affected with cd. D. Siberian husky carrying the cd-affected chromosome. Only the affected chromosome is presented in the diagram. Distances from the mutation are represented in the bottom scale and are drawn to scale Discussion This is a powerful tool when the dogs that are chosen are the least related to each other within a breed, as well as across different breeds. The shared affected haplo- type observed in the purebred affected AM was reduced from 3.53 Mb segment to 1.0–1.5 Mb when compared to affected MAS, and then further reduced to 0.5–1.04 Mb when compared to a third breed, the Siberian husky. One day-blind AM was genotyped normal for the CNGB3-deletion mutation. We do not have pedigree infor- mation or access to DNA samples from its close relatives; therefore, with these limitations we can only suggest heterogeneity for day-blindness in the AM breed, though non-genetic basis for that particular dog is not excluded. Linkage disequilibrium (LD) mapping has become a useful tool for genomic studies and has been previously used in studies of inherited canine retinal diseases [28-30]. Page 8 of 12 Yeh et al. BMC Genetics 2013, 14:27 http://www.biomedcentral.com/1471-2156/14/27 Yeh et al. BMC Genetics 2013, 14:27 http://www.biomedcentral.com/1471-2156/14/27 As exemplified in the case study presented here, dogs which share the same ancestral mutated chromosome, also share the same haplotype within a region flanking the mutation, a region that is in LD with the disease. This is especially powerful when the same disease is observed in more than one breed [28,31,32]. The results in this study suggest that CNGB3 deletion is an ancestral mutation that originated from a dog that served as a common founder. Although most of the dogs carrying the achromatopsia mutation possess an arctic lineage, we have also found it in the MAS whose distinct physical characteristic, func- tional, and genetic background suggest a different breed origin. The MAS breed was developed in the late 1960s as smaller Australian shepherds were selectively bred in order to achieve the desired size. Contrary to the name of the breed, Australian shepherds were relatively recently developed in the western United States in the 19th and early 20th century. In contrast, the AM is thought to be among the more ancient breeds [20]. During the time of the Klondike Gold Rush in the 19th century, the AM became valuable and was frequently crossbred with other breeds, possibly including the Australian shepherd. Achromatopsia was first observed in an inbred strain of the AM in the 1960s [15-18]. Conclusion We found genomic CNGB3 deletion in breeds not previ- ously known to carry the mutation and showed that there is a founder effect. Our findings suggest that this mutation and the resulting day-blindness are likely present in other canine breeds that have not yet been identified with this disorder, such as the Siberian husky and other arctic breeds. We also reported the occurrence of CNGB3-deletion- achromatopsia in a new canine breed, the MAS. Discussion gene MDR1: Several collie-related breeds and two sight hounds, not expected to share collie ancestry, were found to segregate the affected allele which was determined to be IBD since it was conserved among these affected breeds [35]. Another example is the mutation causing progressive rod-cone degeneration (prcd) in over 20 dog breeds representing all the breed groups defined by the American Kennel Club (herding, hound, working, terrier, toy, sporting and non-sporting as well as miscellaneous) [28]. This apparent disparate association is similar to our findings of the identical CNGB3 mutation in the MAS and three arctic breeds. Day-blindness is a rather rare finding in canines with only CNGB3-achromatopsia having been thoroughly characterized. Based on eye certification data [36] and our DNA testing results over the past 20 years, we suspect that CNGB3-achromatopsia has become rare or even non- existent in North American AMs, but it is still reported in Australia [27,37]. Except for a transgenic CNGB3-knockout mouse [38], the dogs discussed here represent the only known animal model of CNGB3-achromatopsia, the most common form of the disease in humans [9,12]. Naturally occurring achromatopsia based on mutations in other genes have been described in Awassi sheep (CNGA3) [39,40] and in mice (CNGA3, GNAT2 and PDE6C) [41-43]. These animals provide valuable models for translational research, including the development of new therapies [14,41,44]. The Alaskan sled dogs are a population of dogs with a northern breed ancestry and were developed through the selection and breeding of several dog breeds based on their athletic abilities. They are mixed breed dogs comprised of several different lineages and can be separated into two clusters, sprint and distance, based on their racing style [33]. It was found that the AM and Siberian husky contributed to enhanced endurance, the pointer and saluki contributed to enhanced speed, and the Anatolian shepherd contributed to work ethic [33]. The presence of both AM and Siberian husky in their ancestry suggests that the mutated cd-allele in these sled dogs could have been contributed by either breed. While it is established that the AM, Siberian husky, and Alaskan sled dog are closely related and belong within the same ancestral cluster [20,21,33], the relation of AM to MAS is less clear. Therefore, based on breed history, it is difficult to determine when a mixture between any of these breeds occurred that introduced the mutation. Determining the deletion breakpoints and establishing a diagnostic test This device consists of a bright (200 kcd/m2) red (630 nm) and blue (480 nm) LED light for the stimulation of canine long- and medium-wavelength-absorbing (L/M) cones and melanopsin-containing intrinsically photosensitive retinal ganglion cells (ipRGCs), respectively [22]. Con- striction of pupil was assessed during illumination with each light for 10 seconds with a 30-second recovery period between tests. and approved by the University of Pennsylvania IACUC. Primers were designed to amplify genomic fragments within the predicted region where the deletion had taken place, based on the CanFam 2 assembly (http://genome. ucsc.edu/cgi-bin/hgGateway; Table S1 in Additional file 1). Products were evaluated for presence/absence and size by comparing the two samples. Targeted sequences were initially chosen within the CNGB3 gene neighboring sequences and subsequently narrowed down to pinpoint the breakpoints. PCR products were sequenced using the Applied Biosystems Automated 3730 DNA analyzer (Applied Biosystems, Foster City, CA, USA), and analyzed using Sequencher 4.2.2 software (Gene Codes Corporation, Ann Arbor, MI, USA). After establishing the breakpoints, primers were designed to identify cd-affected, cd-carrier and normal dogs by PCR, amplifying the normal and mutated alleles in separated reactions as well as in one multiplex PCR reaction. One primer pair (CNGB3_4F: CGACTCTATCTCCTGTGG CTCT, CNGB3_4R: ATTTGTCAGTTTCTGCTTCTCC), which is located within the deleted sequence and therefore is specific to the normal allele, would amplify a 242 bp fragment from a normal chromosome. The second primer pair (D_A_3F: CAAAGTCGGACCCTTTATGTG, D_A_3R: GGCCAAATAGTAGTTCCTGAAA), which is located in sequences flanking the deletion and therefore is specific to the affected allele, would amplify a 289 bp fragment only from an affected chromosome, since on a normal chromosome these primers are more than 404 kb apart (Figure 2). A carrier dog would present both products. PCR products were visualized on a 1.8% agarose gel stained with ethidium bromide. Ten purebred AMs were screened. Those included one dog diagnosed with day-blindness and its parents, one PRA suspicious dog, one dog with day-blindness, and five dogs not known to have day-blindness. The DNA of one day-blind MAS was tested for the presence of the CNGB3-deletion mutation. AM-derived cd-affected and cd-carrier colony dogs, as well as normals, were used as controls. PCR products using D_A_3F/D_A_3R primers were se- quenced and aligned for comparison, using SequencherW 4.2.2 Software. In order to perform ophthalmic examinations, the pupils were dilated by pharmacologic mydriasis using 1% tropicamide ophthalmic solution. Electroretinography Rod and cone photoreceptor mediated retinal function was evaluated by electroretinography. Standard Ganzfeld scotopic and photopic electroretinograms (ERGs) were recorded from the purpose-bred colony dogs under general anesthesia using previously described testing procedures [14]. In the MAS described in this study, the pupils were dilated with 1% tropicamide ophthalmic solution. ERGs were recorded under sedation with intravenous acepromazine maleate (0.01 mg/kg) and butorphanol tartrate (0.1 mg/kg). Two subdermal platinum needle electrodes (Grass Technologies, Astro-Med, Inc., West Warwick, RI, USA) were placed on the forehead between the two eyes (reference) and over the occipital protuberance (ground). The ocular surface was anes- thetized with 0.5% proparacaine ophthalmic solution before the placement of a Kooijman/Damhof ERG lens (Acrivet, Henningsdorf, Germany). This lens combines the corneal contact lens electrode for recording with a white LED light source for retinal stimulation. The responses were recorded by use of the RETIport ERG system (Acrivet, Henningsdorf, Germany). Single rod and mixed rod-cone responses were recorded after Determining the deletion breakpoints and establishing a diagnostic test The anterior segments of the dogs’ eyes were examined by slit-lamp biomicroscopy (Kowa SL-14). Fundic examinations were performed with a portable binocular indirect ophthalmoscope (Keeler All Pupil II; Keeler Instruments, Broomall, PA, USA) and a condensing lens (Pan Retinal 2.2D; Volk Optical, Mentor, OH, USA). Refraction occurred by streak retinoscopy in both the horizontal and vertical meridians using the hand held Welch Allyn retinoscope and Luneau Skiascopy Rack Set (Wilson Ophthalmic, Mustang, OK). Ocular surfaces were anesthetized with 0.5% proparacaine ophthalmic solution for measurement of the intraocular pressures by use of an applanation tonometer (Tono-Pen XL; Reichert, Depew, NY, USA). Determining the deletion breakpoints and establishing a diagnostic test DNA was extracted from blood of a normal and an AM-colony cd-affected dog using QIAmp DNA Blood Mini Kit (Qiagen, Valencia, CA, USA), following manufac- turer protocol. This research colony is maintained at the Retinal Disease Studies Facility of the University of Pennsylvania (Kennett Square, PA, USA) and supported by the National Eye Institute, NIH (R01-EY006855) and a Foundation Fighting Blindness (FFB) Center grant (see review on the establishment of the colony by Sidjanin and colleagues [13]). All procedures involving animals were done in compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research There are similar reports of the founder effect in other genetic diseases in dogs. Founder effect may be more obvious in more closely related breeds. Examples include the mutation in ADAMTS17, which causes primary lens luxation in many breeds, most of which are terriers or breeds with terrier co-ancestry [32], and a 7.8-kb deletion in the non-homologous end-joining factor 1 (NHEJI1) that is responsible for collie eye anomaly in several breeds, most of which fall into a cluster of collie-like dogs [34]. Further example includes multidrug sensitivity, which is caused by a mutation in the canine multidrug resistant Yeh et al. BMC Genetics 2013, 14:27 http://www.biomedcentral.com/1471-2156/14/27 Page 9 of 12 Yeh et al. BMC Genetics 2013, 14:27 http://www.biomedcentral.com/1471-2156/14/27 blue light in a dark room. The responses to white light were elicited with a finoff transilluminator (Welch Allyn, Skaneateles Falls, NY, USA) and a slit lamp biomicroscope (Kowa SL-14; Kowa Co. Ltd., Tokyo, Japan). The Melan- 100 system (Biomed Vision Technologies, Ames, IA, USA) was used as a source for the colored light. This device consists of a bright (200 kcd/m2) red (630 nm) and blue (480 nm) LED light for the stimulation of canine long- and medium-wavelength-absorbing (L/M) cones and melanopsin-containing intrinsically photosensitive retinal ganglion cells (ipRGCs), respectively [22]. Con- striction of pupil was assessed during illumination with each light for 10 seconds with a 30-second recovery period between tests. blue light in a dark room. The responses to white light were elicited with a finoff transilluminator (Welch Allyn, Skaneateles Falls, NY, USA) and a slit lamp biomicroscope (Kowa SL-14; Kowa Co. Ltd., Tokyo, Japan). The Melan- 100 system (Biomed Vision Technologies, Ames, IA, USA) was used as a source for the colored light. Additional files 20 minutes of dark adaptation by use of single flash stimuli of 0.03 cd.s/m2 and 3 cd.s/m2, respectively. A total of 20 responses were averaged each at intervals of 10 and 2 seconds, respectively. A 10 minute light adaptation was completed with light from the Kooijman/Damhof ERG lens (25 cd/m2). Subsequently, single cone and 28-Hz cone flicker responses were recorded using flash stimuli of 3 cd.s/m2 intensity. Twenty traces were averaged for both photopic responses at 2-second intervals. Additional file 1: Table S1. Primer names, sequences and locations. A. Primers used to establish the breaking points of the cd mutation and the corresponding presence or absence of the PCR product in cd-affected dog. In bold are the primers flanking the deletion used to identified the sequence of the affected chromosome and identify the accurate breaking point in the DNA. B. Primers used to establish the affected haplotype linked to the mutation in a ~20 Kb interval. C. Primers used to establish the minimum LD between the affected alleles in the AM, the MAS and the Siberian husky in a 3.93 Mb interval. Table S2 Affected and normal haplotypes in the cd-locus in AM and MAS. Abbreviations: del, deletion; wt, wildtype; mut, CNGB3-deletion mutation. Table S3 Genotype results of five dogs to total eight cd-affected chromosomes from three different breeds. Boxed in red is the minimal LD, 0.913-1.45 Mb long and observed across all affected chromosomes. Highlighted in colors are the informative SNPs with different colors to each allele. Abbreviations: del, deletion; wt, wildtype; mut, CNGB3-deletion mutation. Clinical examination of dogs Day-blindness was diagnosed by behavioral vision testing under photopic and scotopic light conditions. In colony dogs, the visual performance under different light condi- tions was evaluated by use of an obstacle-avoidance test as previously described [45]. For the privately owned MAS behavioral vision testing was performed by observing the dog maneuver around objects and its ability to find toys under different light conditions. The retinal function of this day-blind MAS was first evaluated by assessment of the pupillary light reflexes with diffuse white, red, and Yeh et al. BMC Genetics 2013, 14:27 http://www.biomedcentral.com/1471-2156/14/27 Page 10 of 12 Yeh et al. BMC Genetics 2013, 14:27 http://www.biomedcentral.com/1471-2156/14/27 Authors’ contributions CYY and AMK carried out all clinical examination and diagnostics, and wrote the initial draft of the manuscript. OG carried out the molecular genetics study and drafted part of the manuscript. AVK participated in deletion characterization and edited the manuscript. DH, AK, and HJH collaborated in phenotyping dogs and sample collection. SPK participated in the design and coordination of the study and helped draft the manuscript. CYY, OG, AMK, GMA conceived of the study and participated in its design and coordination. All authors read and approved the final manuscript. Establishing IBD status with the genomic CNGB3-deletion in the cd-affected dogs and determining the minimal linkage disequilibrium (LD) GMA and SPK have a financial interest in the development of genetic testing/diagnostic test and own equity in a company (OptiGen, LLC, Ithaca, NY, USA). The remaining authors have declared that no conflict of interest exists. The affected haplotype surrounding the deletion point was established by sequencing PCR amplicons using seven primer pairs (Table S1B in Additional file 1). Eight dogs were sequenced and compared to confirm IBD: one mixed breed cd-affected AM-colony dog [13], one purebred cd-affected AM, one purebred cd-carrier AM, two purebred AMs not carrying the cd mutation, one normal boxer, one normal MAS, and the day-blind MAS affected by cd. Screening other breeds for the presence of the genomic CNGB3-deletion Blood was collected from all the dogs and genomic DNA extracted as described above. In addition to the testing of the privately owned, day-blind MAS, the following strategy was pursued to screen canine breeds for the CNGB3-deletion mutation: DNA samples were pooled for PCR reaction in the Australian shepherds (118 dogs from 22 countries, in 12 pooled samples), MAS (49 dogs from six countries, in five pooled samples), Siberian huskies (57 dogs from 13 countries, in six pooled samples), Samoyeds (60 dogs from seven countries, in six pooled samples) and Alaskan sled dogs (38 dogs; 23 sprints runners and 15 distance runners). Two pooled controls were used as a proof of principle of the sensitivity of the pooling strategy: one cd-affected dog pooled with nine normal dogs, and one cd-carrier dog pooled with nine normal dogs. Within each breed, dogs were chosen such that there were no grandparents in common. Additional file 2: Movie showing the cd-affected MAS trying to fetch a ball under day-light conditions. The dog could only find the ball by smell, not by sight. Abbreviations ADAMTS17: A disintegrin and metalloproteinase with thrombospondin motif 17; AM: Alaskan malamute; cd: Cone degeneration; CFA: Canis familiaris autosome; CNBD1: Cyclic nucleotide binding domain containing 1; CNGA3: Alpha subunit of cone cyclic-nucleotide gated channel; CNGB3: Beta subunits of cone cyclic-nucleotide gated channel; GNAT2: Alpha subunit of cone transducin; IBD: Identical by descent; LD: Linkage disequilibrium; MAS: Miniature Australian shepherd; MDR1: Multidrug resistance gene 1; MMP16: matrix metallopeptidase 16; NHEJI1: Non-homologous end-joining factor 1; PDE6C: Alpha subunit of cone cyclic guanosine monophosphate (cGMP) phosphodiesterase; PDE6H: Gamma subunit of cone cyclic guanosine monophosphate (cGMP) phosphodiesterase; PRA: Progressive retinal atrophy; prcd: progressive rod-cone degeneration; SLC7A13: solute carrier family 7 (cationic amino acid transporter, y + system) member 13; WWP1: WW domain containing E3 ubiquitin protein ligase 1. Acknowledgements and funding In order to determine the expansion of the cd-associated haplotype with the genomic CNGB3-deletion, a set of 32 primer pairs (Table S1C in Additional file 1) was designed to amplify regions within a 3.93-Mb interval flanking the mutation (3.03 Mb distal to the mutation: 32,670,680 to mutation, and 0.5 Mb proximal to the mutation: mutation to 36,605,630). Five dogs carrying cd-affected alleles from three different breeds were genotyped: one AM cd-carrier, one AM cd-affected, one cd-affected AM-colony dog, one cd-affected MAS, and one cd-carrier Siberian husky. A normal boxer was sequenced to serve as a reference. This study was supported by NIH grants R01-EY019304, R01-EY006855, P30-EY001583, T32-RR007063 and the Foundation Fighting Blindness (FFB). We thank the team (Nicole MacLaren and Justin Dees) at Eye Care for Animals in Salt Lake City, UT (USA) for allowing us to use their facility. 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Meridian, ID: American College of Veterinary Ophthalmologists; 2010. 13. Sidjanin DJ, Lowe JK, McElwee JL, Milne BS, Phippen TM, Sargan DR, Aguirre GD, Acland GM, Ostrander EA: Canine CNGB3 mutations establish cone degeneration as orthologous to the human achromatopsia locus ACHM3. Hum Mol Genet 2002, 11:1823–1833. 37. Stanley RG, Acland GM, Vingrys A, Hardman C, Turner A, Smith REI, Hughes I: Day blindness in Alaskan malamutes in Australia: Clinical and electroretinographic findings. In Proceedings of the 29th Annual Meeting of the American College of Veterinary Ophthalmologists: 21–24 October 1998; Seattle, WA. 1998:22. 14. Komaromy AM, Alexander JJ, Rowlan JS, Garcia MM, Chiodo VA, Kaya A, Tanaka JC, Acland GM, Hauswirth WW, Aguirre GD: Gene therapy rescues cone function in congenital achromatopsia. Hum Mol Genet 2010, 19:2581–2593. 38. Yeh et al. BMC Genetics 2013, 14:27 http://www.biomedcentral.com/1471-2156/14/27 Yeh et al. BMC Genetics 2013, 14:27 http://www.biomedcentral.com/1471-2156/14/27 College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA. 4Department of Animal Sciences, College of Agricultural, Consumer and Environmental Sciences, The University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA. 5Willow Creek Pet Center, Cottonwood Heights, UT 84093, USA. 6Eye Care for Animals, Salt Lake City, UT 84106, USA. 7Bovine Functional Genomics Laboratory, Agricultural Research Services, United States Department of Agriculture, Beltsville, MD 20705, USA. 8OptiGen, LLC, Ithaca, NY 14850, USA. 17. Rubin LF: Hemeralopia in Alaskan Malamute pups. J Am Vet Med Assoc 1971, 158:1699–1701. 18. Rubin LF: Clinical features of hemeralopia in the adult Alaskan malamute. J Am Vet Med Assoc 1971, 158:1696–1698. 19. 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Michaelides M, Hunt DM, Moore AT: The cone dysfunction syndromes. Br J Ophthalmol 2004, 88:291–297. 1. Michaelides M, Hunt DM, Moore AT: The cone dysfunction syndromes. Br J Ophthalmol 2004, 88:291–297. 2. Thiadens AA, den Hollander AI, Roosing S, Nabuurs SB, Zekveld-Vroon RC, Collin RW, De Baere E, Koenekoop RK, van Schooneveld MJ, Strom TM, et al Homozygosity mapping reveals PDE6C mutations in patients with early- onset cone photoreceptor disorders. Am J Hum Genet 2009, 85:240–247. 23. Jacobs GH, Deegan JF 2nd, Crognale MA, Fenwick JA: Photopigments of dogs and foxes and their implications for canid vision. Vis Neurosci 1993, 10:173–180. 24. Neitz J, Geist T, Jacobs GH: Color vision in the dog. Vis Neurosci 1989, 3:119–125. 3. Kohl S, Coppieters F, Meire F, Schaich S, Roosing S, Brennenstuhl C, Bolz S, van Genderen MM, Riemslag FC, European Retinal Disease Consortium, et al A nonsense mutation in PDE6H causes autosomal-recessive incomplete achromatopsia. Am J Hum Genet 2012, 91:527–532. 3. Kohl S, Coppieters F, Meire F, Schaich S, Roosing S, Brennenstuhl C, Bolz S, van Genderen MM, Riemslag FC, European Retinal Disease Consortium, et al: A nonsense mutation in PDE6H causes autosomal-recessive incomplete achromatopsia. Am J Hum Genet 2012, 91:527–532. 25. Nalefski EA, Falke JJ: The C2 domain calcium-binding motif: structural and functional diversity. Protein Sci 1996, 5:2375–2390. 26. Rizo J, Sudhof TC: C2-domains, structure and function of a universal Ca2 + −binding domain. J Biol Chem 1998, 273:15879–15882. 4. Kohl S, Baumann B, Rosenberg T, Kellner U, Lorenz B, Vadala M, Jacobson SG, Wissinger B: Mutations in the cone photoreceptor G-protein alpha-subunit gene GNAT2 in patients with achromatopsia. Am J Hum Genet 2002, 71:422–425. 4. Kohl S, Baumann B, Rosenberg T, Kellner U, Lorenz B, Vadala M, Jacobson SG, Wissinger B: Mutations in the cone photoreceptor G-protein alpha-subunit gene GNAT2 in patients with achromatopsia. Am J Hum Genet 2002, 71:422–425. 27. Seddon JM, Hampson EC, Smith RI, Hughes IP: Genetic heterogeneity of day blindness in Alaskan Malamutes. Anim Genet 2006, 37:407–410. 5. Author details 1 1Department of Clinical Studies, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. 2Department of Small Animal Clinical Sciences, College of Veterinary Medicine, Michigan State University, East Lansing, MI 48824, USA. 3James A. Baker Institute for Animal Health, Page 11 of 12 Yeh et al. BMC Genetics 2013, 14:27 http://www.biomedcentral.com/1471-2156/14/27 References Ding XQ, Harry CS, Umino Y, Matveev AV, Fliesler SJ, Barlow RB: Impaired cone function and cone degeneration resulting from CNGB3 deficiency: down-regulation of CNGA3 biosynthesis as a potential mechanism. Hum Mol Genet 2009, 18:4770–4780. 15. Aguirre GD, Rubin LF: The electroretinogram in dogs with inherited cone degeneration. Invest Ophthalmol 1975, 14:840–847. 39. Shamir MH, Ofri R, Bor A, Brenner O, Reicher S, Obolensky A, Averbukh E, Banin E, Gootwine E: A novel day blindness in sheep: epidemiological, behavioural, electrophysiological and histopathological studies. Vet J 2010, 185:130–137. 16. Aguirre GD, Rubin LF: Pathology of hemeralopia in the Alaskan malamute dog. Invest Ophthalmol 1974, 13:231–235. Page 12 of 12 Page 12 of 12 Yeh et al. BMC Genetics 2013, 14:27 http://www.biomedcentral.com/1471-2156/14/27 Yeh et al. BMC Genetics 2013, 14:27 http://www.biomedcentral.com/1471-2156/14/27 40. Reicher S, Seroussi E, Gootwine E: A mutation in gene CNGA3 is associated with day blindness in sheep. Genomics 2010, 95:101–104. 41. Pang JJ, Deng WT, Dai X, Lei B, Everhart D, Umino Y, Li J, Zhang K, Mao S, Boye SL, et al: AAV-mediated cone rescue in a naturally occurring mouse model of CNGA3-achromatopsia. PLoS One 2012, 7:e35250. 42. Chang B, Grau T, Dangel S, Hurd R, Jurklies B, Sener EC, Andreasson S, Dollfus H, Baumann B, Bolz S, et al: A homologous genetic basis of the murine cpfl1 mutant and human achromatopsia linked to mutations in the PDE6C gene. Proc Natl Acad Sci U S A 2009, 106:19581–19586. 43. Chang B, Dacey MS, Hawes NL, Hitchcock PF, Milam AH, Atmaca-Sonmez P, Nusinowitz S, Heckenlively JR: Cone photoreceptor function loss-3, a novel mouse model of achromatopsia due to a mutation in Gnat2. Invest Ophthalmol Vis Sci 2006, 47:5017–5021. 44. Alexander JJ, Umino Y, Everhart D, Chang B, Min SH, Li Q, Timmers AM, Hawes NL, Pang JJ, Barlow RB, et al: Restoration of cone vision in a mouse model of achromatopsia. Nat Med 2007, 13:685–687. 44. Alexander JJ, Umino Y, Everhart D, Chang B, Min SH, Li Q, Timmers AM, Hawes NL, Pang JJ, Barlow RB, et al: Restoration of cone vision in a mouse model of achromatopsia. Nat Med 2007, 13:685–687. 45. Garcia MM, Ying GS, Cocores CA, Tanaka JC, Komaromy AM: Evaluation of a behavioral method for objective vision testing and identification of achromatopsia in dogs. Am J Vet Res 2010, 71:97–102. doi:10.1186/1471-2156-14-27 Cite this article as: Yeh et al.: Genomic deletion of CNGB3 is identical by descent in multiple canine breeds and causes achromatopsia. BMC Genetics 2013 14:27. Yeh et al. BMC Genetics 2013, 14:27 http://www.biomedcentral.com/1471-2156/14/27 Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit
https://openalex.org/W4389383317	https://periodicos.furg.br/divedu/article/download/15401/10312	Portuguese	null	HORROR HETERONORMATIVO	Revista Diversidade & Educação	2,023	cc-by	12,354	1 Doutorando em Educação. Universidade Luterana do Brasil, Canoas, Rio Grande do Sul, Brasil. RESUMO O presente artigo objetiva analisar as representações de gênero e sexualidade produzidas em They/Them (2022), longa-metragem do diretor estadunidense John Logan, assim como as pedagogias do horror que emergem de sua produção. A partir do campo dos Estudos Culturais, e através de recortes produzidos a partir da adoção de um tripé metodológico, composto por etnografia em tela, análise cultural e metodologia visual crítica, busca-se perceber e evidenciar o horror que se manifesta na heterogeneidade. O artigo evidencia a potencialidade das produções cinematográficas de horror em refletir acerca de questões incômodas e problemáticas contemporâneas. Em They/Them, a manifestação do horror prescinde da figura monstruosa em si, presente em muitas produções clássicas do gênero, apresentando-se através do discurso heteronormativo e de suas consequências para com os sujeitos que transgridem e subvertem as normas. PALAVRAS-CHAVE: Gênero e sexualidade. Heteronormatividade. Representação. ALAVRAS-CHAVE: Gênero e sexualidade. Heteronormatividade. Representação. edagogias do horror. Horror Heteronormativo: representações de gênero e sexualidade e pedagogias do horror no filme They/Them (2022) de John Logan 547 Horror Heteronormativo: representações de gênero e sexualidade e pedagogias do horror no filme They/Them (2022) de John Logan 547 Horror Heteronormativo: representações de gênero e sexualidade e pedagogias do horror no filme They/Them (2022) de John Logan 547 Horror Heteronormativo: representações de gênero e sexualidade e pedagogias do horror no filme They/Them (2022) de John Logan 547 547 HORROR HETERONORMATIVO: REPRESENTAÇÕES DE GÊNERO E SEXUALIDADE E PEDAGOGIAS DO HORROR NO FILME THEY/THEM (2022) DE JOHN LOGAN HETERONORMATIVE HORROR: REPRESENTATIONS OF GENDER AND SEXUALITY AND PEDAGOGIES OF HORROR IN THE FILM THEY/THEM (2022) BY JOHN LOGAN Lucas Bitencourt Fortes1 ABSTRACT hi i l This article analyzes the representations of gender and sexuality produced in They/Them (2022), a feature film by American director John Logan, as well as the pedagogies of horror that emerge from its production. From the field of Cultural Studies, and through clippings produced from the adoption of a methodological tripod, composed of screen ethnography, cultural analysis and critical visual methodology, we seek to perceive and highlight the horror that manifests itself in heterogeneity. The article highlights the potential of horror film productions to reflect on uncomfortable and problematic contemporary issues. In They/Them, the manifestation of horror dispenses with the monstrous figure itself, present in many classic productions of the genre, presenting itself through heteronormative discourse and its consequences for subjects who transgress and subvert norms. j g KEYWORDS: Gender and sexuality. Heteronormativity. Representation. Horror pedagogies. * * * 2 Sigla de Lésbicas, Gays, Bissexuais, Transexuais/Transgêneros/Travestis, Queer, Intersexual, Assexual, Pansexual, etc. RESUMEN Este artículo tiene como objetivo analizar las representaciones de género y sexualidad producidas en They/Them (2022), largometraje del director estadounidense John Logan, así como las pedagogías del horror que se desprenden de su producción. Desde el Revista Diversidade e Educação, v. 11, n. 1, p. 547-578, 2023. Doi: 10.14295/de.v11i1.15401 E-ISSN: 2358-8853 Horror heteronormativo: representações de gênero e sexualidade e pedagogias do horror no filme They/Them (2022) de John Logan Horror heteronormativo: representações de gênero e sexualidade e pedagogias do horror no filme They/Them (2022) de John Logan 548 campo de los Estudios Culturales, ya través de extractos producidos a partir de la adopción de un trípode metodológico, compuesto por etnografía de pantalla, análisis cultural y metodología visual crítica, buscamos percibir y evidenciar el horror que se manifiesta en la heterogeneidad. El artículo destaca el potencial de las producciones cinematográficas de horror para reflexionar sobre temas contemporáneos incómodos y problemáticos. En They/Them, la manifestación del horror prescinde de la propia figura monstruosa, presente en muchas producciones clásicas del género, presentándose a través del discurso heteronormativo y sus consecuencias para los sujetos que transgreden y subvierten las normas. PALABRAS-CLAVE: Género y sexualidad. Heteronormatividad. Representación. Pedagogías del horror. Revista Diversidade e Educação, v. 11, n. 1, p. 547-578, 2023. Doi: 10.14295/de.v11i1.15401 Introdução O presente artigo parte das provocações produzidas pelo presente dossiê, sobretudo pensando o quanto determinados grupos, muitas vezes minoritários, encontram-se vulneráveis, marginalizados e sujeitos as mais diferentes formas de violência. Parte, ainda, do entendimento de que a mídia se constitui em uma potente ferramenta de visibilização de problemáticas da contemporaneidade, oportunizando outros olhares a respeito de temas e sujeitos ora silenciados. Com isso, o foco da discussão proposta recai sobre o lugar ocupado pela população LGBTQIAP+2 na sociedade brasileira. Essa que vem ganhando cada vez mais destaque em diversas esferas, sejam elas sociais, políticas e acadêmicas, embora deva-se dizer ainda não o suficiente, em conta da dura e violenta realidade a qual é Revista Diversidade e Educação, v. 11, n. 1, p. 547-578, 2023. Doi: 10.14295/de.v11i1.15401 E-ISSN: 2358-8853 Revista Diversidade e Educação, v. 11, n. 1, p. 547-578, 2023. Doi: 10.14295/de.v11i1.15401 Horror Heteronormativo: representações de gênero e sexualidade e pedagogias do horror no filme They/Them (2022) de John Logan 549 submetida, e que pode ser confirmada a partir de dados e pesquisas recentes que apontam para uma crescente violência contra essa população (OBSERVATÓRIO, 2022). Muito desta crescente violência pode ser atribuída à lógica que envolve a heteronormatividade, que empurra a população LGBTQIAP+ à marginalização e exclusão, enquanto aprova a heterossexualidade como norma e/ou padrão social (SOUZA; SILVA; SANTOS, 2016). A partir da imposição desta raiz heteronormativa, se passa a atribuir significados positivos às pessoas que aderem a tal norma, assim como a seus símbolos e seus produtos culturais. Em contraposição, atribui-se significados negativos ao que se relaciona às pessoas homossexuais, seus símbolos e produtos culturais (ANUÁRIO, 2022). Conforme destacam Pinho e Pulcino, as heterossexualidades, tidas como naturais: [...] constituem-se em uma matriz que conferirá sentido às diferenças entre os gêneros. Da mesma forma, a reiteração dos papéis de gênero garante o discurso das heterossexualidades. A esta ordem social naturalizada, chamamos heteronormatividade. Sendo assim, a sociedade em que vivemos pode ser classificada como heteronormativa. (2016, p. 673). [...] constituem-se em uma matriz que conferirá sentido às diferenças entre os gêneros. Da mesma forma, a reiteração dos papéis de gênero garante o discurso das heterossexualidades. A esta ordem social naturalizada, chamamos heteronormatividade. Sendo assim, a sociedade em que vivemos pode ser classificada como heteronormativa. (2016, p. 673). Neste estudo, compreende-se a mídia como meio ou espaço por onde uma determinada mensagem é transmitida. 4 Entre os trabalhos de John Logan cabe destacar a criação da série de horror Penny Dreadful e participação nos roteiros de Gladiador (2000), de Ridley Scott, O Aviador (2004), de Martin Scorsese, e Sweeney Todd: O Barbeiro Demoníaco da Rua Fleet (2007), de Tim Burton. 3 No Brasil a produção manteve o nome original acrescentando o subtítulo “O Acampamento”. 4 Introdução Dentro desta concepção, têm-se o rádio, a imprensa, a televisão, a música, os jornais, as revistas, o cinema, por exemplo, como exemplos de mídias (KELLNER, 2001). Para o referido autor, o cinema pode ser entendido como um meio a partir do qual representações são produzidas e pedagogias emergem, indo além de uma definição que contemple unicamente seu teor estético, artístico e/ou de entretenimento popular (FORTES, 2023). Assim, soma-se às provocações produzidas pelo dossiê e à compreensão em torno da mídia, e mais propriamente do cinema, o interesse de pesquisa por representações e pedagogias no cinema de horror. Com estas inspirações, debruçamo- nos sobre a recente produção de horror They/Them3 (2022), do diretor e roteirista estadunidense John Logan4, cuja produção é sua estreia como diretor. A história acompanha um grupo de jovens que é enviado para um acampamento de terapia da E-ISSN: 2358-8853 Revista Diversidade e Educação, v. 11, n. 1, p. 547-578, 2023. Doi: 10.14295/de.v11i1.15401 Horror heteronormativo: representações de gênero e sexualidade e pedagogias do horror no filme They/Them (2022) de John Logan Horror heteronormativo: representações de gênero e sexualidade e pedagogias do horror no filme They/Them (2022) de John Logan 550 conversão5 chamado Whistler Camp, cuja missão é a de adaptar a sexualidade de adolescentes LGBTQIAP+ aos padrões heterossexuais, contando ainda com o surgimento da figura de um misterioso assassino mascarado. Trata-se de “um filme de horror cujo tema é bastante polêmico e pontual, já que se trata de um acampamento que promete corrigir a sexualidade não heteronormativa de seus detentos” (PEREIRA, 2023, n.p.). Quando se pensa essa relação entre produções de horror e questões de gênero e sexualidade, alguns filmes destacam-se, embora não necessariamente abordem a temática de forma direta e evidente. Para estes filmes surge o termo Queer Horror, isto é, produções de horror desenvolvidas por diretores e/ou produtores que se identificam como queer6, e que, consequentemente, contribuem para representar seus medos e ansiedades como indivíduos queer (ELLIOTT-SMITH, 2016). Destaca-se que o conceito pode ser compreendido como fluído e abrangente, não sendo necessariamente dirigido ou produzido por artistas queer, assim, o termo pode ser utilizado para definir “produções que tratam abertamente de temas da comunidade ou que são intricadamente codificadas. Produções destinadas ao público queer ou que são adotadas e até mesmo posteriormente ressignificadas como seus símbolos” (DARKSIDE, 2022, n.p.). 6 Queer (em português: excêntrico, insólito, esquisito, bizarro) é uma palavra-ônibus proveniente do inglês usada para designar pessoas que não seguem o modelo de heterossexualidade ou da binaridade de gênero. 5 Também chamada de terapia de reorientação sexual ou terapia reparativa, compreende um conjunto de métodos que visam eliminar a orientação sexual de um indivíduo que não condiz com a heterossexualidade. 6 métodos que visam eliminar a orientação sexual de um indivíduo que não condiz com a heterossexualidade. 6 Queer (em português: excêntrico, insólito, esquisito, bizarro) é uma palavra-ônibus proveniente do inglês usada para designar pessoas que não seguem o modelo de heterossexualidade ou da binaridade de gênero. Introdução Assim, “ainda que não seja explicitada a intenção de alguns cineastas em abordar questões de gênero e sexualidade de uma forma direta, a teoria queer possibilita que certos filmes sejam analisados sob esse ponto de vista” (SOUZA, 2015, n.p.). Uma forma de compreender o conceito é relacioná-lo a enredos “em que um personagem é LGBT e a problemática tem a ver com a sexualidade (CETRONE, 2021). Embora haja filmes que possam ser considerados como de Queer Horror, cabe destacar que a mera presença de um personagem LGBT, por exemplo, por si só não qualifica um filme como pertencente ao gênero. É necessário que “a obra potencialmente problematize a questão e não a trate como mero adereço” (STANCKI, 2019). De tal forma, muitas produções de horror podem servir de alegoria para expor a condição a qual indivíduos que não se sujeitam a restrição e ao controle de uma sociedade heteronormativa são expostos, mesmo que em muitas delas o uso de estereótipos negativos seja evidente (BENSHOFF, 1997). Assim, partindo de um olhar 5 Também chamada de terapia de reorientação sexual ou terapia reparativa, compreende um conjunto de métodos que visam eliminar a orientação sexual de um indivíduo que não condiz com a heterossexualidade. Revista Diversidade e Educação, v. 11, n. 1, p. 547-578, 2023. Doi: 10.14295/de.v11i1.15401 E-ISSN: 2358-8853 Horror Heteronormativo: representações de gênero e sexualidade e pedagogias do horror no filme They/Them (2022) de John Logan 551 mais abrangente quanto às produções que podem encaixar-se na temática, têm-se alguns clássicos do cinema que carregam em seus subtextos certas mensagens, como, por exemplo, Nosferatu (1922), dirigido por Friedrich Wilhelm Murnau, e, ainda, Frankenstein (1931) e A Noiva de Frankenstein (1935), ambos de James Whale, com personagens que não são aceitos por serem considerados diferentes (DARKSIDE, 2022). Destaca-se que: Com o passar dos anos, diversos filmes continuaram representando personagens queer, seja de forma implícita ou explícita. A lista é longa e variada, com algumas representações mais positivas que outras [...]. Nos anos 1970, o horror queer continuava em alta, beneficiando-se do fim do código Hays e da censura em 1968. A trilogia Karnstein da produtora britânica Hammer inspirou-se em Carmilla de Le Fanu para produzir Atração Mortal (1970), Luxúria de Vampiros (1971) e As Filhas de Drácula (1971). Os três filmes foram considerados „ousados‟ para a época por sua representação explícita da lesbianidade. 7 Sigla que denomina um conjunto de práticas consensuais envolvendo bondage e disciplina, dominação e submissão, sadomasoquismo e outros tipos de comportamento sexual humano relacionados. Pat Mills (ESPIÑEIRA, 2022), que são bons exemplos de produções que podem, de alguma forma, ser enquadradas dentro do Queer Horror ou que ao menos flertam com a temática. Exemplificando algumas outras produções: Pat Mills (ESPIÑEIRA, 2022), que são bons exemplos de produções que podem, de alguma forma, ser enquadradas dentro do Queer Horror ou que ao menos flertam com a temática. Exemplificando algumas outras produções: Faca no Coração, filme de 2018 do diretor Yann Gonzalez, é ambientado no mundo da pornografia gay e povoado por personagens queer. A comédia de horror Freaky: No Corpo de Um Assassino brinca com identidade de gênero ao fazer uma adolescente trocar de corpo com um assassino em série. A francesa Julia Ducournau ganhou a Palma de Ouro com Titane, um filme que questiona sexualidade, corpo e humanidade, enquanto o último Pânico trouxe Jasmine Savoy Brown como Mindy, a primeira personagem assumidamente gay da franquia [...]. O recente Morte, Morte, Morte, da diretora Halina Reijn, traz no seu centro a sexualidade fluida e o questionamento de rótulos da geração Z. Já o fenômeno da Netflix, a trilogia Rua do Medo não só é protagonizada por personagens queer e centrada no romance de duas mulheres, como também questiona as expectativas do próprio público, ao brincar com as convenções de representação de sexualidade no cinema de horror (DARKSIDE, 2022, n.p.). Ao atentar o olhar para produções brasileiras, têm-se o curta-metragem J de Jesus (2013), de Dennison Ramalho, segmento da antologia O ABC da Morte 2 (2013), no qual o espectador acompanha o sequestro do filho de um político conservador, a mando de seu próprio pai, para que então passe por uma sessão de exorcismo em função de sua sexualidade (FORTES, 2022). Há comentários e reflexões interessantes no longa-metragem As Boas Maneiras (2018), de Juliana Rojas e Marco Dutra, no qual, apesar de não ser o foco da produção, aborda o relacionamento lésbico entre as personagens Ana e Clara, produzindo uma série de simbologias e metáforas para pensar o isolamento ao qual determinados indivíduos estão sujeitos em conta de sua orientação sexual ou identidades de gênero (CETRONE, 2021). O recente Verão Fantasma (2022), de Matheus Marchetti, cuja narrativa acompanha dois adolescentes que se apaixonam e investigam o desaparecimento de uma criança (DARKSIDE, 2022). Revista Diversidade e Educação, v. 11, n. 1, p. 547-578, 2023. Doi: 10.14295/de.v11i1.15401 Introdução p p p p ç p Em 1975, foi a vez do fenômeno The Rocky Horror Picture Show. Abordando temas como liberdade sexual, bissexualidade, androginia e expressões de gênero, o musical criou uma comunidade de fãs e ganhou um lugar cativo em nossos corações. O doutor Frank N. Furter, o cientista louco que na verdade é uma travesti do planeta Transexual, tornou-se um símbolo da comunidade queer, convidando todos a celebrarem e abraçarem suas identidades e sexualidades. A mensagem era simples: „Não sonhe, SEJA!‟ (DARKSIDE, 2022, n.p.). Outras produções que podem ser destacadas são A Hora do Pesadelo 2: A Vingança de Freddy (1985), de Jack Sholder, no qual o roteiro pode ser encarado como uma tentativa de realizar um comentário social sobre a homossexualidade (CETRONE, 2021; SOUZA, 2015) e Hellraiser: Renascido do Inferno (1987), de Clive Barker, cujo universo criado pelo diretor, que é assumidamente homossexual, têm como base a estética e a prática BDSM7 (CETRONE, 2021). Destacam-se, também, filmes como Raça das Trevas (1990), de Clive Barker, O Silêncio dos Inocentes (1991), de Jonathan Demme, Entrevista com o Vampiro (1994), de Neil Jordan, Almas Gêmeas (1994), de Peter Jackson, nos anos 90 (GODOY, 2020a), e Alta Tensão (2003), de Alexandre Aja, Hellbent (2004), de Paul Etheredge, O Filho de Chucky (2004), de Don Mancini, Deixa Ela Entrar (2008), de Tomas Alfredson, a partir dos anos 2000 (GODOY, 2020b). Mais recentemente, Um Estranho no Lago (2013), de Alain Guiraudie, Lyle (2014), de Stewart Thorndike, Espiral (2019), de Kurtis David Harder, e The Retreat (2021), de 7 Sigla que denomina um conjunto de práticas consensuais envolvendo bondage e disciplina, dominação e submissão, sadomasoquismo e outros tipos de comportamento sexual humano relacionados. Revista Diversidade e Educação, v. 11, n. 1, p. 547-578, 2023. Doi: 10.14295/de.v11i1.15401 E-ISSN: 2358-8853 Horror heteronormativo: representações de gênero e sexualidade e pedagogias do horror no filme They/Them (2022) de John Logan 552 Revista Diversidade e Educação, v. 11, n. 1, p. 547-578, 2023. Doi: 10.14295/de.v11i1.15401 E-ISSN: 2358-8853 Pat Mills (ESPIÑEIRA, 2022), que são bons exemplos de produções que podem, de alguma forma, ser enquadradas dentro do Queer Horror ou que ao menos flertam com a temática. Exemplificando algumas outras produções: Ainda a partir de uma perspectiva ampla quanto a produções que podem enquadrar-se de alguma forma ao Queer Horror, destacam-se os curtas-metragens Jonas Banhado em Sangue (2017), de Mateus Bandeira, Você é Diferente (2017), de George Pedrosa, e O Mistério da Carne (2018), de Rafaela Camelo, além dos longas-metragens Mate-me Por Favor (2015), de Anita Rocha da Silveira, e Canto dos Ossos (2020), de Jorge Polo e Petrus de Bairros (MARQUES, 2022). E-ISSN: 2358-8853 Horror Heteronormativo: representações de gênero e sexualidade e pedagogias do horror no filme They/Them (2022) de John Logan Horror Heteronormativo: representações de gênero e sexualidade e pedagogias do horror no filme They/Them (2022) de John Logan 553 A produção selecionada para fins de análise e discussão no presente texto, têm claras inspirações em clássicos filmes Slasher8, como as franquias Sexta-Feira 13, com a figura de Jason Voorhees, A Hora do Pesadelo, com a figura de Freddy Krueger, Pânico, com a figura de Ghostface, e mais recentemente, Terrifier, com a figura de Art. Sobretudo, atenta-se para as similaridades de They/Them com Sexta-Feira 13, que também se passa em um acampamento e apresenta a figura de um assassino mascarado. Cabe destacar a possível inspiração no documentário da Netflix chamado Pray Away (2021), de Kristine Stolakis, que traz os relatos de ex-líderes e sobreviventes da chamada terapia de conversão (WALTER, 2022). Atenta-se ainda para uma motivação de cunho pessoal do diretor, conforme destacou em nota para a imprensa quando do anúncio do filme: „They/Them’ é algo que está germinando dentro de mim durante toda a minha vida. Eu amo filmes de terror desde quando me entendo por gente, acho que porque os monstros representam „o outro‟. Como uma criança gay, eu sentia muita afinidade com aqueles que eram diferentes, fora do comum, proibidos. Eu quis fazer um filme que celebre as identidades queers, com personagens que eu nunca vi enquanto crescia. (FANTINEL, 2022, n.p.). A ideia central da produção, por si só, já proporciona bases sólidas para uma produção de horror, contribuindo para reflexões e discussões importantes na contemporaneidade. De fato, as produções de horror em si carregam o potencial de representar e produzir pedagogias acerca dos mais diversos temas, incluindo-se aquelas relacionadas a gênero e sexualidade (FORTES, 2022, 2023). 8 Subgênero de filmes de horror que quase sempre envolve assassinos psicopatas que matam aleatoriamente. Revista Diversidade e Educação, v. 11, n. 1, p. 547-578, 2023. Doi: 10.14295/de.v11i1.15401 E-ISSN: 2358-8853 8 Subgênero de filmes de horror que quase sempre envolve assassinos psicopatas que matam aleatoriamente. Pat Mills (ESPIÑEIRA, 2022), que são bons exemplos de produções que podem, de alguma forma, ser enquadradas dentro do Queer Horror ou que ao menos flertam com a temática. Exemplificando algumas outras produções: O crítico de cinema Diego Ferraz, apesar de apontar aspectos que podem ser compreendidos como negativos no tocante a construção e desenrolar da narrativa de They/Them, também aponta elementos importantes, sobretudo considerando a discussão proposta neste artigo. Como salienta, trata-se de: [...] uma obra que conversa com um público seleto e só funciona para ele. Quem sofreu ou entende de maneira empática as questões opressoras do preconceito sexual vai captar várias mensagens, mesmo que muitas cheguem de maneira bem panfletária e didática, quase como um telecurso. Por outro lado, um público que precisaria de fato ouvir e se chocar com essa mensagem nunca será atingido, pelo modo raso e frio que são tratadas coisas tão importantes. 8 Subgênero de filmes de horror que quase sempre envolve assassinos psicopatas que matam aleatoriamente. Revista Diversidade e Educação, v. 11, n. 1, p. 547-578, 2023. Doi: 10.14295/de.v11i1.15401 E-ISSN: 2358-8853 Horror heteronormativo: representações de gênero e sexualidade e pedagogias do horror no filme They/Them (2022) de John Logan 554 Horror heteronormativo: representações de gênero e sexualidade e pedagogias do horror no filme They/The (2022) de John Logan O que me deixa feliz é ver que talvez esse filme abra fronteiras pra novas abordagens similares, talvez de maneiras mais inteligentes e eficazes, mostrando que as narrativas evoluem e é possível sim ter terror com todo tipo de personagens representados, nem que sejam para morrer ao fugir de um assassino ao correr de costas nas florestas. (FERRAZ, 2022, n.p.). Com estas considerações introdutórias, buscou-se a contextualização do tema e do artefato cultural sobre o qual versam as discussões empreendidas. Na continuidade, apresenta-se o percurso teórico-metodológico, dando relevo a alguns conceitos que nos servem como ferramentas analíticas. Revista Diversidade e Educação, v. 11, n. 1, p. 547-578, 2023. Doi: 10.14295/de.v11i1.15401 Percurso teórico-metodológico e conceitos norteadores Implementamos nossa análise a partir do campo dos Estudos Culturais (EC) que se fundamenta em uma compreensão ampla no tocante à cultura, compreendendo-a como central na contemporaneidade, abrangendo todos os aspectos da vida social e, portanto, interessado em todas as práticas sociais de significação (DU GAY et al, 1997; HALL, 2016). A partir dos EC, o cinema é compreendido como um artefato cultural que, além de ser produzido na cultura, abarca o potencial de afetá-la, considerando as representações que são produzidas e as pedagogias que dele emergem (DUARTE, 2002; FABRIS, 2008; GIROUX, 2013). Para fins metodológicos, faz-se uso de um tripé composto pela etnografia em tela (BALESTRIN, 2011), com o intuito de produzir recortes da produções, atentando- se para aspectos que não seriam perceptíveis se analisada a produção como um todo; pela análise cultural (WILLIAMS, 2003), visando compreender os significados e valores implícitos e explícitos que encontram-se presentes na cultura específica a partir da qual a produção surge; pela metodologia visual crítica (ROSE, 2001) que implica uma postura crítica em relação às imagens visuais as quais se analisa; e do produtor das mesmas, considerando a subjetividade do pesquisador. Dentre os conceitos norteadores, a representação é compreendida como a produção de sentido através da linguagem, ocorrendo a partir de signos e símbolos (HALL, 2016). No tocante ao que se propõe, atenta-se para a linguagem cinematográfica e as representações produzidas através de seu uso. Por pedagogia cultural, entende-se as formas de ser e de compreender o mundo que emergem dos mais diversos âmbitos, transcendendo os lugares educacionais tradicionais, sendo, neste E-ISSN: 2358-8853 Horror Heteronormativo: representações de gênero e sexualidade e pedagogias do horror no filme They/Them (2022) de John Logan 555 momento de expansão do que se entende como pedagogia, por exemplo, os museus, anúncios publicitários, músicas, jogos eletrônicos e, sobretudo, o cinema (CAMOZZATO, 2012). Relacionando-se intimamente com o conceito e pedagogias culturais, têm-se as pedagogias do horror - fruto da expansão da pedagogia, enquanto campo, bem como do caráter educativo que se expande, como vontade de educar a todas as dimensões da vida social. Percurso teórico-metodológico e conceitos norteadores Temos, portanto, pedagogias que emergem de produções cinematográficas pertencentes ao gênero de horror, que, por sua vez, apresentaram determinadas particularidades, como a aprendizagem a partir da figura monstruosa, a reflexão relativa ao contexto em que a produção analisada surge, bem como as possíveis lições contidas na exteriorização de medos, angústias e traumas dos personagens, podendo estes serem aprendidos pelo espectador (FORTES, 2023). Relativo ao horror como gênero cinematográfico, identificamos ser ele compreendido como um gênero que se relaciona com os medos e ansiedades de uma sociedade, cujas representações produzidas contribuem para reflexões acerca dos medos e preocupações nela vigentes. Além disso, ele é marcado por aquilo que é reconhecido como aterrorizante, ao mesmo tempo em que oportuniza uma combinação do que é familiar e inesperado ao espectador (PHILIPS, 2005). Ao pensar gênero e sexualidade, aponta-se para construções sociais que ocorrem através de aprendizagens e práticas, desenvolvidas por um conjunto de instâncias sociais e culturais tais como a igreja, escola, família, entre outros, dentro de um processo inacabado e contínuo. Ou seja, processos de subjetivação que incidem sobre os sujeitos ao longo de toda a vida (LOURO, 2008). Cabe destacar que, embora se interrelacionem, identidade sexual e de gênero se distinguem. Isso é, um sujeito masculino ou feminino pode ser heterossexual, homossexual ou bissexual, por exemplo. Relacionado a estes conceitos, têm-se a heteronormatividade, compreendida como a ordem social naturalizada a partir da matriz heterossexual. Trata-se da presunção de que, para que os corpos estejam coerentes eles devem ser estáveis, tanto na questão sexual quanto na de gênero, fundamentando-se na relação masculino e feminino, assim como na prática da heterossexualidade (BUTLER, 2003). Após este momento, sobre o percurso teórico-metodológico e os conceitos que nortearão a pesquisa, passamos para a análise da produção, com vistas no horror produzido a partir da heteronormatividade. Revista Diversidade e Educação, v. 11, n. 1, p. 547-578, 2023. Doi: 10.14295/de.v11i1.15401 E-ISSN: 2358-8853 Horror heteronormativo: representações de gênero e sexualidade e pedagogias do horror no filme They/Them (2022) de John Logan 5 Horror heteronormativo: representações de gênero e sexualidade e pedagogias do horror no filme They/Them (2022) de John Logan Horror heteronormativo: representações de gênero e sexualidade e pedagogias do horror no filme They/Them (2022) de John Logan 556 Revista Diversidade e Educação, v. 11, n. 1, p. 547-578, 2023. Doi: 10.14295/de.v11i1.15401 Desmembrando o horror heteronormativo de They/Them Na parte inicial da produção, após o espectador presenciar o assassinato de uma mulher que dirigia um carro à noite por uma estrada, embora sem entender as motivações do que levariam a isto, vê-se um grupo de jovens que chegam em um ônibus ao acampamento Whistler Camp, momento em que são recebidos por Owen Whistler (interpretado por Kevin Bacon), o responsável pelo local, e a sua equipe. O nome do acampamento e o sobrenome de Owen, assim como outros elementos ao longo da produção, evidenciam que o local representa um negócio da família há muitos anos, sendo Owen, na atualidade da trama, o responsável. Embora as falas de Owen soem em um tom amigável, um tanto brincalhão, e de acolhimento, é nítido o discurso heteronormativo, como quando diz “Eu não posso fazer vocês virarem héteros. Para mim, tudo bem ser gay. Se vocês são felizes do jeito que são” (Figura 1). Estas falas devem ser compreendidas a partir não unicamente do que é dito, mas dos possíveis significados que o enunciado produz, em contextos diversos. Nesse sentido, quando diz “Para mim, tudo bem ser gay”, outra pergunta poderia ser feita, como: Por qual razão não estaria bem ser gay ou qual seria o problema em ser? FIGURA 1: Boas-vindas. Fonte: They/Them, 2022. FIGURA 1: Boas-vindas. Fonte: They/Them, 2022. Se em um primeiro momento Owen deixa claro que não há o interesse de mudar nenhum daqueles jovens, posteriormente, tal sentido é deslocado através de um discurso E-ISSN: 2358-8853 Horror Heteronormativo: representações de gênero e sexualidade e pedagogias do horror no filme They/Them (2022) de John Logan 557 de base psicológica (ao estilo autoajuda), como por exemplo, no tocante à aceitação perante a sociedade e, sobretudo, a aceitação deles mesmos. Falas como “imagino que alguns de vocês estejam aqui porque, de alguma forma, não estão felizes” e “Talvez vocês não se encaixem. As pessoas zombam de vocês. Talvez queiram encontrar um novo tipo de paz” (Figura 2). Deve-se atentar a estas falas, quando Owen atribui aos próprios jovens a inquietude ou busca por mudarem e encontrarem uma saída para sua suposta infelicidade ou para a não aceitação por parte da sociedade. Desse modo, é reforçada a heteronormatividade, deslocando-se para o sujeito um problema que estaria na sociedade, que impõe uma norma sexual e posiciona as demais sexualidades como desviantes. FIGURA 2: Discurso com bases heteronormativas. Fonte: They/Them, 2022. Revista Diversidade e Educação, v. 11, n. 1, p. 547-578, 2023. Doi: 10.14295/de.v11i1.15401 Desmembrando o horror heteronormativo de They/Them FIGURA 2: Discurso com bases heteronormativas. Fonte: They/Them, 2022. Mesmo que possam soar como receptivas e compreensivas, as falas de Owen carregam o que pode ser compreendido como violência simbólica, expressa através da linguagem. Primeiramente deve-se compreender que “a linguagem é produtora de normalizações e significados” (SEFFNER; FIGLIUZZI, 2011, p. 54). Com isso, afirma- se que a violência se manifesta de diferentes formas, não unicamente em ações e casos mais evidentes e habitualmente pensados, mas, por outro lado, deve-se considerar “uma forma ainda mais fundamental de violência que pertence à linguagem enquanto tal, à imposição de um certo universo de sentido” (ZIZEK, 2014, p. 17). Revista Diversidade e Educação, v. 11, n. 1, p. 547-578, 2023. Doi: 10.14295/de.v11i1.15401 E-ISSN: 2358-8853 Horror heteronormativo: representações de gênero e sexualidade e pedagogias do horror no filme They/Them (2022) de John Logan Horror heteronormativo: representações de gênero e sexualidade e pedagogias do horror no filme They/Them (2022) de John Logan 558 Julga-se pertinente destacar alguns pontos da discussão a respeito do surgimento dos discursos de verdades acerca da sexualidade. Tais discursos surgem em momentos histórico e sociais específicos, com finalidade de normatizar as práticas sexuais de acordo com determinados padrões. Assim, muito do que se têm como verdade no tocante ao sexo nada mais é do que uma construção feita de múltiplos discursos que produzem supostas verdades (FOUCAULT, 2017a, 2017b). Tal compreensão é importante para pensar o quanto a violência que emerge e é produzida a partir das falas de Owen, possui suas próprias contradições. Pois, trata-se de um discurso construído e tomado como verdade absoluta, embora, conforme Foucault, a verdade seja produzida a partir de regimes discursivos que tornam certo enunciado verdadeiro, em contextos, tempos e circunstâncias variáveis. Após o discurso de boas-vindas, da apresentação dos membros da equipe, e de explicar como será o funcionamento do acampamento, Owen então aponta as cabanas nas quais os recém-chegados ficarão, sendo o da sua direita o dos meninos e o da esquerda o das meninas, estabelecendo, assim, uma norma a ser seguida. Contudo, enquanto a maioria do grupo caminha em direção a uma das cabanas, uma pessoa fica parada sem tomar uma decisão. Trata-se de Jordan (interpretado por Theo Germaine), protagonista que é transsexual e pessoa não binária (Figura 3). FIGURA 3: Meninos e meninas. FIGURA 3: Meninos e meninas. FIGURA 3: Meninos e meninas. Fonte: They/Them, 2022. Fonte: They/Them, 2022. E-ISSN: 2358-8853 Horror Heteronormativo: representações de gênero e sexualidade e pedagogias do horror no filme They/Them (2022) de John Logan Horror Heteronormativo: representações de gênero e sexualidade e pedagogias do horror no filme They/Them (2022) de John Logan 559 A fala de Owen e a distinção entre uma cabana para os meninos e outra para as meninas direciona os jovens a posicionarem-se quanto às suas identidades de gênero. Com isso, instala-se “um processo que, supostamente, deve seguir um determinado rumo ou direção. Neste caso, a afirmativa mais do que uma descrição, pode ser compreendida como uma definição ou decisão sobre um corpo” (LOURO, 2004, p. 15). Contudo, compreende-se que ao mesmo tempo que se impõe algo, também se abre espaço para transgressões, subversões e resistências (LOURO, 2004). O simples ato de Owen em definir uma cabana para meninos e uma cabana para meninas, além de regular e delimitar as opções, ou mesmo estabelecer fronteiras e limites, também abre espaço para que Jordan se manifeste. Esse momento da produção possibilita que pensemos na problemática do que surge primeiro, se a regulação ou o gênero. Conforme Butler “um gênero que preexiste a sua regulação, ou é o caso de que, sendo sujeito à regulação, o sujeito generificado emerge, produzido a partir de e por meio dessa forma particular de sujeição? Não é a sujeição o processo pelo qual a regulação produz gênero?” (2014, p. 251). Ainda, conforme Butler [...] um discurso restritivo sobre gênero que insista no binarismo homem e mulher como a maneira exclusiva de entender o campo do gênero atua no sentido de efetuar uma operação reguladora de poder que naturaliza a instância hegemônica e exclui a possibilidade de pensar sua disrupção. (2014, p. 254). Na sequência da narrativa fílmica, a primeira atividade a qual o grupo de jovens é submetido é uma roda de conversas, que pode ser vista também como uma espécie de terapia em grupo. Além dos jovens, encontram-se neste momento o responsável pelo acampamento, Owen, e sua esposa, Cora Whistler (interpretada por Carrie Preston), a psicóloga do acampamento, que toma nota do que é dito pelos jovens (Figura 4). É então que Owen dá início à dinâmica, questionando-os: “Quero começar com uma pergunta simples. Por que vocês estão aqui?”. Revista Diversidade e Educação, v. 11, n. 1, p. 547-578, 2023. FIGURA 3: Meninos e meninas. Doi: 10.14295/de.v11i1.15401 E-ISSN: 2358-8853 Horror heteronormativo: representações de gênero e sexualidade e pedagogias do horror no filme They/Them (2022) de John Logan 560 FIGURA 4: Terapia em grupo. Fonte: They/Them, 2022 FIGURA 4: Terapia em grupo. Fonte: They/Them, 2022 Observam-se dois tipos de posicionamento dos jovens durante a terapia em grupo. Alguns mostram-se bem resolvidos no tocante a seu gênero e sexualidade, embora toda violência, preconceito e pressão familiar que possam sofrer, estando ali muito mais para fins de “agradar” seus familiares e, com isso, não serem “expulsos de casa” (tal como afirma uma das jovens), ou ainda, para emanciparem-se legalmente de seus pais. Alguns outros jovens não se mostram tranquilos quanto à sua sexualidade ou gênero, e dizem ter procurado o acampamento para não serem vistos como uma “aberração”, ou para “ser como todo mundo”, ou ainda, “ser invisível” ou simplesmente “ser amada” (Figura 04). Revista Diversidade e Educação, v. 11, n. 1, p. 547-578, 2023. Doi: 10.14295/de.v11i1.15401 E-ISSN: 2358-8853 Horror Heteronormativo: representações de gênero e sexualidade e pedagogias do horror no filme They/Them (2022) de John Logan Horror Heteronormativo: representações de gênero e sexualidade e pedagogias do horror no filme They/Them (2022) de John Logan 561 FIGURA 4: Aceitação e negação. Fonte: They/Them, 2022 FIGURA 4: Aceitação e negação. FIGURA 4: Aceitação e negação. Fonte: They/Them, 2022 Fonte: They/Them, 2022 A negação manifestada pelos jovens evidencia o horror ao qual estão sendo submetidos nos mais diversos âmbitos, como, por exemplo, no destaque feito a uma das instâncias por eles mencionada: a própria família. Essa que juntamente escola, igreja, instituições legais e médicas, são importantes para o processo constitutivo quando o assunto é gênero e sexualidade (LOURO, 2008). Como salienta a autora: A construção dos gêneros e das sexualidades dá-se através de inúmeras aprendizagens e práticas, insinua-se nas mais distintas situações, é empreendida de modo explícito ou dissimulado por um conjunto inesgotável de instâncias sociais e culturais. É um processo minucioso, sutil, sempre inacabado. Família, escola, igreja, instituições legais e médicas mantêm-se, por certo, como instâncias importantes nesse processo constitutivo. Por muito tempo, suas orientações e ensinamentos pareceram absolutos, quase soberanos. (LOURO, 2008 p. 18). Nesta direção, o filme remete ao conflito psíquico a que muitos indivíduos estão sujeitos, entre o que as pessoas pensam que elas deveriam ser e como elas realmente são e desejam viver as suas vidas. Revista Diversidade e Educação, v. 11, n. 1, p. 547-578, 2023. Doi: 10.14295/de.v11i1.15401 das zonas de policiamento, tanto a obediência, quanto a transgressão e subversão da ordem estão presentes. Uma das formas de transgredir ou subverter encontra-se justamente na paródia, sob uma forma crítica (LOURO, 2004). Como salienta Louro: Quem subverte e desafia a fronteira apela, por vezes, para o exagero e para a ironia, a fim de tornar evidente a arbitrariedade das divisões, dos limites e das separações. Por isso, a paródia que arremeda os „nativos‟ do „outro‟ lado, que embaralha seus códigos com os „desse lado‟, que mistura e confunde as regras, que combina e distorce as linguagens é tão perturbadora. Ela se compraz da ambigüidade, da confusão, da mixagem. Para as fronteiras constantemente vigiadas dos gêneros e da sexualidade, a crítica paródica pode ser profundamente subversiva. (2004, p. 20). Quem subverte e desafia a fronteira apela, por vezes, para o exagero e para a ironia, a fim de tornar evidente a arbitrariedade das divisões, dos limites e das separações. Por isso, a paródia que arremeda os „nativos‟ do „outro‟ lado, que embaralha seus códigos com os „desse lado‟, que mistura e confunde as regras, que combina e distorce as linguagens é tão perturbadora. Ela se compraz da ambigüidade, da confusão, da mixagem. Para as fronteiras constantemente vigiadas dos gêneros e da sexualidade, a crítica paródica pode ser profundamente subversiva. (2004, p. 20). Em determinada cena, os jovens conhecem um pouco mais as atividades propostas pelo acampamento, divididas entre atividades das meninas e atividades dos meninos. As meninas se reúnem na cozinha para fazer tortas, que conforme a diretora de atividades Sarah (interpretada por Hayley Griffith) explica: “Hoje vamos explorar papéis tradicionais. Ao nos comportarmos de certo modo, reconhecermos e aceitarmos certos comportamentos sociais, talvez venhamos a apreciar o valor emocional de reforçar esses comportamentos e a ficar confortáveis e sentir que pertencemos a um grupo. Então, meninas, vamos fazer uma torta”. Enquanto isso, os meninos, reunidos em uma área aberta com alvos a certa distância, recebem espingardas, para que possam exercitar a prática de tiro. Conforme Owen, que está acompanhado do diretor esportivo Zane (interpretado por Boone Platt): “Duas coisas controlam este mundo. A Biologia e a História. Eu sei que não é bom dizer isto hoje em dia, mas os homens evoluíram naturalmente para serem maiores e mais fortes do que as mulheres. Há um motivo para isso. Nossos antepassados eram caçadores. Eles tinham que perseguir as presas e subjugá-las. Era caçar ou passar fome. Isso está no nosso DNA, senhores. Estamos destinados a ser assassinos. É nossa obrigação biológica. E nenhuma quantidade de civilização, progresso, e humanismo liberal pode alterar essa verdade genética. Revista Diversidade e Educação, v. 11, n. 1, p. 547-578, 2023. Doi: 10.14295/de.v11i1.15401 FIGURA 3: Meninos e meninas. Por vezes, estes indivíduos têm uma sensação de vergonha, questionamentos quanto ao seu próprio valor e, sobretudo, ódio de si mesmos (ANTUNES, 2016). Isso ocorre em virtude do esforço “em mostrar continuidades e homogeneidades em nossa vida sexual e de gênero, bem como em nossos ideais de corpo” (SEFFNER; FIGLIUZZI, 2011, p. 52). A falta de apoio, seja por parte da Revista Diversidade e Educação, v. 11, n. 1, p. 547-578, 2023. Doi: 10.14295/de.v11i1.15401 E-ISSN: 2358-8853 Horror heteronormativo: representações de gênero e sexualidade e pedagogias do horror no filme They/Them (2022) de John Logan 562 família, seja do meio social mais amplo em que vivem, e que se compreende através das falas dos jovens, promovem a internalização do preconceito e da violência que sofrem (ALBUQUERQUE et al, 2016). Embora não se trate de uma cena em que o horror heteronormativo se manifeste, têm-se um outro importante momento em que uma forma de resistência ao que vem sendo imposto se faz presente, novamente através da figura de Jordan. Em determinado momento da narrativa fílmica, descobre-se que Alexandra (interpretada por Quei Tann) é transsexual e que não havia informado isto à Owen e ao restante do acampamento, algo afirmado como um fato grave, por Owen. Assim, ao descobrir, o diretor faz com que, pela manhã, Alexandra troque de cabana, passando da cabana das meninas para a cabana dos meninos. Após um discurso exaltado e ofensivo, Owen deixa a mensagem “todos lá na frente. 10 min para a chamada. E, por favor, se vistam de maneira adequada” (Figura 5) O que se sucede pode vir a ser uma resposta para a pergunta: O que é vestir-se adequadamente? FIGURA 5: Ato de resistência. Fonte: They/Them, 2022 FIGURA 5: Ato de resistência. Fonte: They/Them, 2022 Em resposta, e solidariedade, ao que ocorreu, Jordan se apresenta para a chamada com trajes tidos como femininos, um vestido florido e colorido. O sorriso estampado na cara de Jordan pode ser compreendido como uma forma de subversão da ordem proferida ou mesmo de enfrentamento à Owen. Cabe pensar que nas fronteiras E-ISSN: 2358-8853 Horror Heteronormativo: representações de gênero e sexualidade e pedagogias do horror no filme They/Them (2022) de John Logan 563 das zonas de policiamento, tanto a obediência, quanto a transgressão e subversão da ordem estão presentes. Uma das formas de transgredir ou subverter encontra-se justamente na paródia, sob uma forma crítica (LOURO, 2004). Como salienta Louro: Revista Diversidade e Educação, v. 11, n. 1, p. 547-578, 2023. Doi: 10.14295/de.v11i1.15401 das zonas de policiamento, tanto a obediência, quanto a transgressão e subversão da ordem estão presentes. Uma das formas de transgredir ou subverter encontra-se justamente na paródia, sob uma forma crítica (LOURO, 2004). Como salienta Louro: Não podemos discutir com a ciência. E a ciência nos diz que devemos passar esse DNA necessário para nossos descendentes, através das mulheres. Engravidando as mulheres e tendo filhos. Isso é ciência. E é História. Então, quem tem instinto assassino?” (Figura 6). Revista Diversidade e Educação, v. 11, n. 1, p. 547-578, 2023. Doi: 10.14295/de.v11i1.15401 E-ISSN: 2358-8853 Horror heteronormativo: representações de gênero e sexualidade e pedagogias do horror no filme They/Them (2022) de John Logan FIGURA 6: Saberes e habilidades específicas para cada um. Fonte: They/Them, 2022 FIGURA 6: Saberes e habilidades específicas para cada um. Fonte: They/Them, 2022 O que a referida cena possibilita que se pense é sobre a normalização da conduta dos meninos e das meninas, isto é, a distinção que se faz dos saberes e habilidades adequadas a cada um (LOURO, 1997). É muito claro, a partir do desenrolar da cena e das falas que se fazem presentes, a designação de um lugar de docilidade, vinculado a afazeres domésticos, para as meninas, enquanto aos homens ocorre uma associação para a violência, como sinônimo de uma masculinidade ideal. Assim, no “campo do gênero e da sexualidade, certas coisas são adequadas para meninos, mas não são para meninas, e vice-versa” (SEFFNER; FIGLIUZZI, 2011, p. 48). Trata-se de uma formar de contribuir para o processo de masculinização ou de feminilização com o qual se deva comprometer e se identificar (LOURO, 2004). Nas cenas em destaque, os artefatos culturais tomam um repertório naturalizado de posições de gênero e o fazem funcionar. Mas o próprio exagero das cenas que remetem aos lugares sociais femininos e masculinos pode gerar no espectador certa resistência, operando, no sentido inverso àquele que supostamente se propôs na construção da cena. Uma das cenas em que o horror se manifesta de forma extremamente incômoda é durante a sessão de terapia individual de Jordan com a psicóloga Cora. Em uma postura semelhante à de Owen, mantêm um diálogo inicial que busca, de certa forma, acolher e compreender as dificuldades enfrentadas. Cora busca compreender como Jordan se sente, ao mesmo tempo em que lhe tranquiliza, fazendo saber como deve estar se sentindo (Figura 7). E-ISSN: 2358-8853 Horror Heteronormativo: representações de gênero e sexualidade e pedagogias do horror no filme They/Them (2022) de John Logan FIGURA 7: Falso acolhimento. Fonte: They/Them, 2022 FIGURA 7: Falso acolhimento. Revista Diversidade e Educação, v. 11, n. 1, p. 547-578, 2023. Doi: 10.14295/de.v11i1.15401 9 Considerada uma forma de tratamento psiquiátrico ou psicológico em que o paciente é exposto a estímulos enquanto simultaneamente é sujeitado a alguma forma de desconforto. das zonas de policiamento, tanto a obediência, quanto a transgressão e subversão da ordem estão presentes. Uma das formas de transgredir ou subverter encontra-se justamente na paródia, sob uma forma crítica (LOURO, 2004). Como salienta Louro: Fonte: They/Them, 2022 A mobilização do sentido de decepção – ainda que apresentada de forma interrogativa – enlaça o significado de homossexualidade ao do desvio. Na sequência da narrativa, através de falas e olhares, a psicóloga vai aos poucos evidenciando ódio o desprezo para com Jordan, destacando que é na verdade uma farsa, uma forma de tornar-se especial. Contudo, ainda assim, nunca conhecerá o amor: “Não importa o que você faça, nunca será bom o suficiente. Você tenta se tornar especial. Essa coisa que você inventou é uma farsa. Eles nunca vão amar você. Ninguém vai” (Figura 8). Revista Diversidade e Educação, v. 11, n. 1, p. 547-578, 2023. Doi: 10.14295/de.v11i1.15401 Revista Diversidade e Educação, v. 11, n. 1, p. 547-578, 2023. Doi: 10.14295/de.v11i1.15401 E-ISSN: 2358-8853 Horror heteronormativo: representações de gênero e sexualidade e pedagogias do horror no filme They/Them (2022) de John Logan FIGURA 8: O ódio se manifesta. Fonte: They/Them, 2022 FIGURA 8: O ódio se manifesta. FIGURA 8: O ódio se manifesta. Fonte: They/Them, 2022 A violência dessas palavras ultrapassa a tela e afeta o espectador. O fato de Jordan receber passivamente todo o discurso de ódio que se manifesta na fala da psicóloga, intensifica o teor de desconforto produzido na cena. Pode-se conceber este momento como uma manifestação e exemplo de violência simbólica (ZIZEK, 2014), embora seja importante frisar que a recepção e a produção de significados nunca é linear e fixa, podendo esses significados ser subvertidos, problematizados ou recusados. Também se trata de um primeiro exemplo, de outros que são perceptíveis ao longo da produção, de como sujeitos considerados transgressores, ou que subvertem as categorias estabelecidas e impostas, são percebidos como incompressíveis ou doentes (LOURO, 2004). A violência transcorre de outras formas, conforme o desenrolar da narrativa. Em um dos escritórios do acampamento, Jordan depara-se com fotos que estavam guardadas em uma gaveta, elas mostram jovens com diversas marcas em seus corpos, indícios de terem sofrido violência física. Pelo aspecto de algumas fotos, mais envelhecidas e em preto e branco, até outras com aspecto de novas e coloridas, há o indicativo de que há uma linha de continuidade, relativa à moralização pela violência, que ocorre no acampamento. O olhar de Jordan demonstra um evidente espanto frente ao que é visto (Figura 9). E-ISSN: 2358-8853 Revista Diversidade e Educação, v. 11, n. 1, p. 547-578, 2023. das zonas de policiamento, tanto a obediência, quanto a transgressão e subversão da ordem estão presentes. Uma das formas de transgredir ou subverter encontra-se justamente na paródia, sob uma forma crítica (LOURO, 2004). Como salienta Louro: Doi: 10.14295/de.v11i1.15401 Horror Heteronormativo: representações de gênero e sexualidade e pedagogias do horror no filme They/Them (2022) de John Logan 5 (2022) de John Logan (2022) de John Logan (2022) de John Logan FIGURA 9: Violência subjetiva. Fonte: They/Them, 2022 FIGURA 9: Violência subjetiva. FIGURA 9: Violência subjetiva. Fonte: They/Them, 2022 A partir disso, a produção apresenta um outro tipo de violência, essa a mais visível e de fácil percepção. Trata-se da violência subjetiva, diretamente visível e que pode emergir, por exemplo, de “agentes sociais, indivíduos maléficos, aparelhos repressivos disciplinados e multidões fanáticas: a violência subjetiva é tão somente a mais visível das três” (ZIZEK, 2014, p. 25). Concebe-se, que tal violência subjetiva vai emergir ao longo do filme justamente a partir das raízes criadas pela violência simbólica produzida a partir do discurso heteronormativo. Entende-se que, embora alguns setores sociais possam manifestar uma crescente no tocante à aceitação do que difere da norma, outros setores podem posicionar-se e manifestar o contrário, o que pode vir a culminar em atos de extrema agressão e de violência física (LOURO, 2008). A violência subjetiva se concretizará na terapia de aversão9 a qual Stuart (interpretado por Cooper Koch) é submetido. Após ser seduzido e enganado por outro jovem do acampamento, Stuart é amordaçado e amarrado em uma cadeira de frente a uma tela, por Owen e outros membros do acampamento. O lugar é uma cabana mais distante das cabanas centrais do acampamento, fazendo com que Stuart encontre-se absolutamente sozinho. A naturalidade com que Owen lida com o que está por vir é chocante, percebendo como algo normal e necessário. Como ele mesmo destaca, Stuart está em uma situação até mesmo confortável, pois no passado tudo seria pior: “Na Revista Diversidade e Educação, v. 11, n. 1, p. 547-578, 2023. Doi: 10.14295/de.v11i1.15401 E-ISSN: 2358-8853 Horror heteronormativo: representações de gênero e sexualidade e pedagogias do horror no filme They/Them (2022) de John Logan 568 época do meu avô, você estaria numa mesa de lobotomia. Era assim que tratávamos pessoas com a sua doença”. Em seguida, imagens são mostradas à Stuart, primeiramente de mulheres e depois de homens, a cada vez que um homem surge na tela, Stuart é submetido a uma descarga elétrica que está colocada em seu peito. Por algum tempo, Stuart e os espectadores são submetidos a uma sessão de violência e horror (Figura 10). FIGURA 10: Terapia de aversão. Revista Diversidade e Educação, v. 11, n. 1, p. 547-578, 2023. Doi: 10.14295/de.v11i1.15401 das zonas de policiamento, tanto a obediência, quanto a transgressão e subversão da ordem estão presentes. Uma das formas de transgredir ou subverter encontra-se justamente na paródia, sob uma forma crítica (LOURO, 2004). Como salienta Louro: Fonte: They/Them, 2022 FIGURA 10: Terapia de aversão. Fonte: They/Them, 2022 Conforme destacado anteriormente, atribui-se aos sujeitos que ultrapassam os limites estabelecidos marcadores como o da “patologia” (LOURO, 2004). Nos termos empregados pelo personagem Owen, “pessoas com a sua doença”, evidenciam o vínculo entre homossexualidade e patologia. Historicamente, conforme o conceito de instinto vai tornando-se central, isto a partir da metade do século XIX, “qualquer desvio de seu curso „normal‟, isto é, o direcionamento heterossexual, passa a ser objeto de diagnóstico” (GARCIA; MATTOS, 2019, p. 53). Assim, na percepção da não continuidade e homogeneidade no tocante ao gênero e à sexualidade, muitos indivíduos negam “desvios” ou “desejos estranhos” que possam manifestar-se. No limite disto, nega-se “o direto de existência a outros” (SEFFNER; FIGLIUZZI, 2011, p. 52). Ou seja, qualquer manifestação “contrária aos padrões heteronormativos pode vir a ser alvo de violência física, sexual e/ou psicológica (ALBUQUERQUE et al, 2016, p. 101). Referente à prática ao qual Stuart é submetido, ela parte da percepção de que a suposta Revista Diversidade e Educação, v. 11, n. 1, p. 547-578, 2023. Doi: 10.14295/de.v11i1.15401 E-ISSN: 2358-8853 Horror Heteronormativo: representações de gênero e sexualidade e pedagogias do horror no filme They/Them (2022) de John Logan 569 patologia que o acomete é passível de reversão, sendo ela entendida como resultante de uma falha no ambiente ao qual o sujeito se encontraria inserido (GARCIA; MATTOS, 2019). Embora não se tenha dissertado a respeito até o presente momento, cabe debruçar-se sobre a figura monstruosa presente na produção. Ao desenrolar da narrativa, o espectador se depara com a figura de um assassino que vai deixando corpos ensanguentados pelo acampamento, até que finalmente ele tem sua identidade revelada. Em confronto com Owen, que se encontra no chão, com a figura mascarada apontando- lhe uma faca, o assassino revela sua verdadeira face, trata-se da enfermeira Molly (interpretada por Anna Chlumsky), que há pouco tempo trabalhava no acampamento (Figura 11). FIGURA 11: Revelando a figura monstruosa. Fonte: They/Them, 2022 FIGURA 11: Revelando a figura monstruosa. Fonte: They/Them, 2022 Sem compreender o que ocorre, Owen questiona a razão daquilo. É então revelado, por Molly, de que sua presença ali e os assassinatos são decorrentes de um desejo de vingança. Seu nome verdadeiro é Angie Phelps, que anos atrás, ainda muito jovem, esteve no acampamento sendo submetida a todo tipo de horror. Como salienta em sua fala: “Angie Phelps. Você lembra dela? das zonas de policiamento, tanto a obediência, quanto a transgressão e subversão da ordem estão presentes. Uma das formas de transgredir ou subverter encontra-se justamente na paródia, sob uma forma crítica (LOURO, 2004). Como salienta Louro: Ela era uma boa menina na época. Antes de tudo isto, de você colocá-la naquela cadeira e queimar tudo que ela tinha de bom” (Figura 12). As palavras de Molly, ou de Angie, revelam que possivelmente ela Revista Diversidade e Educação, v. 11, n. 1, p. 547-578, 2023. Doi: 10.14295/de.v11i1.15401 E-ISSN: 2358-8853 Horror heteronormativo: representações de gênero e sexualidade e pedagogias do horror no filme They/Them (2022) de John Logan 570 passou pelo mesmo tratamento que a pouco Stuart havia sido submetido, a terapia de aversão. FIGURA 12: O ódio produz monstros. Fonte: They/Them, 2022 FIGURA 12: O ódio produz monstros. Fonte: They/Them, 2022 Alguns autores vão compreender que a figura monstruosa é um elemento necessário para produções de horror (CARROLL, 1999), contudo, deve-se expandir a compreensão em torno desta figura, a fim de não limitar o gênero de horror. Assim, a figura do monstro pode estar presente em uma produção do gênero sendo um ser incompreensível, que rompe com o que concebe como normal na narrativa, ou ainda, como uma ameaça natural. Têm-se, assim, monstros que vão desde zumbis, vampiros, alienígenas, demônios, fantasmas, mas também, animais excessivamente agressivos, assassinos em série e psicopatas (PUPPO, 2012). Com essa compreensão mais abrangente, torna-se possível compreender a figura de Molly, ou Angie, como pertencente ao grupo de figuras monstruosas que se encontram nas produções do gênero de horror. Contudo, sua compreensão deve ir um pouco além, buscando visualizar o que sua presença na produção suscita. Conforme vai se evidenciando ao passar de suas falas, percebe-se o quanto tudo que Molly viveu no acampamento lhe causou sérias consequências e lhe fez um mal, aparentemente irreversível. Como diz em referência a si mesma: “Quando Angie saiu daqui, só tinha tentativas de suicídio, a solidão, o ódio de si mesma, os remédios, os médicos e a raiva, tanta raiva...” (Figura 13). Tal percepção sobre a sua pessoa, Revista Diversidade e Educação, v. 11, n. 1, p. 547-578, 2023. Doi: 10.14295/de.v11i1.15401 E-ISSN: 2358-8853 Horror Heteronormativo: representações de gênero e sexualidade e pedagogias do horror no filme They/Them (2022) de John Logan 571 contribui para pensar e problematizar a construção da figura monstruosa nas produções cinematográficas de horror. FIGURA 13: Problematizando a figura monstruosa. Fonte: They/Them, 2022 FIGURA 13: Problematizando a figura monstruosa. das zonas de policiamento, tanto a obediência, quanto a transgressão e subversão da ordem estão presentes. Uma das formas de transgredir ou subverter encontra-se justamente na paródia, sob uma forma crítica (LOURO, 2004). Como salienta Louro: Fonte: They/Them, 2022 Fonte: They/Them, 2022 Primeiramente deve-se compreender que o monstro em questão, essa figura violenta e vingativa que mata os membros do acampamento, é fruto do que se pode chamar de metamorfose, em decorrência da plasticidade destrutiva, explosiva, inerente ao ser humano. Tal conceito têm fundamento no pensamento da autora Catherine Malabou, ao compreender que, através de graves traumatismos ou acidentes, o caminho de um sujeito então se bifurca e produz um novo ser, que coabita com o antigo e toma, então seu lugar. Trata-se de uma “forma nascida do acidente, nascida por acidente, uma espécie de acidente. Uma estranha raça. Um monstro cuja aparição nenhuma anomalia genética permite explicar” (MALABOU, 2014, p. 9). Conforme Malabou específica em seu trabalho: Fui testemunha de transformações desse tipo, mesmo se não deformavam os rostos, mesmo se procediam menos diretamente de acidentes reconhecidos como tais. Menos espetaculares, menos brutais, nem por isso deixavam de ter o poder de começar um fim, de deslocar o sentido de uma vida. Em certo casal que não conseguia se recuperar de uma infidelidade. Numa mulher de alta sociedade cujo filho rompeu com tudo brutalmente, deixou sua família para morar ilegalmente no norte da França. Num colega que foi viver no Texas achando que seria feliz lá. Em muitas pessoas, no centro da França, Revista Diversidade e Educação, v. 11, n. 1, p. 547-578, 2023. Doi: 10.14295/de.v11i1.15401 E-ISSN: 2358-8853 Fui testemunha de transformações desse tipo, mesmo se não deformavam os rostos, mesmo se procediam menos diretamente de acidentes reconhecidos como tais. Menos espetaculares, menos brutais, nem por isso deixavam de ter o poder de começar um fim, de deslocar o sentido de uma vida. Em certo casal que não conseguia se recuperar de uma infidelidade. Numa mulher de alta sociedade cujo filho rompeu com tudo brutalmente, deixou sua família para morar ilegalmente no norte da França. Num colega que foi viver no Texas achando que seria feliz lá. Em muitas pessoas, no centro da França, vista Diversidade e Educação, v. 11, n. 1, p. 547-578, 2023. Doi: 10.14295/de.v11i1.15401 E-ISSN: 2358-88 Revista Diversidade e Educação, v. 11, n. 1, p. 547-578, 2023. Doi: 10.14295/de.v11i1.15401 Revista Diversidade e Educação, v. 11, n. 1, p. 547-578, 2023. das zonas de policiamento, tanto a obediência, quanto a transgressão e subversão da ordem estão presentes. Uma das formas de transgredir ou subverter encontra-se justamente na paródia, sob uma forma crítica (LOURO, 2004). Como salienta Louro: Doi: 10.14295/de.v11i1.15401 E-ISSN: 2358-8853 Horror heteronormativo: representações de gênero e sexualidade e pedagogias do horror no filme They/Them (2022) de John Logan 572 Horror heteronormativo: representações de gênero e sexualidade e pedagogias do horror no filme They/Them (2022) de John Logan onde morei muito tempo, que perderam seu trabalho quando estavam na faixa dos cinquenta anos durante a crise de 1985, em professores de zonas difíceis, em doentes de Alzheimer. O que era impressionante em todos esses casos era que a metamorfose operada, por explicável que fosse quanto as suas causas (desemprego, problemas relacionais, doenças), era absolutamente surpreendente quanto a seus efeitos, e se tornava portanto incompreensível, deslocando a posteriori a causalidade, rompendo os laços etiológicos. Essas pessoas tinham subitamente se tornado estrangeiras para si mesmas pelo fato de não poderem fugir. Não é, ou apenas, que elas estivessem quebradas, oprimidas pela mágoa ou pelo infortúnio, não, elas tinham se tornado pessoas novas, outras, reengendradas, pertencentes a uma espécie diferente. Como se tivessem sofrido, de fato, um acidente. (MALABOU, 2014, p. 18). Há, assim, esse choque destrutivo ao qual Molly foi submetida, quando mais jovem, às práticas corretivas postas em operação no acampamento. De tal forma, Molly, de uma boa menina, torna-se uma assassina. Deve-se, todavia, ir além de um pensamento simplista de bem ou mal, pois a figura monstruosa a qual Molly representa está inserida, e emerge, a partir de uma complexa matriz de relações existentes. Isto significa pensar que o monstro nasce em “encruzilhadas metafóricas, como a corporificação de um certo momento cultural – de uma época, de um sentimento e de um lugar [...]. O corpo monstruoso é pura cultura” (COHEN, 2000, p. 26, 27). Para entender a figura monstruosa representada por Molly, deve-se considerar seu contexto de emergência. Isso é, deve-se pensar, por um lado, na violência a que foi submetida, como forma de ação monstruosa operando sobre si. Neste sentido para supostamente corrigi-la e, por outro lado, na constituição da figura do monstro, que deseja aniquilar e não corrigir. Cohen salienta: O monstro é a diferença feita de carne; ele mora no nosso meio. Em sua função como Outro dialético ou suplemento que funciona como terceiro termo, o monstro é uma incorporação do Fora, do Além – de todos aqueles locais que são retoricamente colocados como distantes e distintos, mas que se originam no Dentro. Revista Diversidade e Educação, v. 11, n. 1, p. 547-578, 2023. Doi: 10.14295/de.v11i1.15401 das zonas de policiamento, tanto a obediência, quanto a transgressão e subversão da ordem estão presentes. Uma das formas de transgredir ou subverter encontra-se justamente na paródia, sob uma forma crítica (LOURO, 2004). Como salienta Louro: Qualquer tipo de alteridade pode ser inscrito através (construído através) do corpo monstruoso, mas, em sua maior parte, a diferença monstruosa tende a ser cultural, política, racial, econômica, sexual. (COHEN, 2000, p. 32). O monstro é a diferença feita de carne; ele mora no nosso meio. Em sua função como Outro dialético ou suplemento que funciona como terceiro termo, o monstro é uma incorporação do Fora, do Além – de todos aqueles locais que são retoricamente colocados como distantes e distintos, mas que se originam no Dentro. Qualquer tipo de alteridade pode ser inscrito através (construído através) do corpo monstruoso, mas, em sua maior parte, a diferença monstruosa tende a ser cultural, política, racial, econômica, sexual. (COHEN, 2000, p. 32). Uma das lições que a produção cultural constitui é a ideia de que verdades únicas podem gerar figuras monstruosas através de suas imposições. Do mesmo modo, estas figuras monstruosas podem contribuir para refletir e debater acerca da diferença e da violência (FORTES, 2023). O monstro, dentro do que propõe a produção, pode ser Revista Diversidade e Educação, v. 11, n. 1, p. 547-578, 2023. Doi: 10.14295/de.v11i1.15401 Revista Diversidade e Educação, v. 11, n. 1, p. 547-578, 2023. Doi: 10.14295/de.v11i1.15401 E-ISS E-ISSN: 2358-8853 Horror Heteronormativo: representações de gênero e sexualidade e pedagogias do horror no filme They/Them (2022) de John Logan 573 concebido como o que não está no seu lugar, aquele que não pode ser, o irrecuperável, que nasce na transgressão de fronteiras e limites estabelecidos. Dentro das fronteiras de gênero ou de sexualidade, estes sujeitos são vistos como diferentes e desviantes (LOURO, 2004), e, em uma mesma lógica, esse sujeito pode vir a ser transformado em um monstro, em conta do novo que se impõe (COHEN, 2000). O que deve ser tomado em questão é que sua transformação em um monstro não se limita a uma compreensão, como salientado anteriormente, entre bem e mal, ou bom e ruim. A figura do monstro, por si só, resiste “a qualquer classificação construída com base em uma hierarquia ou em uma oposição meramente binária, exigindo, em vez disso, um „sistema‟ que permita a polifonia, a reação mista (diferença na mesmidade, repulsão na atração) e a resistência à integração” (COHEN, 2000, p. 31). Deve-se, sim, tomar a figura monstruosa como resultado de algo maior e mais complexo. das zonas de policiamento, tanto a obediência, quanto a transgressão e subversão da ordem estão presentes. Uma das formas de transgredir ou subverter encontra-se justamente na paródia, sob uma forma crítica (LOURO, 2004). Como salienta Louro: A figura de Molly pode ser compreendida como uma manifestação de resistência frente a heteronormatividade, uma espécie de resistência e subversão às normas regulatórias (LOURO, 2004), mesmo que com o uso de violência extrema. Ela corporifica um tipo de reação movida pelo sentimento de vingança. Mas ela pode também encarnar a possibilidade de fazer cessar a violência que, no limite, pode ser experimentada pelo espectador. O filme revela um horror pouco presente nas telas de produções cinematográficas do gênero, além de produzir uma subversão daquilo que se marca, desde o início da trama, como sendo a figura monstruosa, e que por si só, contribui para uma série de reflexões e problemáticas. Considerações Finais O percurso da análise proposta não se resume a analisar a estética da produção They/Them, isto é, não houve o intuito de analisar o filme e dizer simplesmente se ele é bom ou ruim. Pelo contrário, o objetivo foi o de analisar as representações de gênero e sexualidade produzidas na referida produção, e quais pedagogias objetivamente dela emergem. Pode-se observar que há uma variedade de representações no tocante a diversidade de gênero e sexual, contudo, e o mais importante, percebe-se o quanto estas representações estão alinhadas à imposição da heteronormatividade, estando em constante regulação por ela. A “virada” acontece quando a violência faz emergir o monstro – este que está fora de controle e emprega o assassinato como forma de Revista Diversidade e Educação, v. 11, n. 1, p. 547-578, 2023. Doi: 10.14295/de.v11i1.15401 E-ISSN: 2358-8853 Horror heteronormativo: representações de gênero e sexualidade e pedagogias do horror no filme They/Them (2022) de John Logan 574 vingança. O cenário é típico de uma produção de horror, com um claro teor de violência, desconforto e medo frente a tudo ao que os jovens presentes no acampamento são submetidos. Estão eles sujeitos a diversos tipos de violência, que os afetam das mais diferentes formas, causando, conforme pode-se perceber pelo desfecho do filme, consequências irreparáveis. A narrativa a qual o espectador é exposto não se difere muito dos constantes casos vistos em noticiários, nos quais a população LGBTQIAP+ é submetida diariamente diante do horror fruto da heteronormatividade. Compreende-se, assim, a produção mais do que mero entretenimento, também como um meio a partir do qual uma mensagem é produzida e pedagogias emergem. Algo que caminha ao direcionamento da proposta do dossiê, de compreender o espaço educacional, e assim o cinema, como uma forma de enfrentamento às múltiplas violências. Assim, a violência e o horror presentes na produção podem ser compreendidas não apenas como meras formas de continuidade, mas de denúncia e resistência, e sobretudo, como meio de fazer com que se reflita sobre problemáticas e grupos que por muito tempo foram silenciados. Algumas lições, ou pedagogias, emanam do horror exposto na produção. Uma delas, trata da imposição de verdades únicas, ou a imposição da heteronormatividade que produz monstros, ou seja, o entendimento de que há seres desviantes, indivíduos que fogem à regra e que devem ser corrigidos. Problematizando um pouco mais essa lição, atenta-se ao fato de que a imposição da heteronormatividade confunde as fronteiras estabelecidas. Considerações Finais É possível indagar: quem, de fato, é o monstro? Por fim, e além do exposto até então, deve-se conceber a potencialidade das produções do gênero, que vão além do que o senso comum pode conceber, servindo como um meio pelo qual uma série de problemáticas, das mais diversas, podem ser trazidas à reflexão, como no presente caso, o horror da heteronormatividade. Referências ALBUQUERQUE, Grayce Alencar; PARENTE, Jeanderson Soares; BELÉM, Jameson Moreira; GARCIA, Cintia de Lima. Violência psicológica em lésbicas, gays, bissexuais, travestis e transexuais no interior do Ceará, Brasil. Saúde Debate. Rio de Janeiro, v. 40, n. 109, p. 100-111, Abr./Jun. 2016. ANTUNES, Pedro Paulo Sammarco. Homofobia internalizada: o preconceito do homossexual contra si mesmo. Tese (Doutorado). Programa de Estudos Pós-graduados em Psicologia Social, Pontifícia Universidade Católica de São Paulo, 2016. Revista Diversidade e Educação, v. 11, n. 1, p. 547-578, 2023. 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Fora do armário: as novas representatividades do horror queer no cinema. Monografia (Graduação). Curso de Comunicação Social da Faculdade de Comunicação da Universidade de Brasília. Brasília, 2022. FABRIS, Eli Terezinha Henn. Em cartaz: o cinema brasileiro produzindo sentidos sobre escola e trabalho docente. Tese (Doutorado). Programa de Pós-Graduação em Educação, Universidade Federal do Rio Grande do Sul. Porto Alegre, 2005. FANTINEL, Fernanda. “They/Them”: Adolescentes são mandados para campo de conversão LGBTQIA+ e dão de cara com serial killer. Hugo Gloss. Publicado em: 23 de junho de 2022. Disponível em: <https://hugogloss.uol.com.br/filmes/they-them- adolescentes-sao-mandados-para-campo-de-conversao-lgbtqia-e-dao-de-cara-com- serial-killer-assista-ao-trailer/>. Acesso em: 19 de março de 2023. FERRAZ, Diego. They/Them (2022). Boca do Inferno. Publicado em: 25 de setembro de 2022. Disponível em: <https://bocadoinferno.com.br/criticas/2022/09/they-them- 2022/>. 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Rio de Janeiro, São Paulo: Editora Paz e Terra, 2017b. GARCIA, Marcos Roberto Vieira; MATTOS, Amana Rocha. “Terapias de Conversão”: Histórico da (Des) Patologização das Homossexualidades e Embates Jurídicos Contemporâneos. Psicologia: Ciência e Profissão, v.39, n. 3, p. 49-61, 2019. GIROUX, Henry Armand. Praticando Estudos Culturais nas faculdades de educação. In: SILVA, Tomaz Tadeu da (org.). Alienígenas na Sala de Aula: uma introdução aos estudos culturais em educação. 11ª ed. Referências Petrópolis, Rio de Janeiro: Editora Vozes, 2013. p. 83-100. GODOY, Iam. Horror Queer – Parte 5: Anos 1990 – Novo Cinema Queer e Papais- Vampiro Gays. Boca do Inferno. Publicado em: 02 de abril de 2020a. Disponível em: <https://bocadoinferno.com.br/artigos/2020/04/horror-queer-parte-5-anos-1990-novo- cinema-queer-e-papais-vampiro-gays/>. Acesso em: 18 de abril de 2023. vista Diversidade e Educação, v. 11, n. 1, p. 547-578, 2023. 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Horror no Cinema Brasileiro. Centro Cultural Banco do Brasil, 2012. THEY/THEM. Diretor: John Logan. Roteiro: John Logan. Estados Unidos da América, 2022, 104 min. Revista Diversidade e Educação, v. 11, n. 1, p. 547-578, 2023. Doi: 10.14295/de.v11i1.15401 E-ISSN: 2358-8853 578 Horror heteronormativo: representações de gênero e sexualidade e pedagogias do horror no filme They/Them (2022) de John Logan Horror heteronormativo: representações de gênero e sexualidade e pedagogias do horror no filme They/Them (2022) de John Logan ROSE, Gillian.Visual methodologies: an introduction to the interpretation of visual materials. London: Sage, 2001. SEFFNER, Fernando; FIGLIUZZI, Adriza. Na escola e nas revistas: Reconhecendo pedagogias do gênero, da sexualidade e do corpo. E-ISSN: 2358-8853 Recebido em abril de 2023. Aprovado em julho de 2023. ALL, Stuart. Cultura e Representação. Rio de Janeiro: Ed. PUC-Rio: Apicuri, 2016. Revista FACED, Salvador, nº 19, p. 45-59, jan./jun, 2011. 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Publicado em: 26 de junho de 2019. Disponível em: Publicado em: 26 de junho de 2019. Disponível em: <http://www.aescotilha.com.br/cinema-tv/espanto/representatividade-lgbt-no-cinema- queer-horror/>. Acesso em: 18 de abril de 2023. WALTER, Ed. Crítica - They/Them - O Acampamento (They/Them, 2022). Blog Sangue Tipo B. Publicado em: agosto de 2022. Disponível em: <https://sanguetipob.blogspot.com/2022/08/critica-they-them-2022.html>. Acesso em: 19 de março de 2023. WILLIAMS, Raymond. La larga revolución. Buenos Aires: Nueva Visión, 2003. ZIZEK, Slavoj. Violência: seis reflexões laterais. São Paulo: Boitempo, 2014. Recebido em abril de 2023. Aprovado em julho de 2023. Revista Diversidade e Educação, v. 11, n. 1, p. 547-578, 2023. Doi: 10.14295/de.v11i1.15401 E-ISSN: 2358-8853
https://openalex.org/W2056264205	https://europepmc.org/articles/pmc3231767?pdf=render	English	null	A randomised trial and economic evaluation of the effect of response mode on response rate, response bias, and item non-response in a survey of doctors	BMC Medical research methodology	2,011	cc-by	9,894	Abstract Background: Surveys of doctors are an important data collection method in health services research. Ways to improve response rates, minimise survey response bias and item non-response, within a given budget, have not previously been addressed in the same study. The aim of this paper is to compare the effects and costs of three different modes of survey administration in a national survey of doctors. Methods: A stratified random sample of 4.9% (2,702/54,160) of doctors undertaking clinical practice was drawn from a national directory of all doctors in Australia. Stratification was by four doctor types: general practitioners, specialists, specialists-in-training, and hospital non-specialists, and by six rural/remote categories. A three-arm parallel trial design with equal randomisation across arms was used. Doctors were randomly allocated to: online questionnaire (902); simultaneous mixed mode (a paper questionnaire and login details sent together) (900); or, sequential mixed mode (online followed by a paper questionnaire with the reminder) (900). Analysis was by intention to treat, as within each primary mode, doctors could choose either paper or online. Primary outcome measures were response rate, survey response bias, item non-response, and cost. Results: The online mode had a response rate 12.95%, followed by the simultaneous mixed mode with 19.7%, and the sequential mixed mode with 20.7%. After adjusting for observed differences between the groups, the online mode had a 7 percentage point lower response rate compared to the simultaneous mixed mode, and a 7.7 percentage point lower response rate compared to sequential mixed mode. The difference in response rate between the sequential and simultaneous modes was not statistically significant. Both mixed modes showed evidence of response bias, whilst the characteristics of online respondents were similar to the population. However, the online mode had a higher rate of item non-response compared to both mixed modes. The total cost of the online survey was 38% lower than simultaneous mixed mode and 22% lower than sequential mixed mode. The cost of the sequential mixed mode was 14% lower than simultaneous mixed mode. Compared to the online mode, the sequential mixed mode was the most cost-effective, although exhibiting some evidence of response bias. Conclusions: Decisions on which survey mode to use depend on response rates, response bias, item non- response and costs. Scott et al. BMC Medical Research Methodology 2011, 11:126 http://www.biomedcentral.com/1471-2288/11/126 Scott et al. BMC Medical Research Methodology 2011, 11:126 http://www.biomedcentral.com/1471-2288/11/126 A randomised trial and economic evaluation of the effect of response mode on response rate, response bias, and item non-response in a survey of doctors A randomised trial and economic evaluation of the effect of response mode on response rate, response bias, and item non-response in a survey of doctors Anthony Scott1, Sung-Hee Jeon1, Catherine M Joyce2, John S Humphreys3, Guyonne Kalb1, Julia Witt4 and Anne Leahy1 Correspondence: catherine.joyce@monash.edu 2Department of Epidemiology and Preventive Medicine School of Public Health and Preventive Medicine Monash University, Alfred Hospital Melbourne 3004, Australia Full list of author information is available at the end of the article © 2011 Scott 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. RESEARCH ARTICLE Open Access Background Surveys of medical practitioners can provide important policy-relevant data and information that is often not captured by administrative data or registration data- bases. There is some suggestion that response rates for surveys of medical practitioners may be falling, with important implications for statistical inference, and for the extent to which results can be generalised and used to inform policy [1-3]. There is growing evidence in the literature about the most effective interventions to increase response rates in general population and doctor surveys. Interventions to improve response rates include incentive-based approaches (e.g. money, gifts, lottery and prize draws), design-based approaches (e.g. survey length, follow-up, content) and mode of administration (e.g. paper, internet, interview). Three key factors that influence doctors’ decisions to com- plete a survey are the opportunity cost of their time; their trust that the results will be used appropriately; and the perceived relevance of the survey [4]. Different modes of administration may also influence survey response bias (whether those responding are repre- sentative of the population) and item non-response (the extent to which all questions have been completed) as well as overall survey response rates. Response rates are fre- quently regarded as sentinel indicators of methodological quality in general, and representativeness in particular [15]. Although response rates are often used as a ‘conven- tional proxy’ for response bias, there is in fact no necessary relationship between response rate and response bias [16-19]. Despite this, less than half (44%) of published surveys of doctors discuss response bias, and only 18% provided some analysis of it [20]. Item non-response is also an issue, with respondents less likely to answer sen- sitive questions and some skipping whole sections, depending on how the survey has been designed and administered [21]. High item non-response was found in a web survey, compared to a face-to-face survey, of univer- sity students [22], whilst health professionals who were younger, male, and worked in hospitals were more likely to complete a web survey than a mailed survey [23]. Although the literature about factors influencing response rates is growing, there are three important gaps that this paper aims to address: i) a lack of evidence on the use of mixed mode survey designs; ii) a lack of evidence examining response bias and item non-response, in addi- tion to response rate, and iii) a lack of evidence on the cost-effectiveness of different strategies. Abstract The sequential mixed mode appears to be the most cost-effective mode of survey administration for surveys of the population of doctors, if one is prepared to accept a degree of response bias. Online surveys are not yet suitable to be used exclusively for surveys of the doctor population. * Correspondence: catherine.joyce@monash.edu 2Department of Epidemiology and Preventive Medicine School of Public Health and Preventive Medicine Monash University, Alfred Hospital Melbourne 3004, Australia Full list of author information is available at the end of the article Scott et al. BMC Medical Research Methodology 2011, 11:126 http://www.biomedcentral.com/1471-2288/11/126 Scott et al. BMC Medical Research Methodology 2011, 11:126 http://www.biomedcentral.com/1471-2288/11/126 Page 2 of 12 survey, whilst older respondents are more likely to respond to a mailed survey. Accounting for doctors’ pre- ferences about which survey mode to complete may be important. For example, in a survey of doctors in the United States, paper surveys were preferred to email surveys when they were given the choice [9], and family physicians preferred mail surveys compared to surgeons [10]. The ability of doctors to choose their preferred mode of response to fit with their busy schedules is likely to be important [4,11]. Evidence from non-doctor populations suggests that offering a choice of mode does not increase response rates, but that the sequen- cing or switching of modes (e.g. paper followed by online) may matter [12-14]. A paper examining this for US physicians showed that mail first, followed by a web survey, had a higher response rate than web followed by mail [8]. Methods A randomised trial was conducted as part of the third and final pilot survey for Wave 1 of the Medicine in Australia: Balancing Employment and Life (MABEL) longitudinal cohort/panel study of the dynamics of the medical labour market in Australia, focusing on workforce participation and its determinants among Australian doctors [32]. The first wave of data collection, establishing the baseline cohort for the study, was under- taken in 2008. The questionnaire included eight sections: job satisfac- tion, attitudes to work and intentions to quit or change hours worked; a discrete choice experiment (DCE) exam- ining preferences and trade-offs for different types of jobs; characteristics of work setting (public/private, hospital, private practice); workload (hours worked, on-call arrange- ments, number of patients seen, fees charged); finances (income, income sources, superannuation); geographic location; demographics (including specialty, qualifications, residency); and family circumstances (partner and chil- dren). There were four versions of the survey, each differ- ing slightly in order to tailor them to the type of doctor: GPs, specialists, specialists-in-training, and hospital non- specialists. Although survey length also matters for response rates, the context of the survey and research questions being tested required a long questionnaire, to ensure that sufficient data were collected to adequately test study hypotheses [32]. The length ranged from 58 questions in an eight-page booklet (for specialists-in-train- ing), to 87 questions in a 13-page booklet (for specialists). In all modes, doctors in remote and rural areas (defined using the Rural, Remote and Metropolitan Area (RRMA) classification to doctors in RRMA 6 (Remote centre with population > 5,000) and RRMA7 (Other remote centre with population < 5,000)), mainly GPs, were given a che- que for $100 that was enclosed with the invite letter to recognise both their importance from a policy perspective, and the significant time pressures on these doctors. The purpose was to draw meaningful inferences about recruit- ment and retention in rural and remote areas. Pre-paid monetary incentives, not conditional on response, have been shown to double response rates [25]. The survey described in this paper was also the main pilot survey for the main wave of MABEL, so it was important to pilot the administration of these incentives. However, they did not influence the outcome of this trial as randomisation ensured approximately equal numbers of cheques going out in each arm of the trial. Scott et al. BMC Medical Research Methodology 2011, 11:126 http://www.biomedcentral.com/1471-2288/11/126 Page 3 of 12 4). the costs of the sequential mixed mode will be lower than the simultaneous mixed mode. 4). the costs of the sequential mixed mode will be lower than the simultaneous mixed mode. effect of a lottery for GPs in Canada and found a 6.4% increase in the response rate at a cost of $CAD16 per additional returned survey [28]. Bjertnaes et al examined the effects and costs of the number of reminders in a sur- vey of Norwegian physicians, and found that costs per response increased dramatically with telephone follow up [29]. Erdogan and Baker (2002) examined costs and effects of different methods of follow-up in a sample of advertis- ing agency executives, but compared average costs and effect rather than incremental costs and effect [30]. A study that compared the costs of a mail and email survey in a group of academics found the email survey’s costs were lower but that mail had a 12% higher response rate [31]. h f h d d d d l effect of a lottery for GPs in Canada and found a 6.4% increase in the response rate at a cost of $CAD16 per additional returned survey [28]. Bjertnaes et al examined the effects and costs of the number of reminders in a sur- vey of Norwegian physicians, and found that costs per response increased dramatically with telephone follow up [29]. Erdogan and Baker (2002) examined costs and effects of different methods of follow-up in a sample of advertis- ing agency executives, but compared average costs and effect rather than incremental costs and effect [30]. A study that compared the costs of a mail and email survey in a group of academics found the email survey’s costs were lower but that mail had a 12% higher response rate [31]. Background The use of online and web-based surveys is growing, including those where email is the method of contact. Web-based surveys may seem attractive as there are no printing or data-entry costs, but response bias may be an issue, particularly if older respondents are less likely to respond, and a lack of trust in the security of transmitting information over the internet may reduce response rates and increase item non-response [2]. For doctors, emailed surveys have resulted in lower response rates than mailed surveys [4]. This is also reflected in the use of email in non-doctor populations, where meta analyses have found that web-based surveys, mostly using email contact, have a 10-11% lower response rate compared to other modes [5,6]. This is despite the fact that email surveys that include a weblink reduce the number of steps (and time) to complete a survey. However, evidence also shows that email contact can be impersonal and reduce response rates [7]. For doctors, there is an issue of whether the email will reach the respondent or be initially read by administrative staff who may not forward such emails to respondents, though this may also be an issue if mailed surveys are posted to their work address. There is also a lack of rigorous evidence on the cost- effectiveness of the many different approaches to improve response rates and reduce bias [24]. Email and web surveys may seem cheaper than mailed surveys, and the effects on costs for mixed mode surveys are less clear. Researchers often have limited resources and adoption of all possible measures to increase response rates is usually not possible due to cost constraints and ethical considera- tions, especially when the study population or sample is widely dispersed. For these reasons, researchers must make choices as to which method leads to the largest increase in response rate (or other outcome) for each dollar spent. For example, up-front financial incentives may be the most effective, but are also costly compared with other approaches [7,25-27]. Baron et al examined the Furthermore, the use of different types of mixed mode surveys for doctors has not yet been investigated thor- oughly [4,8]. This is important if, for example, younger respondents are more likely to respond to an online Scott et al. BMC Medical Research Methodology 2011, 11:126 http://www.biomedcentral.com/1471-2288/11/126 Sequential mixed mode Mailed personal invitation letter with: * login details * option to request paper copy Mailed personal invitation letter with: * login details * option to request paper copy Mailed personal invitation letter with: * paper copy & reply paid envelope * option to complete online * option to request paper copy * option to request paper copy Figure 1 Description of mode of initial contact and follow up contact for the three arms of the trial. Directory, which includes all doctors in all States and Territories of Australia and formed our sampling frame. This national database is used extensively for mailing pur- poses (e.g. the Medical Journal of Australia). The Directory is updated regularly using a number of sources. AMPCo receives 58,000 updates to doctors’ details per year, through biannual telephone surveys, and checks medical registration board lists, Australian Medical Association membership lists and Medical Journal of Australia sub- scription lists to maintain accuracy. The directory contains a number of key characteristics that can be used for check- ing the representativeness of the sample and to adjust for any response bias in sample weighting. These characteris- tics include age, gender, location, and job description (used to group doctors into the four types). to the main web page (http://www.mabel.org.au), where they clicked on the ‘Login’ link, which directed them to a login page where they entered their username and pass- word. They were then directed to the first page of the survey. Respondents could save their responses and log- out, and then login again to complete the survey, and they could skip questions. Once logged in, the padlock icon was visible, indicating that the website was secure. The primary outcomes of interest in the trial were response rates, survey response bias with respect to age, gender, doctor type and geographic location, and item- response (the percentage of completed items). A three-arm parallel trial design was used with equal randomisation across arms. The sample size for the trial was calculated to detect a difference of 5% in the response rate at the 95% level of statistical significance, and with a power of 80%. This indicated that a sample of 900 doctors in each arm of the trial would be required, 2,700 doctors in total. Methods BMC Medical Research Methodology 2011, 11:126 http://www.biomedcentral.com/1471-2288/11/126 Online mode Mailed personal invitation letter with: * login details * option to request paper copy As above Sequential mixed mode Mailed personal invitation letter with: * login details * option to request paper copy Mailed personal invitation letter with: * paper copy & reply paid envelope * option to complete online Simultaneous mixed mode Mailed personal invitation letter with: * paper copy & reply paid envelope * option to complete online Mailed personal invitation letter with: * login details * option to request paper copy Figure 1 Description of mode of initial contact and follow up contact for the three arms of the trial. Online mode Mailed personal invitation letter with: * login details * option to request paper copy As above Sequential mixed mode Mailed personal invitation letter with: * login details * option to request paper copy Mailed personal invitation letter with: * paper copy & reply paid envelope * option to complete online Simultaneous mixed mode Mailed personal invitation letter with: * paper copy & reply paid envelope * option to complete online Mailed personal invitation letter with: * login details * option to request paper copy Figure 1 Description of mode of initial contact and follow up contact for the three arms of the trial. Methods The aim of this study is to conduct a randomised trial and economic evaluation of an online survey compared to two types of mixed mode. Our choice of modes reflects the importance to doctors of being able to choose which mode to fill out, and the importance of a personalised mailed letter sent to their preferred mailing address (rather than their work address) as the main mode of contact rather than email. In all three modes, our method of con- tact was by mailed personalised letter. Three response modes were compared (Figure 1): (i) Online mode: a mailed personal invitation letter asked doctors to logon to a secure website to fill out an online version of the ques- tionnaire. Respondents could request a paper copy by phone/fax/email or they could print out a paper question- naire after they logged on to the website. They were sent a reminder letter around three weeks later that again included login details. (ii) Sequential mixed mode: as above, but a paper questionnaire and reply-paid envelope was included with the reminder letter three weeks later; and (iii) Simultaneous mixed mode: a paper questionnaire and reply-paid envelope was sent out with the invitation letter, which also contained login details and so respon- dents could alternatively choose to fill out the survey online if they wished. A reminder letter was sent three weeks later with login details only and no paper survey. Primary outcome measures were response rate, survey response bias and item response. An economic evaluation comparing the costs of each mode of administration was also conducted by applying the results from the trial to the expected costs of the full main wave survey. Our hypotheses are that: 1). online mode will result in a lower response rate and higher item non-response, compared with the two mixed modes; 1). online mode will result in a lower response rate and higher item non-response, compared with the two mixed modes; 2). sequential mixed mode will have a higher response rate than simultaneous mixed mode; 3). the costs of the online mode will be lower than the two mixed modes; and The process of logging in and completing the survey online was kept as simple as possible. Users were directed Page 4 of 12 Scott et al. BMC Medical Research Methodology 2011, 11:126 http://www.biomedcentral.com/1471-2288/11/126 Scott et al. Results Responses were received between March and October 2008. The characteristics of doctors in the three groups for the study sample are shown in Table 1. Although there are some small differences of up to three percentage points, the three groups are broadly similar in terms of key characteristics. The comparison of response rates across modes is shown in Table 2. Response rates were between 6 and 7 percentage points higher for the two mixed modes, compared to online (Table 2). Table 3 shows response rates by mode and doctor type. Specialists had the highest overall response rates and GPs the lowest. Response rates for simultaneous and sequential mixed modes were between 2 and 6 percentage points higher for GPs, and between 10 and 15 percentage points higher for specialists. For hospital non-specialists, the response rate for simultaneous mixed mode was four percentage points higher than online, but four percentage points lower than sequential mixed mode. For specialists in training, the simultaneous mixed mode had the lowest response rate, with the sequential and online modes producing similar results. Analysis included comparisons of response rates; esti- mation of means and proportions of respondents by age, gender, doctor type, and geographic location compared to the doctor population; logistic regression of response bias; and comparisons of the proportion of missing values (item non-response). The statistical significance of the differences between the response rates of the three response modes was analysed using a probit model with response (0/1) as the dependent variable and two dummy variables for response mode, with online as the reference category. The difference between the sequential and simultaneous modes was tested using the restriction that their coefficients be equal. Although respondents were randomly allocated across modes, it is still important to test whether any specific/particular respondent charac- teristics influenced the response rate. The probit model therefore included age, gender, doctor type and geo- graphic location. Survey response bias was examined using a multinomial logit model of respondents (= 1) and the total population of doctors (= 0), with age, gender, geographic location and doctor type as independent vari- ables. For item non-response, a comparison of the pro- portion of completed items was supplemented using generalised linear models that controlled for differences due to age, gender, geographic location and doctor type [33]. Sequential mixed mode This represented just under 5% (2700/54,160 = 0.04985) of all doctors undertaking clinical practice on the Australian Medical Publishing Company’s (AMPCo) Medical A 4.9% stratified random sample of doctors was there- fore taken, with stratification by four doctor types (gen- eral practitioners (GPs), specialists, doctors enrolled in a specialist training program, and non-specialist hospital doctors (including interns and salaried medical officers)), and six rural/remoteness categories (Rural, Remote and Page 5 of 12 Scott et al. BMC Medical Research Methodology 2011, 11:126 http://www.biomedcentral.com/1471-2288/11/126 Metropolitan Area (RRMA) classification). This pro- duced a list of 2,702 doctors. Doctors in this sample were then randomly allocated to a response mode by AS using random numbers generated in STATA. The AMPCo unique identifiers for each of the three groups were sent to AMPCo who conducted the mailing of invitation letters, and survey materials were mailed in late February 2008. Survey invitation letters indicated the University of Melbourne and Monash University as responsible for the survey. AMPCo also provided indivi- dual-level data on the population of doctors so we could examine response bias. Doctors were aware of which mode they had been allocated to on receipt of the invi- tation letter. The survey manager (AL) recorded responses and organised data entry and was blinded to group allocation. SH analysed the data and was not blinded to group allocation. Analysis was by ‘intention to treat’. the paper and web survey, and time liaising with AMPCo and the printers. These costs were therefore not included in the comparison of costs between modes. The expected costs for the main wave 1 survey were estimated based on sending out a survey to all doctors on the AMPCo database (n = 54,168) and using the response rates from the randomised trial to estimate the number of respon- dents. Data on the three primary outcome measures are presented alongside data on costs. Results Analysis of geographic location was based on the Australian Standard Geographic Classification (AGSC) Accessibility and Remoteness Index for Australia (ARIA) [34]. The difference in response rates across modes was sta- tistically significant (Table 4). The table reports the mar- ginal effects of each response mode compared with the online response, and can be interpreted as percentages. Controlling for other factors, the simultaneous mixed mode had a response rate 7 percentage points higher than online, and the sequential mixed mode was 7.7 per- centage points higher than online. The effect of sequen- tial mixed mode was not significantly different from simultaneous mixed mode (c2 = 0.16, p = 0.69). Specia- lists were 16 and 13 percentage points more likely to respond to the simultaneous and sequential mixed modes respectively, than to the online mode. Differences for other types of doctor were not statistically significant. The probit model also controls for differences in age, gender, doctor type and geographic area on response rate. Overall, females were less likely to respond, and the specialists’ response rate was 5.3 percentage points higher than GPs. GPs in outer regional and very remote areas were more likely to respond than those in major cities; this was partly due to a $100 financial incentive provided to doctors in outer regional and very remote areas. The economic evaluation compared the costs of con- sumables (rental of AMPCo list, printing of surveys, let- ters, fax forms, further information fliers and reply-paid envelopes, mail-house processing costs, postage and data entry) across each mode of survey administration. The costs of researcher and staff time were the same for each mode, as each mode required the development of both Scott et al. Results BMC Medical Research Methodology 2011, 11:126 http://www.biomedcentral.com/1471-2288/11/126 Page 6 of 12 Table 1 Group characteristics of full sample Simultaneous mixed Sequential mixed Online Total N (%) 900 N (%) 900 N (%) 902 N (%) 2,702 Doctor type GPs 334 (37.1) 388 (43.1) 369 (40.9) 1,091 (40.3) Specialists 333 (37.0) 318 (35.3) 317 (35.1) 968 (35.8) Hospital non-specialists 161 (17.9) 140 (15.6) 142 (15.7) 443 (16.4) Specialist in training 72 (8.0) 54 (6.0) 74 (8.2) 200 (7.4) Geographic location1 Major city 739 (82.1) 711 (79) 745 (82.6) 2,195 (81.2) Inner regional 116 (12.9) 140 (15.6) 110 (12.2) 366 (13.6) Outer regional 42 (4.7) 40 (4.4) 43 (4.8) 125 (4.6) Remote 1 (0.1) 5 (0.6) 2 (0.2) 8 (0.3) Very remote 2 (0.2) 4 (0.4) 2 (0.2) 8 (0.3) Age Group < = 30 65 (7.2) 64 (7.1) 58 (6.4) 187 (6.9) 30-39 164 (18.2) 150 (16.7) 157 (17.4) 471 (17.4) 40-49 191 (21.2) 200 (22.2) 225 (24.9) 616 (22.8) 50-59 210 (23.3) 212 (23.6) 205 (22.7) 627 (23.2) 60-69 103 (11.4) 123 (13.7) 100 (11.1) 326 (12.1) 70 < = 41 (4.6) 47 (5.2) 44 (4.9) 132 (4.9) missing 126 (14.0) 104 (11.6) 113 (12.5) 343 (12.7) Gender Female 280 (31.1) 308 (34.2) 289 (32.0) 877 (32.4) Male 620 (68.9) 590 (65.6) 613 (68.0) 1,823 (67.5) 1. Australian Standard Geographical Classification (ASGC) classification Remoteness Areas [34]. Table 1 Group characteristics of full sample 1. Australian Standard Geographical Classification (ASGC) classification Remoteness Areas [34]. the population of all doctors in Australia. This was under- taken using a multinomial logit model with four outcomes: simultaneous, sequential, online and population. Table 6 shows the odds ratios for those comparisons and factors that were statistically significant at the 95% level. For example, both mixed modes showed evidence of response bias, whilst the characteristics of online respondents were the same as the population. Those who filled out the simultaneous mixed mode were twice as likely to be spe- cialists when compared to the population, whilst those Doctors allocated to each mode were given the oppor- tunity to complete the survey online or on paper. Table 5 shows that of those allocated to the simultaneous mixed mode, 21% chose to complete the survey online, whilst of those in the online group only 3% requested and filled out a paper survey. Results Doctors allocated to the sequential mixed mode group were more likely to fill out a paper survey (62%) than an online one (38%). Response bias was examined for each mode by compar- ing the characteristics of respondents to each mode with Table 2 Response rates by mode of administration All doctors Simultaneous mixed Sequential mixed Online a) Total 2,702 900 900 902 b) Useable responses (with at least one question answered) 476 175 185 116 c) Refusal (i.e. paper copy returned blank, declined) 11 5 4 2 d) No contact (return to sender) 58 18 15 25 e) No responses 2,133 690 690 753 f) Not eligible (i.e. retired, no longer in clinical practice) 24 12 6 6 Response rate (b/(a-f)) 17.77% 19.71% 20.69% 12.95% Contact rate ((b+c+e))/(a-f)) 97.83% 97.97% 98.32% 97.21% Table 2 Response rates by mode of administration Scott et al. BMC Medical Research Methodology 2011, 11:126 http://www.biomedcentral.com/1471-2288/11/126 Scott et al. BMC Medical Research Methodology 2011, 11:126 http://www.biomedcentral.com/1471-2288/11/126 Page 7 of 12 Page 7 of 12 Table 3 Response rates by mode of administration and doctor type1 (in %) Simultaneous mixed Sequential mixed Online Total GPs 16.01 19.22 13.62 16.34 Specialist 26.23 22.86 11.18 20.17 Hospital non- specialists 16.77 20.71 12.68 16.70 Specialist in training 13.89 18.52 17.57 16.50 1. Response rate for those eligible to respond. Table 3 Response rates by mode of administration and doctor type1 (in %) filling out the sequential mixed mode were more likely to be older and more likely to be in a non-metropolitan area when compared to the population. The results also show that specialists were 2.4 times more likely to complete the simultaneous mixed mode than the online, and that those aged over 60 and in inner regional areas were more likely to complete the sequential mixed mode than the online mode. filling out the sequential mixed mode were more likely to be older and more likely to be in a non-metropolitan area when compared to the population. The results also show that specialists were 2.4 times more likely to complete the simultaneous mixed mode than the online, and that those aged over 60 and in inner regional areas were more likely to complete the sequential mixed mode than the online mode. rginal effects for discrete change of dummy variable from 0 to 1; * P < 0.05, P < 0.01, * P < 0.001; standard errors are in parenth Results The simultaneous mixed mode was more likely to have a higher percentage of items completed than the online in the ‘About You’ and ‘Family’ sections. mode were significantly more likely to complete all ques- tions in the ‘Family’ section compared to those in the online mode (Table 8). The costs of each mode were estimated for the first wave of the survey, which was to be sent out to the population of doctors in Australia (Table 9). The online mode has the lowest total cost, followed by the sequen- tial mixed mode, with simultaneous mixed mode having the highest cost. The total cost of the simultaneous mixed mode is 38% higher than online, and 21% higher than sequential mixed mode. The sequential mixed mode total costs are 14% higher than the online mode. The main sources of cost differences between modes are related to handling and postage of the mail-out, printing of surveys, and data entry for paper copies. Table 9 shows incremental cost-effectiveness ratios with respect to changes in response rate and number of responses. Comparing sequential and simultaneous mixed modes to online, sequential mixed mode was the most cost-effective relative to online. Costs were $6.07 per additional response, and $AUS3, 290 per 1% increase in the response rate, compared to online. Although the main outcomes were similar for the two mixed modes, the sequential mode was cheaper due to lower printing, mailing and data entry costs. Using the sequential mixed mode resulted in total costs which were 21% lower than for the simultaneous mode, with no detrimental impact on response rate, survey response bias, or item non-response. Although the percentage of questions completed overall was 91.4%, only 2.9% of respondents completed every question, and this was lowest for simultaneous mixed mode (1.1%), followed by sequential mixed mode (2.2%), and was highest for online mode (6.9%) (Table 7). These differences were statistically significant (second half of Table 8), with odds ratios of 0.13 for paper mode and 0.23 for mixed mode compared to online. The proportion of respondents completing all questions was similar across the modes for each section. Those using simultaneous Results The order of the sec- tions in the survey is reflected in these tables, with the job satisfaction section coming first. Note that the online sur- vey allowed respondents to skip questions, as would be the case in the paper survey. Overall, the online mode shows the lowest average percentage of items completed, with almost 89% of questions answered compared to around 92% for each of the other modes. This is the case for all sub-sections of the survey, with the section on finances, which includes income questions, having the lowest average percentage of items completed of 80%. This difference is statistically significant, as shown in the first half of Table 8, with odds ratios of 1.48 for paper and 1.53 for mixed mode compared to online. Table 8 also shows statistically significant differences for some sections of the survey. The sequential mixed mode was more likely to have a higher percentage of items completed than the online for the sections on DCE, workload, and location. The simultaneous mixed mode was more likely to have a higher percentage of items completed than the online in the ‘About You’ and ‘Family’ sections. i.e. whether the percentage of items completed = 100% (Table 7). If a question was ‘not applicable’, this was counted as a completed question. The order of the sec- tions in the survey is reflected in these tables, with the job satisfaction section coming first. Note that the online sur- vey allowed respondents to skip questions, as would be the case in the paper survey. Overall, the online mode shows the lowest average percentage of items completed, with almost 89% of questions answered compared to around 92% for each of the other modes. This is the case for all sub-sections of the survey, with the section on finances, which includes income questions, having the lowest average percentage of items completed of 80%. This difference is statistically significant, as shown in the first half of Table 8, with odds ratios of 1.48 for paper and 1.53 for mixed mode compared to online. Table 8 also shows statistically significant differences for some sections of the survey. The sequential mixed mode was more likely to have a higher percentage of items completed than the online for the sections on DCE, workload, and location. Results Item non-response was examined by calculating the average percentage of items completed, and the percen- tage of respondents who completed all relevant questions, Table 4 The effect of mode on response rates (probit regression model) All doctors GPs Specialists Hospital non-specialists Specialists in training Marginal effects (standard error) Simultaneous mixed 0.070* 0.028 0.160* 0.034 -0.030 (0.020) (0.030) (0.036) (0.046) (0.060) Sequential mixed 0.077* 0.053 0.133* 0.072 0.013 (0.020) (0.028) (0.037) (0.049) (0.065) < age30 0.074 0.086 - 0.276 0.108 (0.046) (0.156) - (0.160) (0.166) age30-39 0.037 -0.008 0.029 0.229 0.198* (0.027) (0.040) (0.048) (0.163) (0.085) age50-59 0.054* 0.039 0.058 0.418 0.009 (0.023) (0.031) (0.036) (0.245) (0.144) age60-69 0.002 0.020 -0.018 - - (0.026) (0.038) (0.038) > = age70 -0.028 -0.063 0.005 - - (0.035) (0.042) (0.058) Age missing -0.037 -0.034 -0.080* 0.208 0.127 (0.025) (0.036) (0.039) (0.190) (0.125) Female -0.043 -0.029 -0.066 -0.048 -0.080 (0.017) (0.024) (0.036) (0.037) (0.056) Specialist 0.053 - - - - (0.018) Hospital non-specialist -0.013 - - - - (0.027) Specialist in training 0.009 - - - - (0.032) Inner Regional 0.025 0.024 0.053 -0.050 0.140 (0.023) (0.030) (0.045) (0.054) (0.105) Outer Regional 0.051 0.119* -0.056 - - (0.040) (0.052) (0.064) Remote 0.231 0.354 - - - (0.182) (0.249) Very Remote 0.517 0.542 - 0.459 - (0.163) (0.185) (0.315) N 2702 1091 966 430 196 Log likelihood -1255 -469 -461 -188 -84 c2 (df) 59.7* (16) 28.3 (13) 37.4*** (10) 12.6 (9) 11.3 (8) Pseudo R2 0.03 0.03 0.04 0.04 0.05 Marginal effects for discrete change of dummy variable from 0 to 1; * P < 0.05, P < 0.01, * P < 0.001; standard errors are in parentheses. Table 4 The effect of mode on response rates (probit regression model) Scott et al. BMC Medical Research Methodology 2011, 11:126 http://www.biomedcentral.com/1471-2288/11/126 Scott et al. BMC Medical Research Methodology 2011, 11:126 http://www.biomedcentral.com/1471-2288/11/126 Page 8 of 12 Table 5 Actual mode of response by allocated survey mode Group allocation Simultaneous mixed Sequential mixed Online Total Paper returned (%) 78.86 61.62 3.45 53.78 Online completed (%) 21.14 38.38 96.55 46.22 Total number of surveys 175 185 116 476 Table 5 Actual mode of response by allocated survey mode i.e. whether the percentage of items completed = 100% (Table 7). If a question was ‘not applicable’, this was counted as a completed question. Discussion This study has compared response rates, survey response bias, item non-response, and costs across three modes of Table 6 Response bias for each mode (Odds ratios and 95% CI)1 age50-59 age60-69 Specialist Inner Regional Outer/ remote Simultaneous mixed compared to Population 2.00 (1.39, 2.87) Sequential mixed compared to Population 1.79 (1.16, 2.75) 1.67 (1.01, 2.78) 1.49 (1.00, 2.22) 1.73 (1.01, 2.97) Simultaneous mixed compared to online 2.42 (1.38, 4.26) Sequential mixed compared to online 3.22 (1.26, 8.22) 2.13 (1.02, 4.44) 1. Results from multinomial logit model. Only statistically significant results (P < 0.05) are reported. Full model results available from authors Table 6 Response bias for each mode (Odds ratios and 95% CI)1 1. Results from multinomial logit model. Only statistically significant results (P < 0.05) are reported. Full model results available from authors Scott et al. BMC Medical Research Methodology 2011, 11:126 http://www.biomedcentral.com/1471-2288/11/126 Page 9 of 12 Table 7 Item response by mode and questionnaire section (%) Simultaneous mixed Sequential mixed Online Total Average Percentage of items completed Percentage with 100% completed items Average Percentage of items completed Percentage with 100% completed items Average Percentage of items completed Percentage with 100% completed items Average Percentage of items completed Percentage with 100% completed items Job Satisfaction 99.5 89.7 99.4 88.6 98.5 90.5 99.2 89.5 DCE 94.9 90.3 98.4 96.2 94.8 91.4 96.2 92.9 Work Places 84.6 21.7 86.8 23.2 83.0 22.4 85.1 22.5 Workload 90.3 35.4 91.5 36.8 85.8 44.0 89.7 38.0 Finances 85.8 44.0 85.7 41.1 80.2 44.8 84.4 43.1 Location 92.2 46.9 93.0 50.8 88.6 51.7 91.7 49.6 About You 95.3 80.0 92.8 71.9 88.1 70.7 92.5 74.6 Family 97.9 94.9 96.6 91.4 93.6 85.3 96.4 91.2 Total 92.0 1.1 92.5 2.2 88.5 6.9 91.4 2.9 1. Percentage of items completed: n qi t ∗100 n; the mean of the percentage of completed questions per respondent i, where q is the number of completed questions, t is the total number of questions, and n is the total number of respondents. Table 7 Item response by mode and questionnaire section (%) Simultaneous mixed Sequential mixed Table 7 Item response by mode and questionnaire section (%) conducting a doctor survey. Mailing a letter inviting respondents to complete the questionnaire online, fol- lowed by a mailed reminder letter and paper copy of the survey, was the most cost-effective mode of administra- tion. completed all questions (= 1). Logistic regression model controlling for age, gender, doctor type and rurality * P < 0.05, P < 0.01, Discussion BMC Medical Research Methodology 2011, 11:126 http://www.biomedcentral.com/1471-2288/11/126 Table 9 Effect of mode on survey costs (2008 prices, in $AU) Table 9 Effect of mode on survey costs (2008 prices, in $AU) Table 9 Effect of mode on survey costs (2008 prices, in $AU) Simultaneous mixed Sequential mixed Online Total costs AMPCo rental of list $15,830 $15,830 $15,830 Monetary incentives $60,000 $60,000 $60,000 Web survey costs $4,000 $4,000 $4,000 AMPCO handling and postage $97,157 $72,024 $65,361 Printing of letters, further info, fax sheet, envelopes $17,981 $18,470 $18,458 Printing of surveys $21,284 $19,794 $10,995 Data entry $33,684 $16,630 $6,636 Total $249,936 $206,748 $181,280 Incremental cost effectiveness ratios (compared to online mode) Response rate 19.71% 20.69% 12.95% Estimated number of responses 10,677 11,208 7,015 Change in response rate compared to online 6.76% 7.74% - Change in number of responses compared to online 3,662 4,193 - Change in total cost compared to online $68,656 $25,468 - Additional cost per 1% increase in response rate1 $10,156 $3,290 - Additional cost per additional response2 $18.75 $6.07 - Notes: 1. Δcost/Δ response rate; 2. Δcost/Δ number of responses. Additional cost per additional response2 Notes: 1. Δcost/Δ response rate; 2. Δcost/Δ number of responses. respondents to find a website and login using their user- name and password provided in the letter. Once at the website, they had to go to a login page, enter their details, and were then directed to the beginning of the survey. Though this takes time compared to an email survey with an embedded website link, it does provide a more secure process that may have increased the confidence of respon- dents in the security of the website. We find no support for the hypothesis that offering a simultaneous choice of modes results in lower response rates than a sequenced choice of mode. Literature from non-doctor populations suggests that sequencing may be better than simultaneous choice [12,13]. Though there is a small difference in response rates for both mixed modes, this is not statistically significant. Lower response rates in the online mode arguably reflect the population being surveyed, their familiarity with and trust in the internet, and reliability of access to the inter- net, especially in remote regions of Australia. Most doctors choose to fill out a paper questionnaire, possibly suggest- ing that they are less comfortable with doing a survey online or have issues with sending confidential informa- tion over the internet. Discussion Although online modes were less costly, due to lower printing and data entry costs, and did not exhibit evidence of increased response bias, the response rates and item completion rates were lower than for the sequential and simultaneous mixed modes. The online mode had lower item completion rates in the sections on the DCE, workload, and personal and family characteris- tics. Although the sequential mode is the most cost-effec- tive with respect to response rates, whether this is chosen would depend on the weight given to the existence of response bias when compared to the population of doctors. Table 8 Item non-response by mode (Odds ratio, 95% CI) Job Satisfaction DCE Work Places Workload Finances Location About You Family Total Average Percentage of items completed1 Simultaneous mixed (compared to online) 2.85 1.10 1.14 1.52 1.48 1.46 2.67 3.08** 1.48* [0.86,9.42] [0.42,2.88] [0.83,1.55] [0.92,2.53] [0.94,2.33] [0.98,2.18] [1.42,5.00] [1.32,7.20] [1.03,2.14] Sequential mixed (compared to online) 2.53 3.83* 1.31 1.70* 1.41 1.55* 1.68 1.87 1.53* [0.81,7.91] [1.14,12.95] [0.97,1.76] [1.05,2.76] [0.90,2.20] [1.02,2.37] [0.96,2.94] [0.87,4.03] [1.09,2.17] N 476 476 476 476 476 476 476 476 476. Log likelihood -18.397 -63.804 -149.636 -129.201 -169.354 -106.841 -105.201 -63.197 -107.395 Whether 100% of items were completed2 Simultaneous mixed (compared to online) 0.95 0.96 0.95 0.67 0.98 0.77 1.75 3.25** 0.13* [0.42,2.11] [0.42,2.19] [0.53,1.72] [0.40,1.11] [0.60,1.59] [0.48,1.24] [0.98,3.12] [1.38,7.65] [0.02,0.67] Sequential mixed (compared to online) 0.82 2.64 0.99 0.76 0.84 0.85 1.10 1.86 0.23* [0.37,1.81] [0.97,7.21] [0.55,1.78] [0.46,1.26] [0.51,1.38] [0.53,1.36] [0.64,1.87] [0.90,3.82] [0.07,0.78] N 476 476 459 476 476 476 476 476 459 Log likelihood -156.70 -112.68 -237.51 0.67 -310.12 -320.57 -253.63 -134.05 -51.28 1. Mean proportion of questions completed. A generalised linear model controlling for age, gender, doctor type and rurality. 2. Whether a respondent completed all questions (= 1). Logistic regression model controlling for age, gender, doctor type and rurality * P < 0.05, P < 0.01, * P < 0.001 Table 8 Item non-response by mode (Odds ratio, 95% CI) Page 10 of 12 Scott et al. BMC Medical Research Methodology 2011, 11:126 http://www.biomedcentral.com/1471-2288/11/126 Scott et al. Discussion This is reflected in a higher rate of item non-response for most sections of the survey, espe- cially for the more personal questions. This finding occurred despite assurances about confidentiality and the fact that information was being sent over a secure internet connection. Doctors may also prefer the ‘portability’ of a paper-copy which they can fill out at the office, at home or whilst travelling. Online modes are also becoming more portable (i.e. not confined to the desktop PC) with the use of laptops, touch screen tablets and other mobile devices, so the preference for a paper copy may erode over time. A key issue in relation to survey response is the need to minimise the opportunity cost of survey completion for respondents. The need for internet access and the time it takes to logon needs to be balanced against filling out a paper survey that needs to be posted. A potential reason for the lower online response rate was the need by Response rates in all three arms could be regarded as low, an increasing issue for surveys of doctors [1-3]. It is noteworthy that our comparative analysis with the popu- lation of Australian doctors showed that the mode with the lowest response rate (online) was the most represen- tative, confirming the point noted in the introduction, that response rate and response bias are separate issues and should both be explicitly analysed to ensure appro- priate interpretation. Our study used a diverse sample of doctors with respect to age, specialty and geographic location, increas- ing the generalisability of the results. Although the trial was not designed for sub-group analysis, specialists in training allocated to the online mode had a higher response rate (17.5%) than those allocated to the simulta- neous mixed mode (13.89%), and a similar response rate to those in the sequential mixed mode (18.52%). For those conducting surveys of younger doctors and doctors in training, who are more likely to be familiar with and trusting of the internet, online surveys may be a more desirable option, though item non-response may be an issue. However, specialists had the highest response rate for the simultaneous version (26.2% compared with Page 11 of 12 Page 11 of 12 Page 11 of 12 Scott et al. BMC Medical Research Methodology 2011, 11:126 http://www.biomedcentral.com/1471-2288/11/126 this paper are those of the authors alone. Authors’ contributions AS, CJ, GK, JH, JW, SJ jointly conceived of this work. All authors participated in designing and developing the study instruments and procedures. AS drafted the paper, and SJ maintained the dataset and conducted the statistical analysis. All authors assisted with re-drafting sections of the paper and interpreting results. AL organised the administration of the survey and data entry. All authors approved the final version of the manuscript. AS is the guarantor. Our results are in line with other research showing that lower response rates are likely to result from online sur- veys than mailed surveys [5,6]. Other studies have com- pared mail and online mixed modes in non-doctor samples [12,13]. However, these studies have not exam- ined costs. There are many different types of response mode, and different combinations of mixed modes, that can potentially be used in surveys of doctors. Further research is required in a number of areas. First, compari- sons are needed of modes that offer choice compared to those that do not [14]. Second, all comparisons need to include an examination of the changes in costs. This is mentioned frequently in the literature as a motivation for using online and mixed modes, but there is hardly any evi- dence of the differences in costs. Competing interests ll h h All authors have completed the Unified Competing Interest form at http:// www.icmje.org/coi_disclosure.pdf (available on request from the corresponding author) and declare that none of the authors have financial or non-financial interests that may be relevant to the submitted work. Received: 11 October 2010 Accepted: 5 September 2011 Published: 5 September 2011 Author details 1 1Melbourne Institute of Applied Economic and Social Research, Faculty of Business and Economics The University of Melbourne Level 7, Alan Gilbert Building, 161 Barry Street, Carlton, VIC 3053, Australia. 2Department of Epidemiology and Preventive Medicine School of Public Health and Preventive Medicine Monash University, Alfred Hospital Melbourne 3004, Australia. 3Monash University School of Rural Health PO Box 666 Bendigo Central VIC 3552, Australia. 4Department of Economics 501 Fletcher Argue Building University of Manitoba, Winnipeg, MB R3T 5V5 Canada. Discussion We thank the doctors who gave their valuable time to participate in MABEL, and the other members of the MABEL team for data cleaning and comments on drafts of this paper: Terence Cheng, Daniel Kuehnle, Matthew McGrail, Michelle McIsaac, Stefanie Schurer, Durga Shrestha and Peter Sivey. The study was approved by the University of Melbourne Faculty of Economics and Commerce Human Ethics Advisory Group (Ref. 0709559) and the Monash University Standing Committee on Ethics in Research Involving Humans (Ref. CF07/1102 - 2007000291). De-identified data from MABEL are available from http://www. mabel.org.au. 22.9% for mixed) and the lowest response for the online mode (11.2%). The routine use of exclusively online sur- veys for the population of doctors may therefore be some time off, at least until the current older cohorts have been replaced by younger cohorts. The unit costs of printing and survey administration are likely to vary across geographic locations and companies, though they are not likely to vary across modes within geographic locations, and so should not influence our findings. Printing costs vary greatly with volume, such that for the pilot online mode the unit cost per printed ques- tionnaire (for those requesting a paper survey) was $AUD5.90. However, the unit cost of printing 54,169 paper questionnaires (for the ensuing main wave survey) was $AUD0.32. The relationship between unit costs and volume printed is not linear. The costs of establishing the online survey will also vary across settings, although there are now many low cost survey packages available that cover most needs, some of which can be re-programmed if necessary. Committee on Ethics in Research Involving Humans (Ref. CF07/1102 2007000291). De-identified data from MABEL are available from http://www. mabel.org.au. References 1. Barclay S, Todd C, Finlay I, Grande G, Wyatt P: Not another questionnaire! Maximising the response rate, predicting non-response and assessing non-response bias in postal questionnaire studies of GPs. Family Practice 2002, 19:105-111. 1. Barclay S, Todd C, Finlay I, Grande G, Wyatt P: Not another questionnaire! Maximising the response rate, predicting non-response and assessing non-response bias in postal questionnaire studies of GPs. 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https://openalex.org/W2613493127	https://europepmc.org/articles/pmc6124191?pdf=render	English	null	Insulin‐Stimulated Bone Blood Flow and Bone Biomechanical Properties Are Compromised in Obese, Type 2 Diabetic OLETF Rats	JBMR plus	2,017	cc-by	8,781	ABSTRACT ABSTRACT Type 2 diabetes (T2D) increases skeletal fragility and fracture risk; however, the underlying mechanisms remain to be identiﬁed. Impaired bone vascular function, in particular insulin-stimulated vasodilation and blood ﬂow is a potential, yet unexplored mechanism. The purpose of this study was to determine the effects of T2D on femoral biomechanical properties, trabecular microarchitecture, and insulin-stimulated bone vasodilation by comparison of hyperphagic Otsuka Long-Evans Tokushima Fatty (OLETF) rats with normoglycemic control OLETF rats. Four-week old, male OLETF rats were randomized to two groups: type 2 diabetes (O-T2D) or normoglycemic control (O-CON). O-T2D were allowed ad libitum access to a rodent chow diet and O-CON underwent moderate caloric restriction (30% restriction relative to intake of O-T2D) to maintain normal body weight (BW) and glycemia until 40 weeks of age. Hyperphagic O-T2D rats had signiﬁcantly greater BW, body fat, and blood glucose than O-CON. Total cross-sectional area (Tt.Ar), cortical area (Ct.Ar), Ct.Ar/Tt.Ar, and polar moment of inertia of the mid-diaphyseal femur adjusted for BW were greater in O-T2D rats versus O-CON. Whole-bone biomechanical properties of the femur assessed by torsional loading to failure did not differ between O-T2D and O-CON, but tissue-level strength and stiffness adjusted for BW were reduced in O-T2D relative to O-CON. Micro–computed tomography (mCT) of the distal epiphysis showed that O-T2D rats had reduced percent bone volume, trabecular number, and connectivity density, and greater trabecular spacing compared with O-CON. Basal tibial blood ﬂow assessed by microsphere infusion was similar in O-T2D and O-CON, but the blood ﬂow response to insulin stimulation in both the proximal epiphysis and diaphyseal marrow was lesser in O-T2D compared to O-CON. In summary, impaired insulin-stimulated bone blood ﬂow is associated with deleterious changes in bone trabecular microarchitecture and cortical biomechanical properties in T2D, suggesting that vascular dysfunction might play a causal role in diabetic bone fragility. © 2017 The Authors. JBMR Plus is published by Wiley Periodicals, Inc. on behalf of the American Society for Bone and Mineral Research. KEY WORDS: ANALYSIS/QUANITIFICATION OF BONE; BONE QCT/mCT; ANALYSIS/QUANITIFICATION OF BONE; DXA; ANIMAL MODELS; GENETIC ANIMAL MODEL; ORTHOPAEDICS; BIOMECHANICS; DISEASES AND DISORDERS OF/RELATED TO BONE; OTHER Insulin-Stimulated Bone Blood Flow and Bone Biomechanical Properties Are Compromised in Obese, Type 2 Diabetic OLETF Rats Rebecca K Dirkes, Laura C Ortinau, R Scott Rector, T Dylan Olver, and Pamela S Hinton 1Department of Nutrition and Exercise Physiology, University of Missouri–Columbia, Columbia, MO, USA 2Division of Gastroenterology and Hepatology, Department of Medicine, University of Missouri–Columbia, Columbia, MO, USA 3Research Service, Harry S Truman Memorial VA Hospital, Columbia, MO, USA 4Department of Biomedical Sciences, University of Missouri–Columbia, Columbia, MO, USA ORIGINAL ARTICLE This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. Received in original form March 24, 2017; revised form May 8, 2017; accepted May 10, 2017. Accepted manuscript online May 12, 2017. Address correspondence to: Pamela S Hinton, PhD, 204 Gwynn Hall, Columbia, MO 65211, USA. E-mail: HintonP@missouri.edu JBMR® Plus (WOA), Vol. 1, No. 2, October 2017, pp 116–126 DOI: 10.1002/jbm4.10007 © 2017 The Authors. JBMR Plus Published by Wiley Periodicals, Inc. on behalf of the American Society for Bone and Mineral Research Cortical geometry and trabecular microarchitecture Animals used for assessment of cortical geometry, trabecular microarchitecture, and biomechanical properties were anesthe- tized and euthanized as described in Animal Protocol and Experimental Design. Blood and hind limbs were removed and femurs were cleaned of all soft tissue, wrapped in PBS-soaked gauze, and stored at 80°C for subsequent analysis. Right femurs were scanned using mCT (SIEMENS Inveon Micro-CT scanner; Siemens, Knoxville, TN, USA) at the Biomolecular Imaging Center (Harry S. Truman Memorial VA Hospital, Columbia, MO, USA) in accordance with the guidelines established by Bouxsein and colleagues.(30) Scans were performed at a 31.6-mm3 voxel size, with a peak X-ray potential of 80 kVp and 600 mA. Cortical bone was analyzed at the midpoint of the diaphysis, which was determined using the midpoint between the base of the third trochanter and the distal epiphysis. A 1-mm (500-mm in each direction from the midpoint) region of the diaphysis was used for analysis. A 1-mm region of the distal epiphysis was used for analysis, beginning 1 mm below the end of the distal growth plate. Uniform regions of interest and global thresholds for segmentation were used for each sample. Scans were analyzed using BoneJ software,(31) a subset of ImageJ (NIH, Bethesda, MD, USA; https://imagej.nih. gov/ij/). Trabecular number was calculated from trabecular spacing and thickness (Tb.N ¼ 1/[Tb.Th þ Tb.S]).(32) Introduction In particular, the hemodynamic response to insulin is impaired in T2D.(16) This impaired hemodynamic response to insulin has been observed in numerous tissues, including intestine,(16) brain,(16) nerve,(17) and muscle,(18,19) but, to our knowledge, has not been investigated in bone. In the vascular endothelium, insulin activation of the pI3/Akt pathway(20) promotes vasodilation mediated by endothelial-nitric oxide synthase (eNOS) and reduces synthesis of deleteriousadhesionmolecules(eg,vascularcelladhesionmolecule 1 [VCAM-1]). Thus, the selective insulin resistance of the vascular endothelium that occurs in T2D(21–23) promotes vasoconstriction, adhesion, inﬂammation, and oxidative stress, which lead to cardiovascular disease, neuropathy, retinopathy, and nephropathy. Thus, the present study investigated the effects of T2D on insulin-stimulated bone blood ﬂow and biomechanical proper- ties by comparison of hyperphagic, diabetic Otsuka Long-Evans Tokushima Fatty (OLETF; model of hyperphagia-induced obesity resulting in insulin resistance and T2D(24)) rats (O-T2D) with normoglycemic, normal weight, control OLETF rats (O-CON). We hypothesized that relative to normoglycemic O-CON rats, O-T2D rats would have reduced insulin-mediated bone blood ﬂow and deteriorated trabecular microarchitecture, cortical geometry, and biomechanical properties. Vascular dysfunction is a characteristic feature of T2D. In particular, the hemodynamic response to insulin is impaired in T2D.(16) This impaired hemodynamic response to insulin has been observed in numerous tissues, including intestine,(16) brain,(16) nerve,(17) and muscle,(18,19) but, to our knowledge, has not been investigated in bone. In the vascular endothelium, insulin activation of the pI3/Akt pathway(20) promotes vasodilation mediated by endothelial-nitric oxide synthase (eNOS) and reduces synthesis of deleteriousadhesionmolecules(eg,vascularcelladhesionmolecule 1 [VCAM-1]). Thus, the selective insulin resistance of the vascular endothelium that occurs in T2D(21–23) promotes vasoconstriction, adhesion, inﬂammation, and oxidative stress, which lead to cardiovascular disease, neuropathy, retinopathy, and nephropathy. Introduction dysfunction and impaired bone blood ﬂow.(4) Given the essential role of bone blood ﬂow in the maintenance of healthy bone(5) and the established causal role of reduced blood ﬂow in the pathophysiology of other T2D complications,(6,7) impaired bone vascular function in T2D warrants further investigation. T T he marked and sustained global increase in the prevalence and incidence of type 2 diabetes (T2D) in recent decades is so alarming that some have described this health crisis as a “diabetes pandemic.”(1) The health and economic costs of diabetes are enormous, due largely to the vascular complica- tions of T2D, which result in cardiovascular disease, neuropathy, retinopathy, and nephropathy.(1) A recently recognized compli- cation of T2D is increased fracture risk.(2) Currently, many potential biomechanical and cellular mechanisms for diabetic bone fragility are under investigation.(3) However, one mecha- nism that remains largely unexplored to date, is bone vascular The skeleton accounts for 10% to 15% of resting cardiac output(5) and bone blood ﬂow is essential for nutrient delivery, mineral homeostasis, and maintenance of interstitial ﬂuid ﬂow, which is needed for mechanotransduction.(8) Bone blood ﬂow is also a primary mechanism by which bone resorption and formation are coupled during remodeling.(9) Human and animal data show that bone loss is associated with reduced bone blood ﬂow.(10,11) Cross-sectional and prospective, longitudinal studies 116 experimental period. At 40 weeks of age, following an overnight fast, animals were anesthetized with 0.1 mg/kg ketamine, underwent a dual-energy X-ray absorptiometry scan and a euglycemic hyperinsulinemic clamp, during which tibial blood ﬂow was measured by microsphere infusion. Following the euglycemic hyperinsulinemic clamp,animals wereeuthanizedvia cervical dislocation and exsanguination, and blood and tissues collected (see details for each set of animals used for geometry/ biomechanical properties vs. those used for blood ﬂow). The animal protocol was approved by the IACUC at the University of Missouri, and was conducted in compliance with the guiding principles in the Guide for Care and Use of Laboratory Animals.(29) support a positive association between bone blood ﬂow and bone mass.(12,13) Likewise, both bone blood ﬂow and bone mass decline with age andafter menopause.(14) Femoralblood ﬂow is reduced in Zucker diabetic fatty rats with long-term diabetes in association with reduced endothelium-dependent vasodilation and femoral cancellous and cortical bone mineral density (BMD).(15) Vascular dysfunction is a characteristic feature of T2D. Animal protocol and experimental design We used a longitudinal, parallel study design to test the hypothesis that OLETF-T2D rats would have reduced insulin-stimulated bone blood ﬂow and deteriorated trabecular microarchitecture, cortical geometry, and biomechanical properties. The hyperphagic OLETF rat progressively accumulates excess body fat, which leads to insulin resistance and eventually hyperglycemia and T2D after skeletal maturity.(25) This accurately parallels what occurs in humans who are overweight during adolescence and develop metabolic complications in adulthood.(26) One set of animals (n ¼ 8 per group) was used to evaluate bone blood ﬂow by microsphere infusion and another (n ¼ 8 per group) was used to evaluate trabecular microarchitecture, cortical geometry, and biomechanical properties. Two sets of animals were used because of the interference of the micro- spheres with analysis of the micro–computed tomography (mCT) scans. Four-week old, male, OLETF rats were supplied by Otsuka Pharmaceutical (Tokushima, Japan). Upon arrival, rats were divided into two groups: ad libitum fed (O-T2D) or 30% calorically restricted (O-CON) until 40 weeks of age. We have previously shown that 30% caloric restriction prevents obesity and the development of T2D in hyperphagic OLETF rats,(27) and that O-CON are protected against the detrimental skeletal effects of T2D.(28) Animals were housed individually in a temperature-controlled (21°C) room with a 6:00am–6:00pm light and 6:00pm–06:00am dark cycle maintained throughout the duration of the experiment. All animals were provided standard rodent chow (Formulab 5008; Purina Mills, St. Louis, MO, USA) in clean cages at the beginning of the week. Body mass and food intake were measured weekly throughout the Materials and Methods Animal protocol and experimental design Metabolic outcomes Body mass and food intake were measured weekly throughout the experimental period. Fasting blood samples were taken prior to euthanasia, and serum glucose (Sigma, St. Louis, MO, USA) and insulin (Linco Research, St. Charles, MO, USA) levels were measured using commercially available kits, as described.(27) Whole-body composition and BMD were measured using a Hologic QDR-1000/w DXA scanner calibrated for rats and small animals (Hologic, Bedford, MA, USA; software version 3.6). Isolated femurs were scanned on the same machine (Hologic, Bedford, MA, USA; software version 3.6) for bone-speciﬁc BMD. Thus, the present study investigated the effects of T2D on insulin-stimulated bone blood ﬂow and biomechanical proper- ties by comparison of hyperphagic, diabetic Otsuka Long-Evans Tokushima Fatty (OLETF; model of hyperphagia-induced obesity resulting in insulin resistance and T2D(24)) rats (O-T2D) with normoglycemic, normal weight, control OLETF rats (O-CON). We hypothesized that relative to normoglycemic O-CON rats, O-T2D rats would have reduced insulin-mediated bone blood ﬂow and deteriorated trabecular microarchitecture, cortical geometry, and biomechanical properties. Statistics Independent sample t tests were used to determine differences between O-T2D and O-CON for body mass, fasting insulin, and glucose concentrations. Body-size–dependent bone outcomes (ie, BMD, trabecular microarchitecture, cortical geometry, and biomechanical properties) were adjusted for differences in body size using one-way analysis of covariance (ANCOVA), with ﬁnal body weight as the covariate.(39) The effects of insulin-stimulated blood ﬂow were determined using a two-factor (INSULIN: pre- insulin infusion and post-insulin infusion; and GROUP: O-T2D and O-CON) repeated measures ANOVA (RMANOVA). A GROUP INSULIN interaction with p < 0.15 was considered statistically signiﬁcant to avoid incorrectly concluding that a nonsigniﬁcant test for interaction (ie, p > 0.05) implies a lack of any interaction; and post hoc t tests (pre-insulin infusion versus post-insulin infusion within group and O-T2D versus O-CON within pre-insulin infusion and post-insulin infusion) were used to locate the interaction. One-tailed t tests were used because our hypotheses were directional and p 0.05 was considered statistically signiﬁcant. Insulin-stimulated changes in blood ﬂow and vascular conductance are conventionally reported as percent change. Therefore, percent change in blood ﬂow and vascular conduc- tance were also calculated; and group differences in percent changes were evaluated using the Mann-Whitney U test. This secondary statistical analysis was used to corroborate the results of the two-factor RMANOVA. Data are means SD or adjusted means SE for ANCOVA results (ie, means adjusted for body weight covariate). All statistics were performed using SPSS 23 software (IBM, Armonk, NY, USA). The radioactive microsphere technique is the gold standard method for the measurement of regional blood ﬂow.(34) Microspheres are infused into the systemic circulation and become trapped in the tissue of interest during their ﬁrst pass through the circulation. Consequently, the number of micro- spheres trapped in the tissue is proportional to tissue blood ﬂow. A nonradioactive alternative is the use of stable isotopic tracers that can be assayed by neutron activation.(35) Neutrons penetrate the tissue sample and activate the stable isotope to render it temporarily radioactive, which permits quantiﬁcation using high-resolution radiation counting equipment to gener- ate counts per minute (CPM). Cortical bone biomechanical properties Whole-bone and tissue-level biomechanical properties of the right femur were assessed using torsional loading to failure, as described.(25) Brieﬂy, the proximal and distal ends of the right femur were afﬁxed in cement to a steel holder, with crossbars at each end. The distal crossbar was ﬁxed, while the proximal crossbar was rotated around its long axis at a speed of 10 mm/s with a 100-kg load cell. The machine’s software (Stable Micro Systems, Surrey, UK) measured cable force (F) and applied torque (T). The load-displacement curve from this analysis is 117 INSULIN-STIMULATED BONE BLOOD FLOW/BIOMECHANICS IN OBESE, T2D OLETF RATS 117 117 JBMR® Plus (WOA) ﬂow (mL 100g1 min1) ¼ (0.6 mL min1 CPM1 RBS) (CPMtissue [tissue weight]1) 100, where RBS is the reference blood sample. Adequate mixing of microspheres was assured by comparing bloodﬂow intheright andleft kidneys.Two animalsin the O-T2D group were excluded from the analysis due to insufﬁcient and/or nonuniform perfusion. MAP was used to calculate the measures of vascular conductance (VC; the quotient of bone blood ﬂow and MAP [ﬂow/MAP]) and vascular resistance (VR; the quotient of MAP and bone blood ﬂow [MAP/ﬂow]). analogous with the torque-twist curve, which, along with geometrical data from the analysis of the mCT scans, was used to calculate: maximal torque at fracture (Tmax), torsional stiffness (Ks), shear modulus of elasticity (G), and ultimate tensile strength, or maximal shear stress (Su), as described.(25) Statistics The assumptions of the microsphere infusion technique are satisﬁed when the tech- nique is applied to determination of bone blood ﬂow.(36,37) In addition, the method is reproducible and physiologic changes in bone blood ﬂow are detectable.(38) In the present study, basal and insulin-stimulated bone blood ﬂow were measured by infusion of two distinct microspheres (15 mM diameter) each labeled with a different stable isotope before and after the euglycemic hyperinsulinemic clamp (153samarium and 198gold, respectively; BioPal, Inc. Worcester, MA, USA). To measure basal and insulin-stimulated blood ﬂow, one reference blood sample was taken at baseline (153samarium microspheres), and another was taken after the 60-min euglycemic, hyperinsulinemic clamp (198gold microspheres). The reference blood sample withdrawal began at a rate of 0.6 mL min1 and was followed (10 s later) by infusion of a well-mixed suspension of the respective microspheres and a saline ﬂush (performed over 20 s). Insulin-stimulated bone blood flow Animals used for the assessment of tibial blood ﬂow were anesthetized and then underwent a 60-min euglycemic, hyper- insulinemic clamp as described.(33) Brieﬂy, a catheter (polyeth- ylene-50 tubing) was inserted into the right jugular vein to facilitate constant insulin (10 mU kg min1; 0.400 mIU mL1; Novolin R, Novonordisk, Plainsboro, NJ) and variable glucose (0.4 g mL1; EMD Millipore, Darmstadt, Germany) infusions. The glucose infusion rate was adjusted to maintain euglycemia, which was determined by measurement of blood glucose every 5 min for the ﬁrst 20 min and every 10 min thereafter. A second catheter was inserted into the carotid artery and advanced to the aorta for microsphere infusion. A third catheter was inserted into the caudal artery for withdrawal of the microsphere reference sample and for monitoring mean arterial blood pressure (MAP) by connection of the catheter to a pressure transducer (PX272; Edwards Lifesciences, Irvine, CA, USA). Results O-T2D animals had signiﬁcantly greater body mass (p ¼ 0.004) and body fat percentage (p ¼ 0.001) than O-CON (Table 1). As expected, O-T2D animals had signiﬁcantly greater blood glucose compared to O-CON; however, the two groups had similar insulin concentrations. It is well-established that, at 40 weeks, O-T2D are hypoinsulinemic due to b-cell insufﬁciency,(40–43) whereas O-CON had reduced insulin levels due to energy restriction.(28) In the subset of animals that underwent the hyperinsulinemic, euglycemic clamp, O-T2D had reduced whole- body insulin sensitivity compared to O-CON, as shown by the lower glucose infusion rate (GIR) required to maintain euglycemia (O-T2D ¼ 9 3 mg kg1 min1; O-CON ¼ 23 3 mg kg1 min1; p ¼ 0.001). O-T2D animals had signiﬁcantly higher total body areal BMD than O-CON animals (p ¼ 0.008); there was no group difference in total femoral BMD (p ¼ 0.565). Following the euglycemic, hyperinsulinemic clamp, animals were euthanized as described in Animal Protocol and Experimen- tal Design above, and tibias and kidneys were collected. Tibias were separated into four regions (ie, proximal epiphysis, diaphysis, distal epiphysis, and diaphyseal marrow) for blood ﬂow measures, because regional differences in tibial blood ﬂow have been reported.(15,36) Individual regions of tibias and kidneys were weighed forblood ﬂow calculations. Microsphere content in blood and tissues was measured by neutron activation as counts per minute (CPM) by the microsphere manufacturer (BioPal, Inc. Worcester, MA, USA). Blood ﬂow was calculated as follows: Blood Cortical geometry and trabecular microarchitecture Cortical geometry differed between O-T2D and O-CON animals (Fig. 1A–F). O-T2D tended to have greater Tt.Ar (O-T2D: 118 DIRKES ET AL. JBMR Plus (WOA) Table 1. Body Composition, Metabolic Markers, and Bone Mineral Density in O-CON and O-T2D at 40 Weeks of Age O-CON O-T2D Metabolic outcomesa Body mass (g) 552 39 614 91 Body fat (%) 18.4 3.1 25.9 9.5 Blood glucose (mM) 11.8 0.6 58.3 18 Insulin (pM) 127.54 33.26 85.55 21.02 Areal bone mineral densityb Total body (g/cm2) 0.192 0.002 0.203 0.002 Femur (g/cm2) 0.303 0.005 0.308 0.005 aData are means SD; O-CON (n ¼ 8) and O-T2D (n ¼ 8). bData are means SE adjusted for ﬁnal body weight as a covariate in the ANCOVA; O-CON (n ¼ 8) and O-T2D (n ¼ 8). Signiﬁcantly different than O-CON. Table 1. Body Composition, Metabolic Markers, and Bone Mineral Density in O-CON and O-T2D at 40 Weeks of Age p ¼ 0.003; Fig. 1E),polarmoment ofinertia (O-T2D:29.801 1.511 versus O-CON: 20.335 1.801, p ¼ 0.003; Fig. 1F), and Ct.Ar/Tt.Ar (O-T2D: 28.209 0.640 versus O-CON: 31.646 0.745, p ¼ 0.006; Fig. 1D). There were no differences in Ma.Ar (O-T2D: 3.852 0.097 versus O-CON: 3.938 0.113; Fig. 1C). O-T2D animals had signiﬁcantly lower BV/TV (O-T2D: 0.232 0.016 versus O-CON: 0.287 0.012, p ¼ 0.039; Fig.2D).Tb.Thwas similarinbothgroups (O-T2D: 0.196 0.010 versus O-CON: 0.200 0.008, Fig. 2A); however, Tb.Sp was signiﬁcantly increased (O-T2D: 0.568 0.012 versus O-CON: 0.420 0.010, p ¼ 0.001; Fig. 2B) and Tb.N was signiﬁcantly decreased (O-T2D: 1.313 0.048 versus O-CON: 1.614 0.037, p ¼ 0.004; Fig. 2C) in O-T2D animals compared to O-CON. Conn.D was signiﬁcantly reduced (O-T2D: 9.256 1.653 versusO-CON: 15.498 1.276, p ¼ 0.031;Fig.2E) inO-T2D animals relative to O-CON. Cortical bone biomechanical properties Data are means SE adjusted for body weight; indicates differences between groups (p < 0.05). All groups ar Fig. 2. Trabecular measurements of the femur: trabecular thickness (A); trabecular spacing (B); trabecular number (C); trabecular bone volume to total volume (D); connectivity density (E). Data are means SE adjusted for body weight; indicates differences between groups (p < 0.05). All groups are 40-week-old OLETF rats: O-T2D (n ¼ 8) were allowed ad libitum access to a rodent chow diet and O-CON (n ¼ 6) underwent moderate caloric restriction (30% restriction relative to intake of O-T2D) to maintain normal body weight and glycemia. blood ﬂow in O-CON (post hoc paired t test within O-CON p ¼ 0.049), but not in O-T2D. In the diaphyseal marrow, insulin- stimulated blood ﬂow was lower in O-T2D compared to O-CON (p ¼ 0.016; Fig. 4D). Percent changes in tibial blood ﬂow in response to insulin (Fig. 5A) were consistent with the results of the two-factor RMANOVA. O-CON exhibited a greater increase in blood ﬂow in the proximal epiphysis (p ¼ 0.046), distal epiphysis (p ¼ 0.050), and diaphyseal marrow (p ¼ 0.153) compared to O-T2D. and strength (ultimate tensile strength, Su) were both signiﬁcantly decreased in O-T2D relative to O-CON (O-T2D: 100.138 13.114 versus O-CON: 1985.974 15.635, p ¼ 0.003 and O-T2D: 14.339 1.304 versus O-CON: 20.364 1.555, p ¼ 0.017, respectively; Fig. 3C, D). Cortical bone biomechanical properties O-T2D and O-CON did not differ in whole-bone strength (maximal torque at fracture, Tmax, O-T2D: 168.970 16.277 versus O-CON: 182.645 19.405) or torsional stiffness (K, O-T2D: 280.725 20.503 versus O-CON: 293.146 24.443) (Fig. 3A, B). However, tissue-level stiffness (shear modulus of elasticity, G) 13.688 0.347 versus O-CON: 12.489 0.404, p ¼ 0.051; Fig. 1A). Ct.Ar was signiﬁcantly increased in O-T2D animals (O-T2D: 9.814 0.263 versus O-CON: 8.379 0.307, p ¼ 0.005; Fig. 1B), as was Ct.Th (O-T2D: 0.978 0.021 versus O-CON: 0.849 0.025, g. 1. Cortical measurements of the femur: total area (A); cortical area (B); marrow area (C); cortical-to-total area (D); cortical thickness (E); polar moment f inertia (F). Data are means SE adjusted for body weight; indicates differences between groups (p < 0.05). All groups are 40-week-old OLETF rats: -T2D (n ¼ 8) were allowed ad libitum access to a rodent chow diet and O-CON (n ¼ 6) underwent moderate caloric restriction (30% restriction relative to take of O-T2D) to maintain normal body weight and glycemia. Fig. 1. Cortical measurements of the femur: total area (A); cortical area (B); marrow area (C); cortical-to-total area (D); cortical thickness (E); polar moment of inertia (F). Data are means SE adjusted for body weight; indicates differences between groups (p < 0.05). All groups are 40-week-old OLETF rats: O-T2D (n ¼ 8) were allowed ad libitum access to a rodent chow diet and O-CON (n ¼ 6) underwent moderate caloric restriction (30% restriction relative to intake of O-T2D) to maintain normal body weight and glycemia. INSULIN-STIMULATED BONE BLOOD FLOW/BIOMECHANICS IN OBESE, T2D OLETF RATS 119 JBMR® Plus (WOA) Fig. 2. Trabecular measurements of the femur: trabecular thickness (A); trabecular spacing (B); trabecular number (C); trabecular bone volume to total volume (D); connectivity density (E). Data are means SE adjusted for body weight; indicates differences between groups (p < 0.05). All groups are 40-week-old OLETF rats: O-T2D (n ¼ 8) were allowed ad libitum access to a rodent chow diet and O-CON (n ¼ 6) underwent moderate caloric restriction (30% restriction relative to intake of O-T2D) to maintain normal body weight and glycemia. Fig. 2. Trabecular measurements of the femur: trabecular thickness (A); trabecular spacing (B); trabecular number (C); trabecular bone volume to tota volume (D); connectivity density (E). Insulin-stimulated bone blood flow We observed regional differences in bone blood ﬂow: diaphyseal marrow hadthehighest blood ﬂow,followed by the proximaland distal epiphyses (Fig. 4A, C; Table 2); blood ﬂow was lowest in the diaphysis (Fig. 4B; Table 2). These regional differences are consistent with thosepreviouslyreported innormal(38,44) and T2D animals.(15) Basal blood ﬂow (Fig. 4A–D), VC and VR (Table 2) did not differ between O-T2D and O-CON. Changes in blood ﬂow in response to insulin and group differences to the insulin response could be due to changes in MAP and/or VC. There were signiﬁcant group main effects for MAP (Table 2), such that MAP was greater in O-CON compared to O-T2D (O-T2D: 76.2 2.7 mmHg; O-CON: 90.4 2.3 mmHg; main effects p ¼ 0.002). There was also a signiﬁcant main effect for insulin stimulation, such that MAP was reduced after insulin stimulation (basal: 92.0 2.5 mmHg; insulin: 74.6 3.4 mmHg; main effect p ¼ 0.003). The GROUP-by-INSULIN interaction was not signiﬁcant. Likewise, there was no signiﬁcant group difference in percent change in MAP after insulin stimulation. However, the percent increases in VC in the proximal and distal epiphyses were greater in O-CON than O-T2D (p ¼ 0.06 and p ¼ 0.05, respectively; Fig. 5B). Thus, greater insulin-stimulated increases in VC in O-CON likely accounted for augmented blood ﬂow relative to O-T2D. However, O-T2D and O-CON showed differential blood ﬂow responses to insulin stimulation in the proximal and distal epiphyses; ie, there were signiﬁcant interactions in the two- factor RMANOVA (GROUP INSULIN interaction p values were 0.032 and 0.124, respectively; Fig. 4A, C; Table 2). In support of our hypothesis, in O-T2D blood ﬂow in the proximal epiphysis decreased after insulin-stimulation (post hoc paired t test within O-T2D p ¼ 0.032), such that blood ﬂow after insulin infusion was signiﬁcantly lower in O-T2D compared to O-CON (post hoc t test p ¼ 0.038). In the distal epiphysis, insulin stimulation increased 120 DIRKES ET AL. JBMR Plus (WOA) Fig. 3. Biomechanical properties of the femur: maximal torque (A); torsional stiffness (B); shear modulus of elasticity (C); ultimate tensile strength (D). Data are means SE adjusted for body weight; indicates differences between groups (p < 0.05). Insulin-stimulated bone blood flow All groups are 40-week-old OLETF rats: O-T2D (n ¼ 8) were allowed ad libitum access to a rodent chow diet and O-CON (n ¼ 6) underwent moderate caloric restriction (30% restriction relative to intake of O-T2D) to maintain normal body weight and glycemia. Fig. 3. Biomechanical properties of the femur: maximal torque (A); torsional stiffness (B); shear modulus of elasticity (C); ultimate tensile strength (D). Data are means SE adjusted for body weight; indicates differences between groups (p < 0.05). All groups are 40-week-old OLETF rats: O-T2D (n ¼ 8) were allowed ad libitum access to a rodent chow diet and O-CON (n ¼ 6) underwent moderate caloric restriction (30% restriction relative to intake of O-T2D) to maintain normal body weight and glycemia. Discussion Data are means SE; signiﬁcant interaction (p 0.15) between group and insulin-stimulation in repeated measures ANOVA; #signiﬁcantly different from basal within group (p 0.05, one-tailed paired t test); $signiﬁcantly different from O-CON after insulin stimulation (p 0.05, one-tailed independent sample t test). All groups are 40-week-old OLETF rats: O-T2D (n ¼ 6) were allowed ad libitum access to a rodent chow diet and O-CON (n ¼ 8) underwent moderate caloric restriction (30% restriction relative to intake of O-T2D) to maintain normal body weight and glycemia. Fig. 4. Blood ﬂow measurements of the tibia: proximal epiphysis (A); diaphysis (B); distal epiphysis (C); diaphyseal marrow (D). Data are means SE; signiﬁcant interaction (p 0.15) between group and insulin-stimulation in repeated measures ANOVA; #signiﬁcantly different from basal within group (p 0.05, one-tailed paired t test); $signiﬁcantly different from O-CON after insulin stimulation (p 0.05, one-tailed independent sample t test). All groups are 40-week-old OLETF rats: O-T2D (n ¼ 6) were allowed ad libitum access to a rodent chow diet and O-CON (n ¼ 8) underwent moderate caloric restriction (30% restriction relative to intake of O-T2D) to maintain normal body weight and glycemia. Fig. 4. Blood ﬂow measurements of the tibia: proximal epiphysis (A); diaphysis (B); distal epiphysis (C); diaphyseal marrow (D). Data are means SE; signiﬁcant interaction (p 0.15) between group and insulin-stimulation in repeated measures ANOVA; #signiﬁcantly different from basal within group (p 0.05, one-tailed paired t test); $signiﬁcantly different from O-CON after insulin stimulation (p 0.05, one-tailed independent sample t test). All groups are 40-week-old OLETF rats: O-T2D (n ¼ 6) were allowed ad libitum access to a rodent chow diet and O-CON (n ¼ 8) underwent moderate caloric restriction (30% restriction relative to intake of O-T2D) to maintain normal body weight and glycemia. Table 2. Discussion anesthetized O-CON and O-T2D rats. This result is expected because anesthesia suppresses sympathetic nervous system activity, which increases vasodilation and blood ﬂow. However, the discrepant effects of T2D on basal bone blood ﬂow in our study versus those reported by Stabley and colleagues(15) might also be due to differences in the animal models of T2D. The ZDF rat has altered leptin signaling because of a missense mutation of leptin receptor, and leptin causes vasorelaxation; therefore, animals with reduced leptin signaling might have reduced basal blood ﬂow.(46) Here, we report for the ﬁrst time that insulin-stimulated bone blood ﬂow is compromised in the obese, T2D condition, which also adversely affected bone structural and biomechanical properties. We observed compromised insulin-stimulated vasodilation and blood ﬂow in the proximal and distal epiphyses and in the diaphyseal marrow of T2D animals that was associated with increased vascular conductance. Consistent with our observation, Stabley and colleagues(15) reported reduced nitric oxide–mediated, endothelium-dependent vaso- dilation in isolated femoral principal nutrient arteries in Zucker diabetic fatty (ZDF) rats with a corresponding reduction in bone blood ﬂow. These results suggest that the vascular endothelium of bone, like that of other tissues,(45) becomes insulin resistant in T2D. Additional in vitro experiments with isolated bone blood vessels are needed to deﬁnitively establish that this occurs. Stabley and colleagues(15) also reported reduced basal blood ﬂow in the femur of alert ZDF rats with long-term diabetes compared with nondiabetic animals. In the present study, we did not observe differences in tibia basal blood ﬂow between The results of the present study conﬁrm our previous ﬁndings(25,28) and those of others(2,47,48) that insulin resistance and T2D associated with excess adiposity negatively impact bone health. Speciﬁcally, in the present study, obesity-induced T2D negatively inﬂuenced trabecular microarchitecture and tissue-level cortical strength and stiffness. Trabecular bone volume was decreased with greater trabecular separation and reduced connectivity density in O-T2D rats relative to O-CONs. Although T2D reduced tissue-level strength and stiffness of the femur, whole-bone biomechanical properties were not com- promised, most likely due to compensatory changes in bone 121 121 INSULIN-STIMULATED BONE BLOOD FLOW/BIOMECHANICS IN OBESE, T2D OLETF RATS JBMR® Plus (WOA) Fig. 4. Blood ﬂow measurements of the tibia: proximal epiphysis (A); diaphysis (B); distal epiphysis (C); diaphyseal marrow (D). Discussion Percent change in blood ﬂow (A) and vascular conductance (B) in regions of tibia. Data are means SE; signiﬁcantly different from O-CON. All groups are 40-week-old OLETF rats: O-T2D (n ¼ 6) were allowed ad libitum access to a rodent chow diet and O-CON (n ¼ 8) underwent moderate caloric restriction (30% restriction relative to intake of O-T2D) to maintain normal body weight and glycemia. PE ¼ proximal epiphysis; D ¼ diaphysis; DE ¼ distal epiphysis; M ¼ marrow. geometry, such as increased cortical area and thickness. The effects of T2D on bone health observed in the hyperphagic, obese OLETF rat are consistent with impaired bone micro- architecture and biomechanical properties that occur in obese/ diabetic humans.(49,50) Given the important hemodynamic role of insulin in nutrient delivery, the current data provide novel insight regarding how impaired insulin-stimulated bone blood ﬂow contributes to increased bone fragility. Although the effects of insulin on glucose uptake by skeletal muscle and adipose tissue are well characterized, how insulin affects glucose uptake by bone is not thoroughly understood. Some studies suggest that insulin signaling does not alter expression of glucose transporters.(51,52) However, a recent study of primary osteoblasts showed that GLUT4 expression increases with osteoblast differentiate into mature osteoblasts, and insulin exposure increases GLUT4 expression that is necessary for insulin-stimulated glucose uptake.(53) By contrast, basal glucose uptake appears mediated by insulin-independent GLUT1.(51) Thus, it appears that insulin increases glucose uptake in mature osteoblasts via increased osteoblast expression of GLUT4. Regardless of the direct effects of insulin on glucose uptake via alterations in glucose transporter expression, increased bone blood ﬂow in response to postprandial insulin release increases delivery of oxygen, Moreover, other pathogenic processes associated with endothelial dysfunction might negatively impact bone in T2D. For example, increased inﬂammation,(60) cellular adhe- sion,(61) and oxidative stress(62) might also contribute to the changes in bone structural and material properties that occur in T2D. Impaired bone endothelial function is just one potential mechanism by which T2D results in bone fragility. Discussion Regional Tibial Blood Flow, Vascular Conductance, and Vascular Resistance in Normoglycemic CON and T2D OLETF Rats CON T2D Two-factor RMANOVA Basal Insulin Basal Insulin Group Insulin Group Insulin MAP (mmHg) 98.6 7.7 82.2 15.2 85.3 10.7 67.0 7.8 0.002 0.003 0.841 Proximal epiphysis VC (mL/min/100 g/mmHg) 0.408 0.010 0.582 0.228 0.556 0.211 0.463 0.094 0.821 0.569 0.076 VR (mL/min/100 g)1 2.63 0.83 2.13 1.36 2.15 1.18 2.24 0.48 0.607 0.652 0.511 BF 39.97 9.37 46.01 17.27 48.84 20.95 31.17 7.95 0.643 0.256 0.032 Diaphysis VC (mL/min/100 g/mmHg) 0.143 0.078 0.077 0.024 0.149 0.071 0.071 0.027 0.614 0.086 0.446 VR (mL/min/100 g/ mmHg)1 8.24 4.50 30.39 18.24 7.60 3.66 11.17 3.17 0.359 0.118 0.196 BF 13.35 7.99 6.56 4.63 13.40 6.46 4.99 2.38 0.759 0.099 0.625 Distal epiphysis VC (mL/min/100 g/mmHg) 0.118 0.147 0.257 0.259 0.256 0.178 0.217 0.119 0.294 0.751 0.218 VR (mL/min/100 g/ mmHg)1 27.24 30.65 7.43 6.45 4.00 2.25 5.60 2.39 0.112 0.234 0.167 BF 11.31 13.84 19.97 19.38 19.94 17.31 11.39 3.83 0.879 0.879 0.124 Diaphyseal marrow VC (mL/min/100 g/mmHg) 1.062 0.358 1.488 0.882 0.833 0.615 0.743 0.351 0.208 0.505 0.314 VR (mL/min/100 g/ mmHg)1 1.039 0.348 1.159 1.351 1.614 0.844 1.660 1.014 0.305 0.853 0.934 BF 105.21 38.43 118.39 76.15 77.23 62.94 34.33 21.17 0.164 0.689 0.324 Data are means SD; O-CON (n ¼ 8) and O-T2D (n ¼ 6). VC ¼ vascular conductance; VR ¼ vascular resistance; BF ¼ blood ﬂow. Signiﬁcantly different from basal within group (p 0.05, one-tailed paired t test). Signiﬁcantly different from CON after insulin stimulation (p 0.05), one-tailed independent sample t test. JBMR Plus (WOA) 122 DIRKES ET AL. Fig. 5. Percent change in blood ﬂow (A) and vascular conductance (B) in regions of tibia. Data are means SE; signiﬁcantly different from O-CON. All groups are 40-week-old OLETF rats: O-T2D (n ¼ 6) were allowed ad libitum access to a rodent chow diet and O-CON (n ¼ 8) underwent moderate caloric restriction (30% restriction relative to intake of O-T2D) to maintain normal body weight and glycemia. Discussion PE ¼ proximal epiphysis; D ¼ diaphysis; DE ¼ distal epiphysis; M ¼ marrow. nutrients, growth factors, and hormones. In particular, delivery of circulating growth factors and hormones, the release of which is upregulated in response to feeding, is augmented as a result of insulin-stimulated blood ﬂow. Thus, impaired insulin- stimulated bone blood ﬂow might adversely affect bone due to reduced delivery of nutrients and other compounds that are essential for normal bone. In particular, reduced postprandial availability of insulin and glucose has the potential to have signiﬁcant deleterious skeletal effects because of the critical roles of insulin and glucose in osteoblast and osteoclast development and function. Insulin stimulates osteoblastogenesis and osteoblast differentiation,(54) inhibits osteoblast apoptosis,(51,55) and reduces osteoclastogenesis.(52) Osteoblast-speciﬁc deletion of the insulin receptor impairs bone acquisition in mice, with up to 50% decreases in trabecular bone.(56) Reduced glucose uptake in bone causes decreased bone formation and reduced bone mass in GLUT1 knockout mice.(51) Thus, although the direct effects of insulin on glucose uptake in bone are not entirely clear, insulin has other essential direct actions on osteoblasts and osteoclasts.(57) In the present study, insulin-stimulation increased bone blood ﬂow despite a decrease in MAP in the normoglycemic O-CON rats. Because blood ﬂow is the product of MAP and VC, the increase in bone blood ﬂow observed in the O-CON control animals was due to increased VC, ie, vasodilation, in response to insulin. By contrast, insulin did not increase blood ﬂow or VC in the O-T2D rats. These ﬁndings as well as the reduced NO- mediated vasodilation in femoral primary nutrient arteries isolated from ZDF rats(58) suggest eNOS production or availability is reduced in T2D. In a study of cerebral vascular function in the animals used in the present study to assess tibial blood ﬂow, NOS-dependent vasodilation was reduced, whereas endothelin-1 (ET-1)-mediated vasoconstriction was increased in isolated cerebral arteries of diabetic O-T2D animals.(59) These observations are consistent with our hypothesis that the T2D- associated reduction in insulin-stimulated bone blood ﬂow is due to altered insulin vasodilatory response in the vascular endothelium of T2D animals. Although the response of the bone vascular endothelium to insulin appears altered in T2D, the responsiveness of the bone vascular endothelium to other physiologic stimuli, such as exercise or hormones, known to cause vasodilation and increased blood ﬂow in T2D, has not been studied to date. Fig. 5. Discussion Glucotoxicity,(63) lipotoxicity,(52) and reduced Wnt/b-catenin signaling(64) also contribute to diabetic bone fragility by compromising the osteogenic potential of mesenchymal stem cells,(65) reducing osteoblastogenesis, increasing osteoblast apoptosis, impairing osteoblast function, and increasing osteoclast number and activity.(66) Moreover, accumulation of advanced glycation end-products (AGEs) negatively affect bone’s material properties,(67) impairs osteoblast differentia- tion,(68) and mineralization,(69) and increase sclerostin expres- sion and apoptosis in osteocyte-like cells.(70) Thus, dysfunction of bone endothelial cells is just one of many interrelated mechanisms of diabetic bone fragility. 123 INSULIN-STIMULATED BONE BLOOD FLOW/BIOMECHANICS IN OBESE, T2D OLETF RATS 123 JBMR® Plus (WOA) 4. Fajardo RJ. Is diabetic skeletal fragility associated with microvas- cular complications in bone? Curr Osteoporos Rep. 2017 Feb;15(1): 1–8. 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https://openalex.org/W4220778487	https://findresearcher.sdu.dk/ws/files/201012778/sciadv.abj8360.pdf	English	null	The glucocorticoid receptor associates with the cohesin loader NIPBL to promote long-range gene regulation	Science advances	2,022	cc-by	23,112	Download date: 24. Oct. 2024 University of Southern Denmark 1Laboratory of Receptor Biology and Gene Expression, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA. 2Department of Physics, University of Maryland, College Park, MD 20742, USA. 3High-Throughput Imaging Facility (HiTIF), Center for Cancer Research (CCR), NCI/NIH, Bethesda, MD 20892, USA. 4Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark. 5Institute for Physical Science and Technology, University of Maryland, College Park, MD 20742, USA. Corresponding author. Email: hagerg@exchange.nih.gov (G.L.H.); lorenzo.rinaldi@ nih.gov (L.R.) †These authors contributed equally to this work. The glucocorticoid receptor associates with the cohesin loader NIPBL to promote long-range gene regulation The cohesin complex is central to chromatin looping, but mechanisms by which these long-range chromatin interactions are formed and persist remain unclear. We demonstrate that interactions between a transcription factor (TF) and the cohesin loader NIPBL regulate enhancer-dependent gene activity. Using mass spectrometry, genome mapping, and single-molecule tracking methods, we demonstrate that the glucocorticoid (GC) receptor (GR) interacts with NIPBL and the cohesin complex at the chromatin level, promoting loop extrusion and long-range gene regulation. Real-time single-molecule experiments show that loss of cohesin markedly diminishes the con- centration of TF molecules at specific nuclear confinement sites, increasing TF local concentration and promoting gene regulation. Last, patient-derived acute myeloid leukemia cells harboring cohesin mutations exhibit a reduced response to GCs, suggesting that the GR-NIPBL-cohesin interaction is defective in these patients, resulting in poor response to GC treatment. M O L ECULAR B IOLOG Y Copyright © 2022 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution License 4.0 (CC BY). The glucocorticoid receptor associates with the cohesin loader NIPBL to promote long-range gene regulation Rinaldi, Lorenzo; Fettweis, Gregory; Kim, Sohyoung; Garcia, David A.; Fujiwara, Saori; Johnson, Thomas A.; Tettey, Theophilus T.; Ozbun, Laurent; Pegoraro, Gianluca; Puglia, Michele; Blagoev, Blagoy; Upadhyaya, Arpita; Stavreva, Diana A.; Hager, Gordon L. Citation for pulished version (APA): Rinaldi, L., Fettweis, G., Kim, S., Garcia, D. A., Fujiwara, S., Johnson, T. A., Tettey, T. T., Ozbun, L., Pegoraro, G., Puglia, M., Blagoev, B., Upadhyaya, A., Stavreva, D. A., & Hager, G. L. (2022). The glucocorticoid receptor associates with the cohesin loader NIPBL to promote long-range gene regulation. Science Advances, 8(13), Article eabj8360. https://doi.org/10.1126/sciadv.abj8360 Go to publication entry in University of Southern Denmark's Research Portal Terms of use This work is brought to you by the University of Southern Denmark. Unless otherwise specified it has been shared according to the terms for self-archiving. If no other license is stated, these terms apply: g y y y Unless otherwise specified it has been shared according to the terms for self-archiving. If no other license is stated, these terms apply: • You may download this work for personal use only • You may download this work for personal use only. Y t f th di t ib t th t i l it f y y p g • You may freely distribute the URL identifying this open access version If you believe that this document breaches copyright please contact us providing details and we will investigate your claim. Please direct all enquiries to puresupport@bib.sdu.dk If you believe that this document breaches copyright please contact us providing details and we will investigate your claim. Please direct all enquiries to puresupport@bib.sdu.dk Download date: 24. Oct. 2024 SCIE N C E A D V A NCES \| RESEA R CH A RT ICL E Copyright © 2022 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution License 4.0 (CC BY). INTRODUCTION d complex is still not well understood. Recent biophysical studies revealed that nucleosomes and other protein complexes restrict cohesin translocation (18), suggesting that transcriptional processes and chromatin remodelers could promote the cohesin translocation along the DNA strand (18, 19). Dynamic remodeling of nuclear architecture is paramount during development, stem cell differentiation, and environmental adapta- tion (1, 2). The cohesin complex is a master regulator of genome organization, regulating sister chromatid cohesion, DNA repair, and nuclear topology. Besides its prolonged binding at TAD bound- aries (3–7), cohesin is also present at enhancer-promoter contacts (2, 6–9). Enhancer-promoter interactions are dynamically con- trolled by lineage-specific transcription factors (TFs) that ensure proper phenotypic gene expression. This suggests a possible regulatory connection between TFs and the architectural cohesin complex (2). In mammals, the cohesin complex is formed by pro- tein subunits SMC1a (structural maintenance of chromosomes 1a), SMC3, and RAD21. In addition, a variety of accessory proteins such as NIPBL, Mau2, SA1/SA2, Wapl, and PDS5 regulate cohesin genome functions. The main cohesin subunits SMC1a, SMC3, and RAD21 bind stoichiometrically at chromatin at a 1:1:1 ratio to form the ring-shaped complex. However, a large fraction (60 to 80%) of these subunits are not associated with chromatin, suggesting that cohesin loading is a dynamic process (5, 10–12). SCC2/NIPBL dy- namically loads cohesin to chromatin accessible sites (13), guiding the bidirectional loop extrusion mechanism toward CTCF anchors in an adenosine 5′-triphosphate (ATP)–dependent manner (2, 4, 13–15). The genome localization and nuclear topology functions of the NIPBL-cohesin complex are modulated by tran- scriptional processes (16, 17). However, the mechanism behind the interplay between transcriptional machinery and the cohesin Cohesin complex function and transcriptional processes are in some ways correlated, but acute depletion of the cohesin complex does not alter the global RNA production in mammalian cells (6). Considering that most of these studies are performed under steady- state conditions, they only address the impact of cohesin depletion on the basal transcriptional activity (6, 20). The cohesin complex has been shown to bind at chromatin accessible sites bound by the estrogen receptor (ER) (8, 21) and to modulate ER-mediated tran- scriptional activity (8). Furthermore, the activation of steroid hormone receptors results in changes in hormone-dependent chromatin looping (21, 22), suggesting a profound relationship between steroid hormone receptor actions and genome organization. However, how cohesin regulates gene expression in response to induction is still under discussion (23). NIPBL regulates inducible long-range gene regulation NIPBL regulates inducible long-range gene regulation The cohesin complex, NIPBL, and TFs are all jointly involved in the modulation of the long-range chromatin interactions (2). To assess whether NIBPL regulates GR-bound long-range interactions, we performed GR-HiChIP in NIPBL knockdown cells (Fig. 2, A to C) (28). As expected, given that the GR binds mostly at distal regulatory elements, most of the chromatin interactions bound by GR are enhancer-enhancer interactions and enhancer-promoter interactions (Fig. 2D), while only 2.5% are promoter-promoter interactions. Very few GR-bound interactions engage TAD boundaries marked by the presence of CTCF, suggesting that the GR-bound interactions are independently formed from the structural interactions defined at CTCF TAD boundaries (Fig. 2D). Loss of NIPBL reduced both the number and the strength of GR-bound long-range interactions, identified by FitHiChIP (Fig. 2, B to D, and fig. S3, A to C) (29). As a control, we performed GR-HiChIP experiments in ethanol (EtOH)– treated cells, where GR is mostly cytosolic, finding no significantly detectable interactions (Fig. 2, B to D). These data indicate that NIPBL modulates GR binding to chromatin loops. To examine whether NIPBL regulates GR-mediated RNA transcription, we quantified nascent RNA transcripts for well-known GR target genes in NIPBL knock- down cells. The genes altered in NIPBL knockdown cells are cobound by both GR and NIPBL after dexamethasone treatment (fig. S1, H and I). These experiments revealed a profound dysregulation of GR-directed gene expression (for both activated and repressed genes) in the knock- down cells, suggesting that NIPBL is essential for proper GR-mediated transcriptional activity (Fig. 2G and fig. S3, D to F). Perturbation of the cohesin complex could result in several indirect effects includ- ing changes in cellular morphology and cellular viability. To rule out these secondary effects, we have repeated the siRNA treatment against NIPBL and SMC1a and checked for those potential indirect effects. We did not observe any drastic changes in either morphology or viability, suggesting that the residual SMC1A or NIPBL remaining (after siRNA treatment) in our breast adenocarcinoma cells is enough to maintain our cells in healthy conditions (fig. S3, G and H). To assess the indirect effects of GC treatment toward the cohesin complex and potential action of GC treatment on global recruitment of the cohesin complex, we performed an independent chromatin fractionation assay, followed by Western blot. SCIE N C E A D V A NCES \| RESEA R CH A RT ICL E SCIE N C E A D V A NCES \| RESEA R CH A RT ICL E Downloaded from https://www.science.org on April 11, 2022 /www.science.org on April 11, 2022 Here, we investigate the interplay between the cohesin com- plex and TFs, using the glucocorticoid (GC) receptor (GR), as model system. GR is a nuclear receptor known to translocate from the cytoplasm to the nucleus upon ligand treatment. Previous studies have shown that treatment with GCs strengthens chromatin inter- actions; these could be either stable or dynamic (21, 22, 24). GR binds preestablished chromatin loops enriched for cohesin sub- units Rad21 and Smc3 but depleted by CTCF, suggesting a possi- ble connection between cohesin and GR activity (22). Here, we show that chromatin-bound GR interacts with the cohesin loader NIPBL and demonstrate that GC treatment induces NIPBL re- cruitment to enhancers, promoting cohesin complex chromatin binding. Although most of the current work uses GR as a model TF, we suggest that this mechanism may be applicable to many other tran- scriptional regulators capable of interacting with NIPBL. Together, our data reveal a general mechanism by which TFs regulate the three-dimensional organization of the genome. 1 of 18 Rinaldi et al., Sci. Adv. 8, eabj8360 (2022) 30 March 2022 RESULTS Loop extrusion is triggered in a hormone-dependent manner to GR-binding sites versus those distant from GR peaks. TAD bound- aries near GR peaks showed a much stronger enrichment of cohesin after dexamethasone treatment (fig. S1F). This suggests that the activation of the steroid receptor promotes NIPBL-cohesin chromatin binding and loop extrusion of the cohesin in the direction of CTCF- bound TAD boundaries. To confirm our hypothesis, we performed SMC1a ChIP quantitative polymerase chain reaction (qPCR) after 10, 20, and 60 min of GC treatment to quantify the extent of cohesin chromatin binding and loop extrusion at the TSC22D3 gene locus (Fig. 1H). We found a strong enrichment at 20 min at the GR- bound site, whereas at 60 min, SMC1a was still enhanced at the proximal TAD boundary (Fig. 1I). To confirm this pattern of cohes- in loading, we have repeated the assay investigating the chromatin landscape of multiple GR target genes regulated by long-range inter- actions. Most of the GR-bound enhancers investigated show an acute enrichment of the cohesin subunit SMC1A after only 20 min of dexamethasone compared to 60 min (fig. S2, B to D). This en- richment at GR-bound enhancers was reduced after 60 min, while it was still highly evident at the nearby TAD boundary (fig. S2, B to D). Together, these data indicate that GR and probably other TFs can promote cohesin binding at enhancer sites by a direct inter- action with NIPBL. The cohesin complex is then extruded toward the proximal TAD boundary in an ATP-dependent manner (13). NIPBL regulates inducible long-range gene regulation We did not observe any dexamethasone-dependent global changes of NIPBL or SMC1A at the chromatin fraction (fig. S2A), suggesting that the enhanced chromatin binding of NIPBL and SMC1A is mostly exclusive to chromatin sites bound by GR after dexamethasone treatment. Rinaldi et al., Sci. Adv. 8, eabj8360 (2022) 30 March 2022 RESULTS To uncover novel interacting partners of GR at the chromatin level, we performed quantitative label-free mass spectrometry (MS), chromatin immunoprecipitation–selective isolation of chromatin- associated proteins (ChIP-SICAP) (25), using mouse mammary carcinoma cells treated or untreated with dexamethasone, a steroid hormone known to rapidly translocate the steroid receptor GR to the nucleus and promote its binding to chromatin and gene regula- tory functions (fig. S1A). The cohesin loader NIPBL and the cohesin subunit SMC1a are among the top interactors with the chromatin- bound GR (Fig. 1A). Independent methods, including immuno- precipitation and proximity ligation assay, confirmed the direct interaction between endogenous GR, NIPBL, and the cohesin com- plex (Fig. 1, B and C). To investigate whether the NIPBL-GR interaction increases chromatin binding after hormone activation, we performed two biological replicates of ChIP sequencing (ChIP-seq) for NIPBL and SMC1a before and after 1 hour of dexamethasone treatment. ChIP- seq analysis demonstrated an increased binding of NIPBL and SMC1a upon 1-hour dexamethasone treatment at GR-bound chro- matin elements (Fig. 1, D and E, and fig. S1, B, C, H, and I). Together, these data demonstrate that the steroid hormone receptor GR inter- acts at the chromatin level with the cohesin loader NIPBL and the cohesin arm SMC1a, promoting cohesin chromatin binding at GR sites in a hormone-dependent manner (Fig. 1, D and E). SCIE N C E A D V A NCES \| RESEA R CH A RT ICL E N C E A D V A NCES \| RESEA R CH A RT ICL E 12 345 7 6 8 9 A ChIP- SICAP B C D GR NIPBL EtOH Dex EtOH Dex G siCTRL siNIPBL siSMC1a EtOH GR siCTRL siNIPBL siSMC1a 3815 3446 2085 155 5 32 23 Dex EtOH Flag EtOH Dex SMC1a chr11:101663377-101704782 GR/Nipbl proteomic interaction GR/SMC1a proteomic interaction Q value Q value EtOH 1-hour Dex 1-hour Dex chr11 GR SMC1a CTCF NIPBL RAD21 GR − + − + − + chrX:140,491,608-140,553,226 F TAD boundary TAD boundary Top 3000 GR sites H GR site TAD boundary SMC1a chip ` after dex time course I E Top 3000 GR sites 0.40 0.30 0.20 0.10 0.0 GR NIPBL EtOH Dex EtOH Dex EtOH Dex SMC1a 0.1 0.4 0.5 0.2 0.3 0 0.4 3 1.6 1.2 0.8 0.4 0.0 EtOH Dex 0 100 200 300 Intensity (Nuc.) NIPBL/GR interaction by PLA EtOH Neg Ctrl 0 500 1000 1500 Smc3/GR interaction by PLA Dex EtOH Neg Ctrl NIPBL CTCF − + SMC1a − + − + − + 1 2 3 4 5 7 6 8 9 Arl4d Tsc22d3 Mau2 Nipbl Dex GR Smc3 Smc1 Rad21 PBL and cohesin are loaded onto chromatin in a hormone-dependent manner. (A) GR-cohesin–interacting partners identified by GR ChIP-SICAP. GR inter- ficantly with NIPBL and SMC1a, marked in red. (B) GR-immunoprecipitation (and FLAG-IP) and blotting against SMC1, SMC3, NIPBL, and GR after 1 hour of EtOH ethasone treatment in mouse breast adenocarcinoma cells. (C) Endogenous interaction, measured by proximity ligation assay, between GR and NIPBL (left) and ht). (D) GR, NIPBL, and SMC1a ChIP-seq after 1 hour of EtOH or dexamethasone treatment. (E) Heatmaps representing GR, NIPBL, and SMC1a ChIP-seq at the 3000 GR locations previously identified in 3134 cells before and after 1 hour of 100 nM dexamethasone. Data combine two biological replicates normalized to reads. (F) Heatmap of GR ChIP-seq intensity at the 3000 strongest GR locations [same as (E)] in siCTRL, siSMC1a, and siNIPBL cells. Genomic data are normalized ts per kilobase million (fpkm)] to a total of 10 million reads and further to local tag density (P < 0.000001 from Wilcoxon test). (G) Venn diagram of GR ChIP-seq ntified in two independent replicates of siCTRL, siSMC1a, and siNIPBL cells. (H) Genome browser screenshot of the Tsc22d3 gene locus for the ChIP-seq of GR, MC1A, RAD21, and CTCF. SCIE N C E A D V A NCES \| RESEA R CH A RT ICL E The TAD boundary at the 3′ untranslated region of TSC22D3 gene was used to evaluate cohesin binding and loop extrusion mechanism by GR. (I) Red highlighted regions correspond to PCR products designed to evaluate the loop extrusion process. On the right side, ChIP qRT-PCR for SMC1a after d 20′ d 60′ f d h (d i h h bi l i l li ) p g p , A ChIP- SICAP GR/Nipbl proteomic interaction GR/SMC1a proteomic interaction Q value Q value EtOH 1-hour Dex 1-hour Dex EtOH Mau2 Nipbl Dex GR Smc3 Smc1 Rad21 B Dex EtOH Flag C Dex 0 100 200 300 Intensity (Nuc.) NIPBL/GR interaction by PLA EtOH Neg Ctrl 0 500 1000 1500 Smc3/GR interaction by PLA Dex EtOH Neg Ctrl B A C D GR NIPBL EtOH Dex EtOH Dex GR EtOH Dex SMC1a chr11:101663377-101704782 chr11 Arl4d D E Top 3000 GR sites 0.40 0.30 0.20 0.10 0.0 GR NIPBL EtOH Dex EtOH Dex EtOH Dex SMC1a 0.1 0.4 0.5 0.2 0.3 0 0.4 E GR GR siCTRL siNIPBL siSMC1a EtOH GR F Top 3000 GR sites 3 1.6 1.2 0.8 0.4 0.0 Downloaded from https://www.science.org on April 11, 2022 RL siNIPBL siSMC1a 3815 3446 2085 155 5 32 23 GR site TAD boundary SMC1a chip ` after dex time course 1 2 3 4 5 7 6 8 9 Downloaded from https://www.science.org on April 11, 2022 G 12 345 7 6 8 9 GR SMC1a CTCF NIPBL RAD21 GR − + − + − + chrX:140,491,608-140,553,226 TAD boundary TAD boundary H GR site SMC1 ` after dex I NIPBL CTCF − + SMC1a − + − + − + 1 2 3 4 Tsc22d3 H Fig. 1. NIPBL and cohesin are loaded onto chromatin in a hormone-dependent manner. (A) GR-cohesin–interacting partners identified by GR ChIP-SICAP. GR inter- acts significantly with NIPBL and SMC1a, marked in red. (B) GR-immunoprecipitation (and FLAG-IP) and blotting against SMC1, SMC3, NIPBL, and GR after 1 hour of EtOH or dexamethasone treatment in mouse breast adenocarcinoma cells. (C) Endogenous interaction, measured by proximity ligation assay, between GR and NIPBL (left) and SMC3 (right). (D) GR, NIPBL, and SMC1a ChIP-seq after 1 hour of EtOH or dexamethasone treatment. (E) Heatmaps representing GR, NIPBL, and SMC1a ChIP-seq at the 3000 strongest GR locations previously identified in 3134 cells before and after 1 hour of 100 nM dexamethasone. Downloaded from https://www.science.org on April 11, 2022 To evaluate whether this interaction was important for GR chro- matin binding, we performed GR ChIP-seq upon NIPBL and SMC1a knockdown. We performed small interfering RNA (siRNA) treatment against NIPBL or SMC1A in mammary breast adeno- carcinoma cells. An efficient knockdown, achieved after 72 hours of siRNA transfection, was evaluated by Western blot for both NIPBL and the core cohesin subunit SMC1a (fig. S1G). Loss of NIPBL re- duced GR binding to chromatin, and both the number of GR chro- matin sites and the strength of GR binding were severely compromised (Fig. 1, F and G). To further validate these findings, we performed GR ChIP-seq in SMC1a knockdown cells. In agreement with our siNIPBL results, depletion of SMC1A (72 hours of siRNA treatment) significantly reduced GR binding to chromatin (Fig. 1, F and G). In support of these results, our chromatin fraction data clearly dis- played a decrease in GR accumulation in the chromatin fraction after the knockdown of NIPBL and Smc1a (fig. S2A). These data suggest a multifactorial model whereby the NIPBL-cohesin com- plex associates with transcriptional regulators to induce long-range gene regulation (4, 16, 17, 26). By determining the position of TAD anchors via CTCF motif orientation analysis (27), we observed that GR primarily binds in close proximity to the TAD anchors (fig. S1, D and E). To test whether GR induces cohesin stalling at the TAD boundaries, we quantified the enrichment of cohesin at TAD boundaries proximal 2 of 18 Rinaldi et al., Sci. Adv. 8, eabj8360 (2022) 30 March 2022 SCIE N C E A D V A NCES \| RESEA R CH A RT ICL E SCIE N C E A D V A NCES \| RESEA R CH A RT ICL E Data combine two biological replicates normalized to 10 million reads. (F) Heatmap of GR ChIP-seq intensity at the 3000 strongest GR locations [same as (E)] in siCTRL, siSMC1a, and siNIPBL cells. Genomic data are normalized [fragments per kilobase million (fpkm)] to a total of 10 million reads and further to local tag density (P < 0.000001 from Wilcoxon test). (G) Venn diagram of GR ChIP-seq peaks identified in two independent replicates of siCTRL, siSMC1a, and siNIPBL cells. (H) Genome browser screenshot of the Tsc22d3 gene locus for the ChIP-seq of GR, NIPBL, SMC1A, RAD21, and CTCF. The TAD boundary at the 3′ untranslated region of TSC22D3 gene was used to evaluate cohesin binding and loop extrusion mechanism mediated by GR. (I) Red highlighted regions correspond to PCR products designed to evaluate the loop extrusion process. On the right side, ChIP qRT-PCR for SMC1a after EtOH treatment and 20′ and 60′ of dexamethasone (data points show three biological replicates). 3 of 18 Rinaldi et al., Sci. Adv. 8, eabj8360 (2022) 30 March 2022 SCIE N C E A D V A NCES \| RESEA R CH A RT ICL E FitHiChIP signiﬁcant interactions GR ChIP siNPBL GR-HiCHIP siCTRL ARL4D gene locus GR-HiCHIP TSC22D3 gene locus B D C F E GR ChIP siNPBL siCTRL A G GRHiChIP: Normalized coverage of signiﬁcant interactions siCTRL + dex siNPBL + dex siCTRL + etOH 0 5000 10,000 Number of significant chromatin interactions siCTRL + dex siNPBL + dex siCTRL + etOH 0 50 100 chr1 chr2 chr3 chr4 chr5 chr6 chr7 chr8 chr9 chr10 chr11 chr12 chr13 chr14 chr15 chr16 chr17 chr18 chr19 chrx APA (Z score) enrichment Ethanol siCTRL + dex siNIPBL + dex * * regulates GR-bound long-range interactions and gene regulation. (A) Experimental design of the GR-HiChIP protocol and analys ome browser screenshot of GR-bound long-range interaction identified by FitHiChIP at the TSC22D3 and ARL4D locations, in siCTRL and rmalized at a 5-kb resolution. (D) Top: The number of significant GR long-range interactions identified by FitHiChIP, at a Q value of 0.01 u untreated cells, siCTRL + 1-hour dexamethasone, and siNIPBL + 1-hour dexamethasone. Bottom: The percentages of the GR-bond chro different types of long-range interactions. (E) Quantification intensity of the commonly identified GR-HiChIP long-range interactions iden 1) in siCTRL and siNIPBL samples, at a 5-kb resolution. SCIE N C E A D V A NCES \| RESEA R CH A RT ICL E P < 0.00001 derived from KS test. (G) Nascent RNA quantification, measured by qRT-PCR, of GR target genes (1 hour of EtOH or dexamethasone treatment) after 48 hours of siRNA transfection against nontargeting sequence (siCTRL) or NIPBL. Fold inductions (dexamethasone/EtOH) are normalized on glyceraldehyde-3- phosphate dehydrogenase (GAPDH) nascent RNA. Error bars represent SE, and P value was derived from unpaired Mann-Whitney tests (n = 3 independent biological replicates). ns, not significant. Downloaded from https://www.science.org on April 11, 2022 FitHiChIP signiﬁcant interactions GR ChIP siNPBL GR-HiCHIP siCTRL ARL4D gene locus C D 0 5000 10,000 Number of significant chromatin interactions siCTRL + dex siNPBL + dex siCTRL + etOH D C E GRHiChIP: Normalized coverage of signiﬁcant interactions siCTRL + dex siNPBL + dex siCTRL + etOH * F G 0 50 100 chr1 chr2 chr3 chr4 chr5 chr6 chr7 chr8 chr9 chr10 chr11 chr12 chr13 chr14 chr15 chr16 chr17 chr18 chr19 chrx APA (Z score) enrichment Ethanol siCTRL + dex siNIPBL + dex * * E F Downloaded from https://www.science.org on April 11, 2022 * Fig. 2. NIPBL regulates GR-bound long-range interactions and gene regulation. (A) Experimental design of the GR-HiChIP protocol and analysis pipeline. (B and C) WashU genome browser screenshot of GR-bound long-range interaction identified by FitHiChIP at the TSC22D3 and ARL4D locations, in siCTRL and siNIPBL cells. Loop strength is normalized at a 5-kb resolution. (D) Top: The number of significant GR long-range interactions identified by FitHiChIP, at a Q value of 0.01 using coverage bias parameters, in untreated cells, siCTRL + 1-hour dexamethasone, and siNIPBL + 1-hour dexamethasone. Bottom: The percentages of the GR-bond chromatin interactions stratified into different types of long-range interactions. (E) Quantification intensity of the commonly identified GR-HiChIP long-range interactions identified by FitHiChIP (Q value of 0.01) in siCTRL and siNIPBL samples, at a 5-kb resolution. NIPBL depletion leads to profound reduction of GR-bound chromatin interaction number and looping strength. P < 0.00001 derived from KS test. (F) Aggregate enrichment for each mouse chromosome in the GR-HiChIP datasets in EtOH, siCTRL, and siNIPBL samples. Aggregate peak analysis (APA) measurements (Z score) were calculated on the long-range interactions identified by FitHiChIP (Q value of 0.01) using the juicertools at a 5-kb resolu- tion and VC normalization. P < 0.00001 derived from KS test. SCIE N C E A D V A NCES \| RESEA R CH A RT ICL E NIPBL depletion leads to profound reduction of GR-bound chromatin interaction nu .00001 derived from KS test. (F) Aggregate enrichment for each mouse chromosome in the GR-HiChIP datasets in EtOH, siCTRL, and siNIPBL sa (APA) measurements (Z score) were calculated on the long-range interactions identified by FitHiChIP (Q value of 0.01) using the juicertoo ormalization P < 0 00001 derived from KS test (G) Nascent RNA quantification measured by qRT-PCR of GR target genes (1 hour of EtOH o FitHiChIP signiﬁcant interactions GR ChIP siNPBL GR-HiCHIP siCTRL ARL4D gene locus GR-HiCHIP TSC22D3 gene locus B C GR ChIP siNPBL siCTRL A B Rinaldi et al Sci Adv 8 eabj8360 (2022) 30 March 2022 4 of 18 FitHiChIP signiﬁcant interactions GR ChIP siNPBL GR-HiCHIP siCTRL ARL4D gene locus G D C F E siNPB G GRHiChIP: Normalized coverage of signiﬁcant interactions siCTRL + dex siNPBL + dex siCTRL + etOH 0 5000 10,000 Number of significant chromatin interactions siCTRL + dex siNPBL + dex siCTRL + etOH 0 50 100 chr1 chr2 chr3 chr4 chr5 chr6 chr7 chr8 chr9 chr10 chr11 chr12 chr13 chr14 chr15 chr16 chr17 chr18 chr19 chrx APA (Z score) enrichment Ethanol siCTRL + dex siNIPBL + dex * * * Fig. 2. NIPBL regulates GR-bound long-range interactions and gene regulation. (A) Experimental design of the GR-HiChIP protocol and analysis pipeline. (B and C) WashU genome browser screenshot of GR-bound long-range interaction identified by FitHiChIP at the TSC22D3 and ARL4D locations, in siCTRL and siNIPBL cells. Loop strength is normalized at a 5-kb resolution. (D) Top: The number of significant GR long-range interactions identified by FitHiChIP, at a Q value of 0.01 using coverage bias parameters, in untreated cells, siCTRL + 1-hour dexamethasone, and siNIPBL + 1-hour dexamethasone. Bottom: The percentages of the GR-bond chromatin interactions stratified into different types of long-range interactions. (E) Quantification intensity of the commonly identified GR-HiChIP long-range interactions identified by FitHiChIP (Q value of 0.01) in siCTRL and siNIPBL samples, at a 5-kb resolution. NIPBL depletion leads to profound reduction of GR-bound chromatin interaction number and looping strength. P < 0.00001 derived from KS test. (F) Aggregate enrichment for each mouse chromosome in the GR-HiChIP datasets in EtOH, siCTRL, and siNIPBL samples. Aggregate peak analysis (APA) measurements (Z score) were calculated on the long-range interactions identified by FitHiChIP (Q value of 0.01) using the juicertools at a 5-kb resolu- tion and VC normalization. Acute loss of cohesin impairs inducible TF binding to chromatin Among the significant dex-induced loops, our analysis identi- fied chromatin interactions connecting GR-bound enhancers to the GR target genes Fkbp5 and Tsc22D3 (Fig. 3C and table S5) (24). NIPBL and SMC1A bind these enhancer-promoter contacts in a dexamethasone-dependent manner (Fig. 3C). These interactions are strongly impaired by the loss of NIPBL (Fig. 3C). The TAD boundaries at these loci are already formed before dexamethasone treatment; however, the activation of the GR induces both the formation of enhancer loops and mostly of the architectural stripes connecting the GR site to the TAD boundaries (Fig. 3E and fig. S2F). Architectural stripes are associated to the loop extrusion process (13), where a loop anchor interacts with the entire domain at high frequency, to acti- vate target gene activation (13, 32). To accurately identify these features, we used the Stripenn protocol (32) to analyze our Micro-C data (Fig. 3E). Our analysis, at 5000–base pair (bp) resolution, identified 2300 stripes in control EtOH versus 1703 in control dexamethasone cells. NIPBL knockdown lowered the numbers of stripes to 1157 versus 557 (table S6). A total of 561 architectural stripes (at 5-kb resolution) were specifi- cally bound by the GR (table S6). Dexamethasone treatment significantly strengthened the GR-bound stripes compared to the EtOH control, suggesting that GR chromatin binding promotes a higher frequency of interactions and stimulates the loop extrusion process (Fig. 3, F and G). This dexamethasone-induced activation of the architectural stripes is lost after NIPBL knockdown. Together, these data suggest that GR and NIPBL promote DNA extrusion through the formation of both enhancer-promoter contacts and architectural stripes. To extend our findings, we used the auxin degron system to rapidly deplete the cohesin subunit RAD21 in HCT116 cells (Fig. 5A and fig. S5, A and B) (6, 33). We performed GR ChIP-seq (two indepen- dent biological replicates) after 1 hour of dexamethasone induction in untreated and auxin-treated cells and found that rapid depletion (4 and 24 hours) of RAD21 drastically reduces GR binding to chro- matin (Fig. 5, B and C). At the RNA level, the acute depletion of RAD21 strongly impaired GR-induced transcription of TSC22D3 and several other target genes (Fig. 5D). /www.science.org on April 11, 2022 We further asked whether loss of cohesin could also impair chromatin binding of a noninducible TF. To this end, we performed ChIP-seq for YY1, a noninducible TF known to bind and regulate distal regulatory elements. Ultradeep Micro-C sequencing shows that NIPBL and GR mediate long-range chromatin interactions To further understand how GR and NIPBL mediate long-range inter- actions, we performed Micro-C in two biological replicates (before and after 100 nM dexamethasone) in control and siNIPBL cells. We opted for Micro-C since this method offers great advantages com- pared to in situ Hi-C, given that the use of micrococcal nuclease provides unparallel resolution (Fig. 3A) (30, 31). After filtering for low-count reads across replicates, we identified 29,674 loops in siCtrl control cells. Dexamethasone treatment resulted in an overall loop count of 22,573. In this fraction, 2650 interactions (12%) represent new loops induced by dex, while 6404 interactions (22%) were elimi- nated by dex treatment (Fig. 3, B and C, and table S5). Approximately 50% of the new loops created by dex were sensitive to siNIPBL knock- down (Fig. 3B), while very few of the dex-repressed loops were affected by siNIPBL knockdown (Fig. 3B), confirming the importance of NIPBL regulating nuclear architecture (Fig. 3B) (7, 14). Dex-induced interactions are strongly bound by GR, as detected by ChIP-seq (Fig. 3D). The strong reduction of dex-induced interactions in siNIPBL cells indicates that GR and NIPBL jointly promote chromatin looping in a large fraction of the new loops (Fig. 3, B to D). Since the per- centage of dex-repressed interactions was not altered by NIPBL knock- down (Fig. 3B), it appears that dex-repressed loops are not regulated by NIPBL. Intriguingly, GR may regulate looping repression through novel cofactors identified in our GR ChIP-SICAP analysis, such as HP1-alpha (Cbx5), HP1-gamma (Cbx3), WIZ, EHMT1 (GLP), lamin B receptor, and nuclear receptor corepressor 1 (table S1). Acute loss of cohesin impairs inducible TF binding to chromatin We found that YY1 binding was largely unaffected after 24 hours of auxin treatment, confirming that the cohesin complex depletion has little impact on this stably bound TF (Fig. 5E). To explore whether overall chromatin accessibility and histone acetylation were altered by the acute depletion of RAD21, we performed ATAC-seq (assay for transposase-accessible chroma- tin using sequencing) (34) and H3K27ac ChIP-seq before and after auxin treatment (two independent biological replicates). In agreement with previous studies (6), acute depletion of RAD21 (4 to 24 hours) showed minor changes in chromatin accessibility and histone acetyla- tion (Fig. 5, F and G), indicating that the steady-state transcriptional machinery is largely unaltered by the acute loss of RAD21. Nevertheless, prolonged cohesin deprivation (72 hours) leads to the loss of active chro- matin modifications and cohesin-associated proteins (fig. S5, E and F). Single-molecule nascent RNA-FISH reveals that GR associates with NIPBL to promote long-range gene regulation SCIE N C E A D V A NCES \| RESEA R CH A RT ICL E SCIE N C E A D V A NCES \| RESEA R CH A RT ICL E hybridization chain reaction (HCR) RNA fluorescence in situ hy- bridization (FISH) to probe the expression of nascent RNA from well-known GR target genes Tsc22d3 and Arl4d (Fig. 4, A and B). The results confirm that nascent transcription of both Tsc22d3 and Arl4d genes is impaired approximately two- and fourfold in siRAD21 and siSMC1a knockdown cells, respectively (Fig. 4, C and D, and fig. S4, A to D). With HCR, one can investigate the distribution of gene expression at the single-cell and single-allele level. Thus, we measured the percentage of actively transcribing cells for each con- dition and their allelic contribution (Fig. 4E). Approximately 65% of cells treated with a scrambled siRNA actively transcribe the target genes examined, while in both siSMC1a- and siRAD21-treated cells, less than 40% of these are transcriptionally active (Fig. 4E). Consid- ering the function of the cohesin complex in nuclear architecture, we also investigated whether nuclear positions of the loci are affected. We analyzed the localization of Tsc22d3 and Arl4d loci (measured by HCR) with respect to the nuclear membrane, observing a recruit- ment of active loci to the periphery of the nucleus under all the tested conditions, while finding no difference between control and knock- down cells (fig. S4E). Collectively, our single-molecule nascent RNA-FISH data show that the loss of NIPBL and of the core cohesin subunits markedly affects the GR-mediated gene expression by reducing the number of bursting sites and the intensity of the tran- scriptional activity without affecting localization of target genes in the nucleus. Ultradeep Micro-C sequencing shows that NIPBL and GR mediate long-range chromatin interactions SCIE N C E A D V A NCES \| RESEA R CH A RT ICL E (G) Nascent RNA quantification, measured by qRT-PCR, of GR target genes (1 hour of EtOH or dexamethasone treatment) after 48 hours of siRNA transfection against nontargeting sequence (siCTRL) or NIPBL. Fold inductions (dexamethasone/EtOH) are normalized on glyceraldehyde-3- phosphate dehydrogenase (GAPDH) nascent RNA. Error bars represent SE, and P value was derived from unpaired Mann-Whitney tests (n = 3 independent biological replicates). ns, not significant. 4 of 18 Rinaldi et al., Sci. Adv. 8, eabj8360 (2022) 30 March 2022 Single-molecule nascent RNA-FISH reveals that GR associates with NIPBL to promote long-range gene regulation (E) Contact frequencies, in siCTRL Dex, of the architectural stripe connecting the GR-bo A B 20-KB resolution Chr 1: (Mb) 60 70 80 86 78 70 68 66 72 74 76 Chr 1: (Mb) 5-KB resolution 79 80 84 81 80 82 81 82 83 Chr 1: (Mb) A 20-KB resolution (Mb) 60 70 80 86 78 70 68 66 72 74 76 Chr 1: (Mb) 5-KB resolution 79 80 84 81 80 82 81 82 83 78 74 76 1: b) 5-KB resolution 79 80 84 81 80 82 81 82 83 B 81 82 GR dex NIPBL etOH NIPBL dex SMC1A etOH SMC1A dex CTCF GR HiChIP loops Fkbp5 EtOH Dex siCTRL B E F 2000-bp resolution GR dex NIPBL etOH NIPBL dex SMC1A etOH SMC1A dex CTCF 2000-bp resolution siNIPBL Dex Chr 17: (Kb) 28,440 28,520 GR HiChIP loops Fkbp5 C GR dex NIPBL etOH NIPBL dex SMC1A etOH SMC1A dex CTCF 2000-bp resolution siCTRL Dex siNIPBL Dex G siNIPBL Dex siCTRL EtOH EtOH Dex EtOH Dex siCTRL D siCTRL Dex siNIPBL Dex EtOH Dex D siCTRL EtOH siCTRL Dex siNIPBL Dex siNIPBL EtOH 3 2 1 0 D 2000-bp resolution GR dex NIPBL etOH NIPBL dex SMC1A etOH SMC1A dex CTCF 2000-bp resolution siNIPBL Dex Chr 17: (Kb) 28,440 28,520 GR HiChIP loops Fkbp5 C GR dex NIPBL etOH NIPBL dex SMC1A etOH SMC1A dex CTCF 2000-bp resolution siCTRL Dex siNIPBL Dex siNIPBL Dex siCTRL EtOH EtOH Dex siCTRL D 2000-bp resolution Chr 17: (Kb) 28,440 28,520 C siCTRL EtOH C Downloaded from https://www.science.org on April 11, 2022 G F G G E F E GR Fig. 3. Ultradeep Micro-C sequencing shows that NIPBL regulates GR-mediated long-range interactions. (A) Chromosome contact maps for three successive zoom- ins across mouse chromosome 1 using Micro-C data. Red arrows depict anchors by moustache loop calling, and red boxes are architectural stripes detected by Stripenn. (B) Middle: Total numbers of Micro-C loops in siCTRL EtOH and siCTRL Dex. A total of 2650 loops were identified as dex-induced while 6350 were found as dex-repressed loops. siNIPBL cells showed a reduction of the dex-induced loops: measured by the ratio of the dex-induced loops over the sum of the differential loops and found by comparing siCTRL EtOH versus siCTRL Dex and siNIPBL EtOH versus siNIPBL Dex. There was no change in the dex-repressed loops as measured by the ratio of the dex-repressed loops. Single-molecule nascent RNA-FISH reveals that GR associates with NIPBL to promote long-range gene regulation To validate our findings with a nonsteroidal, inducible TF, we characterized the response of nuclear factor kB (NF-kB) to tumor necrosis factor– (TNF-) stimulation in the RAD21mAID cells (±auxin) by RNA sequencing (RNA-seq; two independent biological replicates). Elegantly, earlier work using the Hct116 RAD21mAID To accurately investigate gene expression changes upon loss of co- hesin at the single-cell level, we used a high-throughput version of 5 of 18 Rinaldi et al., Sci. Adv. 8, eabj8360 (2022) 30 March 2022 SCIE N C E A D V A NCES \| RESEA R CH A RT ICL E N C E A D V A NCES \| RESEA R CH A RT ICL E A B 20-KB resolution Chr 1: (Mb) 60 70 80 86 78 70 68 66 72 74 76 Chr 1: (Mb) 5-KB resolution 79 80 84 81 80 82 81 82 83 Chr 1: (Mb) E F 2000-bp resolution GR dex NIPBL etOH NIPBL dex SMC1A etOH SMC1A dex CTCF 2000-bp resolution siNIPBL Dex Chr 17: (Kb) 28,440 28,520 GR HiChIP loops Fkbp5 C GR dex NIPBL etOH NIPBL dex SMC1A etOH SMC1A dex CTCF 2000-bp resolution siCTRL Dex siNIPBL Dex GR G siNIPBL Dex siCTRL EtOH EtOH Dex EtOH Dex siCTRL D siCTRL EtOH siCTRL Dex siNIPBL Dex siNIPBL EtOH 3 2 1 0 ltradeep Micro-C sequencing shows that NIPBL regulates GR-mediated long-range interactions. (A) Chromosome contact maps for three successive zo s mouse chromosome 1 using Micro-C data. Red arrows depict anchors by moustache loop calling, and red boxes are architectural stripes detected by Stripe le: Total numbers of Micro-C loops in siCTRL EtOH and siCTRL Dex. A total of 2650 loops were identified as dex-induced while 6350 were found as dex-repres NIPBL cells showed a reduction of the dex-induced loops: measured by the ratio of the dex-induced loops over the sum of the differential loops and found ng siCTRL EtOH versus siCTRL Dex and siNIPBL EtOH versus siNIPBL Dex. There was no change in the dex-repressed loops as measured by the ratio of essed loops. (C) Micro-C robustly captures enhancer-promoter contacts driven by the GR. GR, SMC1A, NIPBL, and CTCF ChIP-seq are on top of the contact m alized datasets represent two biological replicates and plotted at 2-kb resolution. (D) Aggregate peak analysis plots of the dex-induced loops. The heatm how the GR ChIP-seq intensity at the dex-induced interactions. Single-molecule nascent RNA-FISH reveals that GR associates with NIPBL to promote long-range gene regulation Downloaded from https://www.science.org on April 11, 2022 Percentage of transcribing cells D Nascent RNA foci intensity E TSC22D3: RNA intensity ARL4D: RNA intensity TSC22D3: Heterogeneity ARL4D: Heterogeneity One Two One Two Spot number/cell Spot number/cell 0.8 1 0.75 * * * * ns ns Sum spot intensity per cell Sum spot intensity per cell lls/well well siCTRL siRAD21 siSMC1a Zero Foci number One Two 16,384 32,768 65,536 131,072 262,144 524,288 * * ns * * ns 16,384 32,768 65,536 131,072 262,144 524,288 Downloaded from https://www.science.org o D Nascent RNA foci intensity TSC22D3: RNA intensity ARL4D: RNA intensity One Two One Two Spot number/cell Spot number/cell * * * * ns ns Sum spot intensity per cell Sum spot intensity per cell siCTRL siRAD21 siSMC1a 16,384 32,768 65,536 131,072 262,144 524,288 * * ns * * ns 16,384 32,768 65,536 131,072 262,144 524,288 Downloaded from https://w D Nascent RNA foci intensity A y TSC22D3: RNA intensity One Two Spot number/cell * * * * ns ns Sum spot intensity per cell 16,384 32,768 65,536 131,072 262,144 524,288 ARL4D: RNA intensity One Two Spot number/cell Sum spot intensity per cell 16,384 32,768 65,536 131,072 262,144 524,288 * * ns * * ns D Sum spot intensity per cell Downloaded from https://www.science.org on April 11, 2022 Spot number/cell Spot number/cell ARL4D: Heterogeneity Spot number/cell Time after dex (hours) 0.8 0.6 0.4 0.2 0 Proportion of cells/well Zero ci ber One Two Percentage of transcribing cells E TSC22D3: Heterogeneity ARL4D: Hetero Time after dex (h Time after dex (hours) 0.8 0.6 0.4 0.2 0 1 0.75 0.5 0.2 0 Proportion of cells/well Proportion of cells/well Zero Foci number One Two Percentage of transcribing cells E TSC22D3: Heterogeneity Time after dex (hours) 1 0.75 0.5 0.2 0 Proportion of cells/well E Percentage of transcribing cells Time after dex (hours) Fig. 4. Cohesin depletion impairs GR gene activation. (A) Schematic illustration of HCR protocol. (B) Representative images of nascent RNA before and after Dex treat- ment. ARL4D images are taken after 1 hour of dexamethasone; TSC22D3 images are taken after 4 hours of Dex treatment. Scale bar, 5 m. (C) Average number of active nascent RNA foci per well at 0, 1, and 4 hours of dex treatment for ARL4D and TSC22D3, after 72 hours of siRNA against SMC1a or RAD21. Left: TSC22D3; right: ARL4D. Single-molecule nascent RNA-FISH reveals that GR associates with NIPBL to promote long-range gene regulation (C) Micro-C robustly captures enhancer-promoter contacts driven by the GR. GR, SMC1A, NIPBL, and CTCF ChIP-seq are on top of the contact maps. All normalized datasets represent two biological replicates and plotted at 2-kb resolution. (D) Aggregate peak analysis plots of the dex-induced loops. The heatmaps below show the GR ChIP-seq intensity at the dex-induced interactions. (E) Contact frequencies, in siCTRL Dex, of the architectural stripe connecting the GR-bound superenhancer to the gene Nsmce2. (F) GR and NIPBL promote the formation of dexamethasone-dependent architectural stripes. Boxplot depicting the significance [−log10(P value)] of all the GR-bound architectural stripes (n = 561), in siCTRL EtOH, siCTRL dex, siNIPBL EtOH, and siNIPBL dex Micro-C data. (G) Heatmap showing the intensity of the architectural stripes (two biological replicates were combined on the level of binned data). 6 of 18 Rinaldi et al., Sci. Adv. 8, eabj8360 (2022) 30 March 2022 SCIE N C E A D V A NCES \| RESEA R CH A RT ICL E N C E A D V A NCES \| RESEA R CH A RT ICL E TSC22D3: Active foci siCTRL siGR siRAD21 siSMC1a ARL4D: Active foci Average number Percentage of transcribing cells C D Nascent RNA foci intensity E B Nascent RNA spots per nucleus Nascent RNA spots per nucleus TSC22D3: RNA intensity ARL4D: RNA intensity TSC22D3: Heterogeneity ARL4D: Heterogeneity ARL4D EtOH Dexamethasone Target mRNA Exon Exon Exon Intron A Probe Sequence specific HCR initiator Sequence specific Amplifier h1 h2 TSC22D3 0 0.25 0.50 0.75 1.00 One Two One Two Spot number/cell Spot number/cell Time after dex (hours) Time after dex (hours) Time after dex (hours) Time after dex (hours) 0.8 0.6 0.4 0.2 0 1 0.75 0.5 0.2 0 active foci/well * * * * ns ns Sum spot intensity per cell Sum spot intensity per cell Proportion of cells/well Proportion of cells/well siCTRL siRAD21 siSMC1a Zero Foci number One Two * 0.25 0.50 0.75 1.00 16,384 32,768 65,536 131,072 262,144 524,288 * * ns * * ns 16,384 32,768 65,536 131,072 262,144 524,288 * * * Cohesin depletion impairs GR gene activation. (A) Schematic illustration of HCR protocol. (B) Representative images of nascent RNA before and after Dex treat- RL4D images are taken after 1 hour of dexamethasone; TSC22D3 images are taken after 4 hours of Dex treatment. Scale bar, 5 m. Single-molecule nascent RNA-FISH reveals that GR associates with NIPBL to promote long-range gene regulation (C) Average number of active RNA foci per well at 0, 1, and 4 hours of dex treatment for ARL4D and TSC22D3, after 72 hours of siRNA against SMC1a or RAD21. Left: TSC22D3; right: TSC22D3 P value is 0.012 for siRAD21 and 0.0005 for siSMC1a, both derived from two-way analysis of variance (ANOVA) test. ARL4D, P < 0.00001 derived from y ANOVA test both for siSMC1a and siRAD21. (D) Nascent RNA intensity of each of the foci in control cells and SMC1a and RAD21 knockdown cells. Left: TSC22D3; RL4D. P < 0.00001 for TSC22D3 and P < 0.001 for ARL4D, derived from two-way ANOVA test for siSMC1a and siRAD21. No statistically significant difference was etween siSMC1a and siRAD21 cells. (E) Percentage of actively transcribing cells measured by HCR before and after dex treatment in control and SMC1 and RAD21 own cells. Left: TSC22D3; right: ARL4D. At 0-hour treatment, almost 100% of cells are not actively transcribing. After 1 or 4 hours of dexamethasone treatment, mately 70% of control cells transcribe nascent RNA of the target genes. In siSMC1a or siRAD21 cells, only 30% of cells actively transcribe nascent RNA for GR-regulated iGR cells, the negative control, show no gene activation after dexamethasone treatment, confirming that nascent RNA foci enrichment is GR dependent. Single-molecule nascent RNA-FISH reveals that GR associates with NIPBL to promote long-range gene regulation Downloaded from https://www.science.org on April 11, 2022 B ARL4D EtOH Dexamethasone Target mRNA Exon Exon Exon Intron A Probe Sequence specific HCR initiator Sequence specific Amplifier h1 h2 TSC22D3 B Target mRNA Exon Exon Exon Intron A Probe Sequence specific HCR initiator Sequence specific Amplifier h1 h2 A B EtOH TSC22D3: Active foci siCTRL siGR siRAD21 siSMC1a ARL4D: Active foci Average number C Nascent RNA spots per nucleus Nascent RNA spots per nucleus ne 0 0.25 0.50 0.75 1.00 Time after dex (hours) Time after dex (hours) active foci/well * 0.25 0.50 0.75 1.00 * * * TSC22D3: Active foci s s s s Average number Nascent RNA spots per nucleus 0 0.25 0.50 0.75 1.00 Time after dex (hours) active foci/well * * C a ARL4D: Active foci Nascent RNA spots per nucleus Time after dex (hours) * 0.25 0.50 0.75 1.00 * Average number active foci/well Percentage of transcribing cells D Nascent RNA foci intensity E TSC22D3: RNA intensity ARL4D: RNA intensity TSC22D3: Heterogeneity ARL4D: Heterogeneity One Two One Two Spot number/cell Spot number/cell Time after dex (hours) Time after dex (hours) 0.8 0.6 0.4 0.2 0 1 0.75 0.5 0.2 0 * * * * ns ns Sum spot intensity per cell Sum spot intensity per cell Proportion of cells/well Proportion of cells/well siCTRL siRAD21 siSMC1a Zero Foci number One Two 16,384 32,768 65,536 131,072 262,144 524,288 * * ns * * ns 16,384 32,768 65,536 131,072 262,144 524,288 ohesin depletion impairs GR gene activation. (A) Schematic illustration of HCR protocol. (B) Representative images of nascent RNA before and after Dex treat- L4D images are taken after 1 hour of dexamethasone; TSC22D3 images are taken after 4 hours of Dex treatment. Scale bar, 5 m. (C) Average number of active RNA foci per well at 0, 1, and 4 hours of dex treatment for ARL4D and TSC22D3, after 72 hours of siRNA against SMC1a or RAD21. Left: TSC22D3; right: SC22D3 P value is 0.012 for siRAD21 and 0.0005 for siSMC1a, both derived from two-way analysis of variance (ANOVA) test. ARL4D, P < 0.00001 derived from ANOVA test both for siSMC1a and siRAD21. (D) Nascent RNA intensity of each of the foci in control cells and SMC1a and RAD21 knockdown cells. Left: TSC22D3; L4D. P < 0.00001 for TSC22D3 and P < 0.001 for ARL4D, derived from two-way ANOVA test for siSMC1a and siRAD21. Single-molecule nascent RNA-FISH reveals that GR associates with NIPBL to promote long-range gene regulation No statistically significant difference was tween siSMC1a and siRAD21 cells. (E) Percentage of actively transcribing cells measured by HCR before and after dex treatment in control and SMC1 and RAD21 wn cells. Left: TSC22D3; right: ARL4D. At 0-hour treatment, almost 100% of cells are not actively transcribing. After 1 or 4 hours of dexamethasone treatment, ately 70% of control cells transcribe nascent RNA of the target genes. In siSMC1a or siRAD21 cells, only 30% of cells actively transcribe nascent RNA for GR-regulated GR cells, the negative control, show no gene activation after dexamethasone treatment, confirming that nascent RNA foci enrichment is GR dependent. SCIE N C E A D V A NCES \| RESEA R CH A RT ICL E A B Auxin = IAA GR ChIP-seq GR ChIP-seq Dex Dex 1 hour 1 hour Untreated 24-hour IAA Untreated 24-hour IAA + dex 4-hour IAA + dex Untreated + dex GR replicate1 H3K27ac HCT116 RAD21mAID A Auxin = IAA GR ChIP-seq GR ChIP-seq Dex Dex 1 hour 1 hour HCT116 RAD21mAID B A Nascent RNA of dex-regulated genes before and after dex and auxin treatment D Fold induction (dex over NT) Nascent RNA of dex-regulated genes before and after dex and auxin treatment +24 hours GR − + + + − − +4 hours Dex IAA 0 1.1 2.3 3.4 4.6 C D D C Rinaldi et al Sci Adv 8 eabj8360 (2022) 30 March 2022 8 of 18 − IAA +24 hours YY-1 0 15.4 E +4 hours 0 16.8 ATAC-seq − +4 hours +24 hours IAA H3K27ac − +4 hours +24 hours IAA +6 hours C1 C2 C3 F G C1 C2 C3 Fig. 5. Cohesin acute depletion impairs GR binding to chromatin. (A) Schematic representation of ChIP-seq experiments after double treatment of dexamethasone and auxin of the HCT116 RAD21mAID cells. (B) Genome browser screenshot of representative GR-bound chromatin location in HCT116 RAD21mAID cells. Two biological replicates are combined for each sample. (C) Heatmap representing GR ChIP-seq intensity at GR chromatin–bound locations (n = 371) identified in HCT116 RAD21mAID cells. Data combine two independent biological replicates. (D) Nascent RNA quantification before and after dexamethasone treatment before and after 6 hours of auxin treatment in RAD21mAID cells. Log10 fold inductions (dexamethasone/EtOH) are normalized on GAPDH mRNA. Five biological replicates are shown for each column. Columns depict mean with error bars representing SD between experiments. P values derived from unpaired Mann-Whitney-Wilcoxon test. (E to G) YY1, H3K27ac ChIP- seq, and ATAC-seq in RAD21mAID cells, before and after auxin treatment. No substantial changes are found in YY1 chromatin binding and chromatin accessibility after acute depletion of cohesin subunit RAD21. Data combine two independent biological replicates each. All heatmaps and genomic data are normalized to a total of 10 million reads and further to local tag density. Homer and bedtools algorithms were used to identify unique and common sites between each experimental condition. Single-molecule nascent RNA-FISH reveals that GR associates with NIPBL to promote long-range gene regulation TSC22D3 P value is 0.012 for siRAD21 and 0.0005 for siSMC1a, both derived from two-way analysis of variance (ANOVA) test. ARL4D, P < 0.00001 derived from two-way ANOVA test both for siSMC1a and siRAD21. (D) Nascent RNA intensity of each of the foci in control cells and SMC1a and RAD21 knockdown cells. Left: TSC22D3; right: ARL4D. P < 0.00001 for TSC22D3 and P < 0.001 for ARL4D, derived from two-way ANOVA test for siSMC1a and siRAD21. No statistically significant difference was found between siSMC1a and siRAD21 cells. (E) Percentage of actively transcribing cells measured by HCR before and after dex treatment in control and SMC1 and RAD21 knockdown cells. Left: TSC22D3; right: ARL4D. At 0-hour treatment, almost 100% of cells are not actively transcribing. After 1 or 4 hours of dexamethasone treatment, approximately 70% of control cells transcribe nascent RNA of the target genes. In siSMC1a or siRAD21 cells, only 30% of cells actively transcribe nascent RNA for GR-regulated genes. siGR cells, the negative control, show no gene activation after dexamethasone treatment, confirming that nascent RNA foci enrichment is GR dependent. 7 of 18 Rinaldi et al., Sci. Adv. 8, eabj8360 (2022) 30 March 2022 SCIE N C E A D V A NCES \| RESEA R CH A RT ICL E +24 hours GR − + + + − − +4 hours Dex IAA 0 1.1 2.3 3.4 4.6 − IAA +24 hours YY-1 0 15.4 D A B E Auxin = IAA GR ChIP-seq GR ChIP-seq Dex Dex 1 hour 1 hour Fold induction (dex over NT) Untreated 24-hour IAA Untreated 24-hour IAA + dex 4-hour IAA + dex Untreated + dex GR replicate1 H3K27ac +4 hours C HCT116 RAD21mAID 0 16.8 ATAC-seq − +4 hours +24 hours IAA H3K27ac − +4 hours +24 hours IAA +6 hours C1 C2 C3 F G C1 C2 C3 Nascent RNA of dex-regulated genes before and after dex and auxin treatment Fig. 5. Cohesin acute depletion impairs GR binding to chromatin. (A) Schematic representation of ChIP-seq experiments after double treatment of dexamethasone and auxin of the HCT116 RAD21mAID cells. (B) Genome browser screenshot of representative GR-bound chromatin location in HCT116 RAD21mAID cells. Two biological replicates are combined for each sample. (C) Heatmap representing GR ChIP-seq intensity at GR chromatin–bound locations (n = 371) identified in HCT116 RAD21mAID cells. Data combine two independent biological replicates. Single-molecule nascent RNA-FISH reveals that GR associates with NIPBL to promote long-range gene regulation (D) Nascent RNA quantification before and after dexamethasone treatment before and after 6 hours of auxin treatment in RAD21mAID cells. Log10 fold inductions (dexamethasone/EtOH) are normalized on GAPDH mRNA. Five biological replicates are shown for each column. Columns depict mean with error bars representing SD between experiments. P values derived from unpaired Mann-Whitney-Wilcoxon test. (E to G) YY1, H3K27ac ChIP- seq, and ATAC-seq in RAD21mAID cells, before and after auxin treatment. No substantial changes are found in YY1 chromatin binding and chromatin accessibility after acute depletion of cohesin subunit RAD21. Data combine two independent biological replicates each. All heatmaps and genomic data are normalized to a total of Downloaded from https://www.science.org on April 11, 2022 SCIE N C E A D V A NCES \| RESEA R CH A RT ICL E SCIE N C E A D V A NCES \| RESEA R CH A RT ICL E As predicted, the TNF-–induced gene response was altered in the auxin-treated cells, supporting our hypothesis that the induc- ible TF NF-kB requires cohesin to properly induce gene expression (Fig. 6, A to D). Most of the TNF- up-regulated genes were differ- entially regulated in both untreated and auxin-treated cells (Fig. 6B), while more than 50% of the TNF- down-regulated genes were not found in the RAD21-depleted cells. This suggests an important role for cohesin to regulate gene repression. The log-fold induction of all genes regulated by TNF treatment stimulation was impaired after RAD21 depletion (Fig. 6, C and D), suggesting an essential role for the cohesin complex to fine-tune gene activation and gene repres- sion. Supporting our findings, previous elegant studies performed in mouse macrophages treated with bacterial lipopolysaccharides (LPSs) showed how the cohesin complex regulates gene expression during hematopoietic stem cell differentiation and self-renewal (23). On the contrary, the loss of CTCF did not alter the gene expression mediated by LPS treatment in human macrophages (35), suggesting that cohesin regulates gene expression, independently of the inter- action with the CTCF at TAD boundaries. y g Acute loss of RAD21 decreased both the chromatin binding and the nuclear confinement fractions (Fig. 7, E and F). However, the fraction of confined molecules was altered to a much greater extent (Fig. 7F). Loss of cohesin diminishes more than 65% of the nuclear confined molecules of GR (Fig. 7F). Nuclear confined regions have been proposed to arise from highly interacting DNA loops, such as TADs, where a high concentration of TFs promotes dynamic tran- scriptional events and chromatin intermingling (3, 39). By the for- mation of these confined regions through their IDRs, TFs could amplify transcriptional output, perhaps by increasing the local con- centration of transcriptional regulators at specific chromatin sites (36). The real-time microscopy results argue strongly that the cohesin complex is functionally implicated in confinement of TFs (Fig. 7G). These findings imply a novel synergism between TF-mediated long- range interactions, nuclear confined regions, and possibly loop extrusion. GC treatment is altered by mutations in the cohesin complex Cohesin subunits are often mutated in several types of cancer. In some forms of acute myeloid leukemia (AML), the cumulative level of mutations in RAD21, SMC1a, SMC3, and STAG2 genes reaches 13% (40). This percentage is even higher in Down syndrome–associated AML (DS-AML), where more than 50% of patients harbor cohesin mutations (40). SCIE N C E A D V A NCES \| RESEA R CH A RT ICL E To evaluate whether cancer mutations in the cohesin genes can alter the response to GCs, we examined the dexamethasone response with AML patient–derived samples. Kasumi cells and CMK-CMY cells are derived from DS-AML–affected patients (Fig. 8A) (41). In both sample sets, the cohesin mutated cells had a much lower response to GC treatment, supporting the model that cohesin modu- lates GR activity and the GC therapeutic response (Fig. 8, B and C). GCs, such as betamethasone and dexamethasone, are beneficial in leukemia treatment when combined with chemotherapy agents (42, 43), likely due to their anti-inflammatory properties. However, our data suggest that the efficacy of the GC treatment may depend on the integrity of the cohesin complex. When the structure of these proteins is altered because of mutations, GC treatment may be in- effective or even deleterious. SCIE N C E A D V A NCES \| RESEA R CH A RT ICL E Downloaded from https://www.science.org on April 11, 2022 Downloaded from https://www.science.org on April 11, 2022 H3K27ac − +4 hours +24 hours IAA +6 hours G C1 C2 C3 − IAA +24 hours YY-1 0 15.4 E +4 hours 0 16.8 ATAC-seq − +4 hours +24 hours IAA C1 C2 C3 F G F E E Fig. 5. Cohesin acute depletion impairs GR binding to chromatin. (A) Schematic representation of ChIP-seq experiments after double treatment of dexamethasone and auxin of the HCT116 RAD21mAID cells. (B) Genome browser screenshot of representative GR-bound chromatin location in HCT116 RAD21mAID cells. Two biological replicates are combined for each sample. (C) Heatmap representing GR ChIP-seq intensity at GR chromatin–bound locations (n = 371) identified in HCT116 RAD21mAID cells. Data combine two independent biological replicates. (D) Nascent RNA quantification before and after dexamethasone treatment before and after 6 hours of auxin treatment in RAD21mAID cells. Log10 fold inductions (dexamethasone/EtOH) are normalized on GAPDH mRNA. Five biological replicates are shown for each column. Columns depict mean with error bars representing SD between experiments. P values derived from unpaired Mann-Whitney-Wilcoxon test. (E to G) YY1, H3K27ac ChIP- seq, and ATAC-seq in RAD21mAID cells, before and after auxin treatment. No substantial changes are found in YY1 chromatin binding and chromatin accessibility after acute depletion of cohesin subunit RAD21. Data combine two independent biological replicates each. All heatmaps and genomic data are normalized to a total of 10 million reads and further to local tag density. Homer and bedtools algorithms were used to identify unique and common sites between each experimental condition. Rinaldi et al., Sci. Adv. 8, eabj8360 (2022) 30 March 2022 8 of 18 SCIE N C E A D V A NCES \| RESEA R CH A RT ICL E and fig. S7, D and E). The chromatin-bound fraction is determined by the DNA binding properties of the TFs, whereas the confined fraction depends on protein-protein interactions through the TF’s intrinsically disordered region (IDR) (36). cells showed that the acute loss of the cohesin complex results in genome-wide loss of chromatin loops (6). However, 6 hours of acute RAD21 depletion did not significantly alter the steady-state transcriptomic profiles. Here, we investigated whether the acute loss of RAD21 was altering transcriptional events driven by induc- ible TFs. Loss of cohesin markedly diminishes nuclear confinement of TFs TFs cooperate with the cohesin complex to modulate long-range gene regulation and nuclear topology. In addition, several studies have investigated the relationship between spatial genome organization and TF biophysics and dynamics (2, 12, 36). To understand the im- pact of cohesin on GR dynamics, we used single-molecule tracking (SMT), a powerful superresolution microscopy method to charac- terize transcriptional dynamics and identify diffusive and chromatin- bound fractions (fig. S7, A to C) (37). We performed SMT experiments using the RAD21mAID cell line transfected with Halo-tagged wild- type GR (GRwt-Halo; fig. S7F) visualized by the JF549 fluorophore after activation with dexamethasone (dex). For optimal balance between fast acquisition, highest signal-to-noise ratio, and minimal localization noise, we acquired the image with two different setups: the “fast acquisition,” where the samples were imaged continuously using 12-ms exposure times, and the “slow acquisition,” where im- ages were taken every 200 ms with an exposure of 10 ms. This dual acquisition of the data allows us to precisely quantify the dynamic of GR molecules from diffusing to slower events such as chromatin binding (36). The trajectories of localized particles from a represent ative cell are shown in Fig. 7A. www.science.org on April 11, 2022 Rinaldi et al., Sci. Adv. 8, eabj8360 (2022) 30 March 2022 SCIE N C E A D V A NCES \| RESEA R CH A RT ICL E III) RNA-seq uster CL1 CL2 CL3 CL4 CL5 CL6 CL7 D C −+ WT IAA6h IAA12h TNFa −+ −+ Attenuated TNFa response in IAA Cluster −2 −1 0 1 2 GH3979 GH3978 GH3975 GH3974 GH3973 GH3972 GH3977 GH3976 GH3971 GH3970 GH3969 GH3968 Cluster No TNFa TNFa WT IAA6h IAA12h WT IAA6h IAA12h CL1 CL2 CL3 CL4 CL5 CL6 CL7 14 25 14 76 46 57 83 D C −+ WT IAA6h IAA12h TNFa −+ −+ Attenuated TNFa response in IAA Cluster −2 −1 0 1 2 GH3979 GH3978 GH3975 GH3974 GH3973 GH3972 GH3977 GH3976 GH3971 GH3970 GH3969 GH3968 Cluster No TNFa TNFa WT IAA6h IAA12h WT IAA6h IAA12h CL1 CL2 CL3 CL4 CL5 CL6 CL7 14 25 14 76 46 57 83 D C −+ WT IAA6h IAA12h TNFa −+ −+ Attenuated TNFa response in IAA GH3979 GH3978 GH3975 GH3974 GH3973 GH3972 GH3977 GH3976 GH3971 GH3970 GH3969 GH3968 Cluster No TNFa TNFa WT IAA6h IAA12h WT IAA6h IAA12h 14 25 14 76 46 57 83 C Downloaded from https://www.science.org on April 11, 2022 Fig. 6. NF-kB response to TNF- treatment is weakened after cohesin loss, suggesting that inducible TFs promote gene expression through the cohesin com- plex. (A) Schematic representation of RNA-seq experiments after double treatment of dexamethasone and auxin of the HCT116 RAD21mAID cells (n = 2 independent biological replicates). (B) Venn diagram showing up-regulated genes (left) and down-regulated genes (right) after 2 hours of TNF treatment in untreated cells, 6-hour IAA-treated cells, and 26-hour IAA-treated HCT116 RAD21mAID cells (II versus I, IV versus III, and VI versus V). Differentially expressed genes (DEGs) were obtained by DESeq2 using threshold of adjusted P < 0.05 and fold change of 1.5. (C) Right: Heatmap summarizing 315 DEGs that are the union of (B). Left: Biological duplicates are shown as two individual bars under each condition. The y axis of each bar represents the average of z-scored values of variance stabilizing transformed gene expression values in each cluster. Red arrows indicate the response to TNFa treatment under WT, IAA6h, and IAA24h based on average expression profiles. CL1 and CL2 show the attenuated suppression response by TNFa under IAA treatment. CL3, CL4, CL5, and CL6 show the attenuated induction response (with various degrees) by TNFa under IAA treatment. CL7 contains genes less affected by IAA. (D) Log2 fold changes estimated from DESeq2 of TNF-–regulated genes (n = 386) in (B). SCIE N C E A D V A NCES \| RESEA R CH A RT ICL E SCIE N C E A D V A NCES \| RESEA R CH A RT ICL E A B D C Auxin = IAA Ethanol RNA-seq 2 hours TNFα 4 hours Auxin = IAA 24 hours Ethanol RNA-seq RNA-seq 2 hours TNFα Ethanol RNA-seq RNA-seq 2 hours TNFα Ethanol RNA-seq (I) (II) (III) (IV) (V) (VI) (IV vs. III) IAA6h + TNF IAA26h + TNF Up-regulated genes after TNF treatment (VI vs. V) NT + TNF (II vs. I) (VI vs. V) Down-regulated genes after TNF treatment NT + TNF IAA6h + TNF IAA26h + TNF (II vs. I) (IV vs. III) −+ WT IAA6h IAA12h TNFa −+ −+ Attenuated TNFa response in IAA Cluster −2 −1 0 1 2 GH3979 GH3978 GH3975 GH3974 GH3973 GH3972 GH3977 GH3976 GH3971 GH3970 GH3969 GH3968 Cluster No TNFa TNFa WT IAA6h IAA12h WT IAA6h IAA12h CL1 CL2 CL3 CL4 CL5 CL6 CL7 14 25 14 76 46 57 83 Fig. 6. NF-kB response to TNF- treatment is weakened after cohesin loss, suggesting that inducible TFs promote gene expression through the cohesin com- plex. (A) Schematic representation of RNA-seq experiments after double treatment of dexamethasone and auxin of the HCT116 RAD21mAID cells (n = 2 independent biological replicates). (B) Venn diagram showing up-regulated genes (left) and down-regulated genes (right) after 2 hours of TNF treatment in untreated cells, 6-hour IAA-treated cells, and 26-hour IAA-treated HCT116 RAD21mAID cells (II versus I, IV versus III, and VI versus V). Differentially expressed genes (DEGs) were obtained by DESeq2 using threshold of adjusted P < 0.05 and fold change of 1.5. (C) Right: Heatmap summarizing 315 DEGs that are the union of (B). Left: Biological duplicates are shown as two individual bars under each condition. The y axis of each bar represents the average of z-scored values of variance stabilizing transformed gene expression values in each cluster. Red arrows indicate the response to TNFa treatment under WT, IAA6h, and IAA24h based on average expression profiles. CL1 and CL2 show the attenuated suppression response by TNFa under IAA treatment. CL3, CL4, CL5, and CL6 show the attenuated induction response (with various degrees) by TNFa under IAA treatment. CL7 contains genes less affected by IAA. (D) Log2 fold changes estimated from DESeq2 of TNF-–regulated genes (n = 386) in (B). SCIE N C E A D V A NCES \| RESEA R CH A RT ICL E Genes are sorted on the basis of the fold change values by TNF treatment in IAA untreated cells to compare the trend of attenuated TNF treatment response in IAA treated cells Downloaded from https://www.science.org on Ap A B Auxin = IAA Ethanol RNA-seq 2 hours TNFα 4 hours Auxin = IAA 24 hours Ethanol RNA-seq RNA-seq 2 hours TNFα Ethanol RNA-seq RNA-seq 2 hours TNFα Ethanol RNA-seq (I) (II) (III) (IV) (V) (VI) (IV vs. III) IAA6h + TNF IAA26h + TNF Up-regulated genes after TNF treatment (VI vs. V) NT + TNF (II vs. I) (VI vs. V) Down-regulated genes after TNF treatment NT + TNF IAA6h + TNF IAA26h + TNF (II vs. I) (IV vs. III) B (IV vs. III) IAA6h + TNF IAA26h + TNF Up-regulated genes after TNF treatment (VI vs. V) NT + TNF (II vs. I) (VI vs. V) Down-regulated genes after TNF treatment NT + TNF IAA6h + TNF IAA26h + TNF (II vs. I) (IV vs. III) B (IV vs. III) IAA6h + TNF IAA26h + TNF Up-regulated genes after TNF treat (VI vs. V) NT ( A B Auxin = IAA Ethanol RNA-seq 2 hours TNFα 4 hours Auxin = IAA 24 hours Ethanol RNA-seq RNA-seq 2 hours TNFα Ethanol RNA-seq RNA-seq 2 hours TNFα Ethanol RNA-seq (I) (II) (III) (IV) (V) (VI) A B (VI vs. V) Down-regulated genes after TNF treatment NT + TNF IAA6h + TNF IAA26h + TNF (II vs. I) (IV vs. DISCUSSION Long-range interactions govern genome organization and function, profoundly altering cellular phenotypes (1). TFs modulate chromatin interactions connecting enhancer and promoter to properly modu- late cell fate (2). Therefore, a clear correlation has been shown between the number and/or strength of chromatin loops and the modulation of transcriptional events (6, 9, 13). Several studies have investigated this from different perspectives. Phase separation, transcriptional hubs, and TAD heterogeneity are all causes/consequences of the tight relationship between looping and transcriptional machineries (44–47). The cohesin ring-shaped structure is central to nuclear architecture and is stably bound to chromatin, but its assembly is promoted dynamically by the cohesin loader NIPBL at chromatin accessible sites (10, 11, 13, 14, 48). As NIPBL does not exhibit sequence-specific DNA binding, the mechanism of its recruitment to chromatin remains elusive. TFs, such as steroid receptors, bind in- accessible chromatin, remodel nucleosomes, and create chromatin In agreement with our genomic data, RAD21-depleted cells (using the auxin degron cells) exhibited a strong reduction of GR chromatin interaction compared to the control cells (Fig. 7B). More precisely, the fraction of diffusive (nonbound) GR molecules was increased by 180% after the loss of RAD21 (Fig. 7B). Next, we applied an analysis based on unsupervised machine learning and Bayesian inference criteria (BIC) (36, 38) to classify the molecular trajectories based on their diffusive properties. Using this approach, we observed two categories of diffusive and two categories of bound molecules. We previously reported that the bound category with the most limited movement represents chromatin binding of TFs (green), while the other is associated with nuclear spatial confinement (red) (Fig. 7, C and D, Rinaldi et al., Sci. Adv. 8, eabj8360 (2022) 30 March 2022 9 of 18 SCIE N C E A D V A NCES \| RESEA R CH A RT ICL E h ll i f i l H2B H l d GR H l j d i h JF Ri h l h h ll i f j i f l i l X position E 35% 65% 64% 36% Unbound Bound GR - NT GR - 24-hour IAA B D 0 0.005 0.01 0.015 0.02 0.025 0.03 0.2 0.4 0.6 1 0.8 1.2 Time lag (s) Conﬁnement Chromatin binding GR - NT GR - 24-hour IAA D 0 0.1 0.2 0.3 0.4 0.5 0.6 0.01 0.02 0.03 0.05 0.04 0.06 Time lag (s) 0.07 Diffusive state 1 Diffusive state 2 Low-mobility states 0 0.005 0.01 0.015 0.02 0.025 0.03 0.2 0.4 0.6 1 0.8 1.2 Time lag (s) Conﬁnement Chromatin binding GR - NT GR - 24-hour IAA 35% 65% 64% 36% Unbound Bound GR - NT GR - 24-hour IAA B C D 0 0.1 0.2 0.3 0.4 0.5 0.6 0.01 0.02 0.03 0.05 0.04 0.06 Time lag (s) 0.07 Diffusive state 1 Diffusive state 2 Low-mobility states C B C F Chromatin binding Unbound Conﬁnement 35% 36% 29% GR - 24-hour IAA 64% 26% 10% GR - NT GR Cohes CTCF NIPBL G GR 24-hour IAA GR - NT Power-law fit 10 Time (s) Shorter Longer F Chromatin binding Unbound Conﬁnement 35% 36% 29% GR - 24-hour IAA 64% 26% 10% GR - NT GR Cohesin CTCF NIPBL G er F Chromatin binding Unbound Conﬁnement 35% 36% 29% GR - 24-hour IAA 64% 26% 10% GR - NT E F Chromatin binding Unbound Conﬁnement 35% 36% 29% GR - 24-hour IAA 64% 26% 10% GR - NT GR 24-hour IAA GR - NT Power-law fit 1 10 Time (s) Survival distribution 1 0.1 0.01 Shorter Longer E F 2 GR 24-hour IAA GR - NT Power-law fit 1 10 Time (s) Survival distribution 1 0.1 0.01 Shorter Longer GR Cohesin CTCF NIPBL G F Downloaded from https://www.science.org on April 11, 2022 E G Fig. 7. Cohesin mediates TF dynamics and nuclear confinement. (A) Time projection of single-particle images from sample SMT experiments after tracking. Left two panels represent the collection of single H2B-Halo and GR-Halo conjugated with JF549. Right panels show the collection of trajectories after superresolution localization and tracking. (B) Bound versus unbound proportions for GR-Halo before and 24 hours after auxin. To rule out the effects of aberrant mitosis, we imaged only rounded and intact nuclei. SCIE N C E A D V A NCES \| RESEA R CH A RT ICL E Genes are sorted on the basis of the fold change values by TNF- treatment in IAA-untreated cells to compare the trend of attenuated TNF- treatment response in IAA-treated cells. accessible sites (26, 49). Other studies have shown that steroid hor- mone receptor activation increases the frequencies of already pre- established loops, and the cohesin complex is important to modulate gene expression driven by steroid receptors (8, 21, 22, 50). Here, we demonstrate that site-specific chromatin binding of the NIPBL- cohesin complex occurs at the chromatin accessible sites driven by the activation of TFs such as nuclear receptors (Fig. 1, E and F), promoting chromatin interactions, loop extrusion, and long-range gene regulation. complex, stabilizing cohesin binding at GR-responsive sites, pro- moting loop extrusion, and strengthening preestablished chroma- tin loops (Fig. 8D) (21, 22, 24, 50). Our work investigated mostly the relationship between the GR and the cohesin loader NIPBL; however, given the interplay between transcriptional regulators and the co- hesin complex to regulate genome organization (2, 8, 16, 17, 21), other TFs may follow the same pattern. Nevertheless, some TFs may modulate long-range gene regulation through other mechanisms. Other studies, however, have shown that multiple chromatin regu- lators, including mediator subunits, chromatin remodelers, and CBP/P300, are associated with the cohesin complex at cohesin- enriched locations, suggesting a multifactorial model where many factors cooperate to regulate long-range gene regulation. However, g g Subsequently, the cohesin complex extrudes DNA in an ATP- dependent manner toward the nearest TAD boundary (Figs. 1H, 3, F and G, and 8D) (13). In this context, our results demonstrate that GR (and most likely other TFs) associates with the NIPBL-cohesin Rinaldi et al., Sci. Adv. SCIE N C E A D V A NCES \| RESEA R CH A RT ICL E 8, eabj8360 (2022) 30 March 2022 10 of 18 N C E A D V A NCES \| RESEA R CH A RT ICL E A 3.0 µm X position Particle tracks H2B 3.0 µm Particle tracks GR X position Single-particle H2B X position Y position 3.0 µm 0 0.1 0.2 0.3 0.4 0.5 0.6 0.01 0.02 0.03 0.05 0.04 0.06 Time lag (s) 0.07 E Diffusive state 1 Diffusive state 2 Low-mobility states 0 0.005 0.01 0.015 0.02 0.025 0.03 0.2 0.4 0.6 1 0.8 1.2 Time lag (s) Conﬁnement Chromatin binding GR - NT GR - 24-hour IAA F 35% 65% 64% 36% Unbound Bound GR - NT GR - 24-hour IAA Chromatin binding Unbound Conﬁnement 35% 36% 29% GR - 24-hour IAA 64% 26% 10% GR - NT B C D GR Cohesin CTCF NIPBL G GR 24-hour IAA GR - NT Power-law fit 1 10 Time (s) Survival distribution 1 0.1 0.01 Shorter Longer 3.0 µm X position Single-particle GR ohesin mediates TF dynamics and nuclear confinement. (A) Time projection of single-particle images from sample SMT experiments after tracking. Left two epresent the collection of single H2B-Halo and GR-Halo conjugated with JF549. Right panels show the collection of trajectories after superresolution localization king. (B) Bound versus unbound proportions for GR-Halo before and 24 hours after auxin. To rule out the effects of aberrant mitosis, we imaged only rounded and uclei. (C) Mean-squared displacement (MSD) versus lag time for the different diffusivity states detected by pEM for GR-Halo-JF549. (D) MSD versus lag time for the nd states of GR-NT (solid line) and GR-IAA after 24 hours of auxin treatment (dashed line). Individual colored tracks in (C) and (D) represent the spatial distribution dynamic state. (E) Dwell time distribution for the chromatin-bound GR-Halo treated with 100 nM dexamethasone, before (blue dots) and after 24 hours of auxin nt (red dots). The binding time to chromatin of GR decreases after cohesin depletion (paired KS test, P value of 7 × 10−140). (F) Unbound, chromatin-bound, and ment fractions of GR-Halo before (top) and after 24 hours of auxin (bottom). The confinement fraction decreases markedly from 29 to 10%, and the chromatin fraction decreases from 36 to 26%. Cohesin depletion has a profound effect on the confinement of GR, already noticeable in (D). (G) Nuclear confined regions ng-range chromatin contacts and high concentration of transcriptional regulators. SCIE N C E A D V A NCES \| RESEA R CH A RT ICL E Loss of cohesin results in weaker TF-chromatin interactions at nuclear confined Downloaded from https://www.science.org on April 11, 2022 SCIE N C E A D V A NCES \| RESEA R CH A RT ICL E i et al., Sci. Adv. 8, eabj8360 (2022) 30 March 2022 E N C E A D V A NCES \| RESEA R CH A RT ICL E 11 o A 3.0 µm X position Particle tracks H2B 3.0 µm Particle tracks GR X position Single-particle H2B X position Y position 3.0 µm 0 0.1 0.2 0.3 0.4 0.5 0.6 0.01 0.02 0.03 0.05 0.04 0.06 Time lag (s) 0.07 E Diffusive state 1 Diffusive state 2 Low-mobility states 0 0.005 0.01 0.015 0.02 0.025 0.03 0.2 0.4 0.6 1 0.8 1.2 Time lag (s) Conﬁnement Chromatin binding GR - NT GR - 24-hour IAA F 35% 65% 64% 36% Unbound Bound GR - NT GR - 24-hour IAA Chromatin binding Unbound Conﬁnement 35% 36% 29% GR - 24-hour IAA 64% 26% 10% GR - NT B C D GR Cohesin CTCF NIPBL G GR 24-hour IAA GR - NT Power-law fit 1 10 Time (s) Survival distribution 1 0.1 0.01 Shorter Longer 3.0 µm X position Single-particle GR Cohesin mediates TF dynamics and nuclear confinement. (A) Time projection of single-particle images from sample SMT experiments after tracking. Left s represent the collection of single H2B-Halo and GR-Halo conjugated with JF549. Right panels show the collection of trajectories after superresolution localiza acking. (B) Bound versus unbound proportions for GR-Halo before and 24 hours after auxin. To rule out the effects of aberrant mitosis, we imaged only rounded nuclei. (C) Mean-squared displacement (MSD) versus lag time for the different diffusivity states detected by pEM for GR-Halo-JF549. (D) MSD versus lag time fo ound states of GR-NT (solid line) and GR-IAA after 24 hours of auxin treatment (dashed line). Individual colored tracks in (C) and (D) represent the spatial distribu ch dynamic state. (E) Dwell time distribution for the chromatin-bound GR-Halo treated with 100 nM dexamethasone, before (blue dots) and after 24 hours of a ment (red dots). The binding time to chromatin of GR decreases after cohesin depletion (paired KS test, P value of 7 × 10−140). (F) Unbound, chromatin-bound ement fractions of GR-Halo before (top) and after 24 hours of auxin (bottom). SCIE N C E A D V A NCES \| RESEA R CH A RT ICL E The confinement fraction decreases markedly from 29 to 10%, and the chrom g fraction decreases from 36 to 26%. Cohesin depletion has a profound effect on the confinement of GR, already noticeable in (D). (G) Nuclear confined reg ong-range chromatin contacts and high concentration of transcriptional regulators. Loss of cohesin results in weaker TF-chromatin interactions at nuclear conf s. A Single-particle H2B X position Y position 3.0 µm 3.0 µm X position Single-particle GR 3.0 µm X position Particle tracks H2B 3.0 µm Particle tracks GR X position 3.0 µm X position Particle tracks H2B 3.0 µm Particle tracks GR X position 3.0 µm X position 3.0 µm X position X position 3.0 µm 0 0.1 0.2 0.3 0.4 0.5 0.6 0.01 0.02 0.03 0.05 0.04 0.06 Time lag (s) 0.07 E Diffusive state 1 Diffusive state 2 Low-mobility states 0 0.005 0.01 0.015 0.02 0.025 0.03 0.2 0.4 0.6 1 0.8 1.2 Time lag (s) Conﬁnement Chromatin binding GR - NT GR - 24-hour IAA F 35% 65% 64% 36% Unbound Bound GR - NT GR - 24-hour IAA Chromatin binding Unbound Conﬁnement 35% 36% 29% GR - 24-hour IAA 64% 26% 10% GR - NT B C D GR Cohesin CTCF NIPBL G GR 24-hour IAA GR - NT Power-law fit 1 10 Time (s) Survival distribution 1 0.1 0.01 Shorter Longer 3.0 µm X position ohesin mediates TF dynamics and nuclear confinement. (A) Time projection of single-particle images from sample SMT experiments after tracking. SCIE N C E A D V A NCES \| RESEA R CH A RT ICL E Patient derived Cancer type Cohesin mutated Mutation type Hotspot CMK DS-derived AMKL / – CMY DS-derived AMKL RAD21 Nonsense c.9C>G Kasumi-1 AML-M2 RAD21 Frameshift c.987insCCGG Kasumi-3 AML-M0 / – Kasumi-6 AML-M2 STAG2 Missense Homozygous G>A T>G A B C D GC treatment depends on the cohesin complex. (A) Cohesin mutations in the myeloid leukemia–derived cells used to evaluate GC treatment. (B) Nascent RNA cation measured by qRT-PCR of GR target genes after 2 hours of dexamethasone treatment in AML patient–derived cells: Kasumi-1 (RAD21mut), Kasumi-3 (cohesin d Kasumi-6 (STAG2mut). Log10 fold inductions (dex/EtOH) are normalized on -actin nascent RNA. (C) Nascent RNA quantification measured by qRT-PCR of GR enes after 2 hours of dexamethasone treatment in the DS-AMKL: CMK (cohesin WT) and CMY (RAD21mut). For both (B) and (C), GC treatment is strongly altered nt-derived cells harboring cohesin mutations. Log10 fold inductions (dex/etOH) are normalized on -actin nascent RNA. Boxplots represent minimum to maximum and error bars represent SE (n = 4 independent biological replicates). P values derived from unpaired t test. (D) Model of GR-NIPBL synergy to promote cohesin tin binding, activation of the loop extrusion, and long-range gene regulation. Downloaded from https://www.science.org on April 1 Patient derived Cancer type Cohesin mutated Mutation type Hotspot CMK DS-derived AMKL / – CMY DS-derived AMKL RAD21 Nonsense c.9C>G Kasumi-1 AML-M2 RAD21 Frameshift c.987insCCGG Kasumi-3 AML-M0 / – Kasumi-6 AML-M2 STAG2 Missense Homozygous G>A T>G A B C B D g 8 GC treatment depends on the cohesin complex (A) Cohesin mutations in the myeloid leukemia derived cells used to evaluate GC treatment (B) Nascent RNA Downloaded from https://www D D A R Downloaded from https://www.science.org on April 11, 2022 Fig. 8. GC treatment depends on the cohesin complex. (A) Cohesin mutations in the myeloid leukemia–derived cells used to evaluate GC treatment. (B) Nascent RNA quantification measured by qRT-PCR of GR target genes after 2 hours of dexamethasone treatment in AML patient–derived cells: Kasumi-1 (RAD21mut), Kasumi-3 (cohesin WT), and Kasumi-6 (STAG2mut). Log10 fold inductions (dex/EtOH) are normalized on -actin nascent RNA. (C) Nascent RNA quantification measured by qRT-PCR of GR target genes after 2 hours of dexamethasone treatment in the DS-AMKL: CMK (cohesin WT) and CMY (RAD21mut). For both (B) and (C), GC treatment is strongly altered in patient-derived cells harboring cohesin mutations. Log10 fold inductions (dex/etOH) are normalized on -actin nascent RNA. SCIE N C E A D V A NCES \| RESEA R CH A RT ICL E (C) Mean-squared displacement (MSD) versus lag time for the different diffusivity states detected by pEM for GR-Halo-JF549. (D) MSD versus lag time for the two bound states of GR-NT (solid line) and GR-IAA after 24 hours of auxin treatment (dashed line). Individual colored tracks in (C) and (D) represent the spatial distribution for each dynamic state. (E) Dwell time distribution for the chromatin-bound GR-Halo treated with 100 nM dexamethasone, before (blue dots) and after 24 hours of auxin treatment (red dots). The binding time to chromatin of GR decreases after cohesin depletion (paired KS test, P value of 7 × 10−140). (F) Unbound, chromatin-bound, and confinement fractions of GR-Halo before (top) and after 24 hours of auxin (bottom). The confinement fraction decreases markedly from 29 to 10%, and the chromatin binding fraction decreases from 36 to 26%. Cohesin depletion has a profound effect on the confinement of GR, already noticeable in (D). (G) Nuclear confined regions favor long-range chromatin contacts and high concentration of transcriptional regulators. Loss of cohesin results in weaker TF-chromatin interactions at nuclear confined regions. 11 of 18 Rinaldi et al., Sci. Adv. 8, eabj8360 (2022) 30 March 2022 SCIE N C E A D V A NCES \| RESEA R CH A RT ICL E SCIE N C E A D V A NCES \| RESEA R CH A RT ICL E Therefore, there is a disconnect between the phenotypes observed by NIPBL muta- tions (slow growth) and the phenotypic changes observed with the cohesin core subunits (fast growth). Last, we show that GC stimulation relies on the status of the co- hesin complex to properly regulate gene targets in AML cell lines known for their high incidence of cohesin gene mutations (Fig. 8, A to C). Although GCs are extensively studied and characterized, the treat- ment of respiratory diseases and their potential usefulness in cancer may be marginalized by mutations in cohesin function. NF-kB gene regulation was also hampered by Rad21-depleted cells (23), suggesting that cohesin associates to a much greater panel of TFs (Fig. 6) (16). Therefore, consideration of the mutational landscape of the patient, especially the presence of cohesin mutations, could help in the eval- uation of possible benefits of GC and/or cytokine treatment. Proteomics GR ChIP-SICAP spectral library generation and data-dependent acquisition All proteomic experimental procedures were already described in detail in our previous work (55). In brief, a fraction (20%) of trypsin- digested peptides from each ChIP-SICAP–obtained samples were combined and used to generate the spectral library for the following liquid chromatography–tandem MS (LC-MS/MS) data-independent acquisition (DIA). The combined peptide mixture was fractionated using high-pH reversed-phase chromatography on an Ultimate3000 high-performance liquid chromatography system (Thermo Fisher Scientific) using a 10-cm-long ACQUITY CSH C18 1.7-m column (Waters). The fractionated peptides, together with the input flow- through fraction, were vacuum-dried in a speed-vac, resolubilized in 9 l of 0.5% AA in water, and used for the nanoLC-MS/MS anal- ysis on a Q Exactive HF-X mass spectrometer coupled with an EASY-nLC 1000 ultrahigh-pressure system (Thermo Fisher Scientific). MS data were acquired using a data-dependent acquisition (DDA) method switching between full scan events and the top 12 MS/MS scans. An automatic gain control target value was set to 3 × 106, and resolution was set to 60,000 for full MS scan events with a scan range of 300 to 1700 mass/charge ratio (m/z) and a maximum ion injection time (IT) of 15 ms. Precursors were fragmented by higher- energy collisional dissociation with a normalized collisional energy of 28%. MS/MS scans were acquired with a resolution of 60,000, maximum IT of 110 ms, and 1.2 m/z isolation window. The ob- tained Thermo .raw files were analyzed using MaxQuant software (version 1.5.2.8, Max Planck Institute of Biochemistry, Martinsried, Germany) and a Mus musculus FASTA file downloaded from Uni- Prot (www.uniprot.org/) in January 2019, supplemented with com- monly observed contaminants. The MaxQuant search settings for maximum missed cleavages were set to 2, peptide mass tolerance to 4.5 parts per million (ppm), and fragment ion tolerance to 20 ppm, and trypsin was chosen as enzyme. Variable modifications were specified to include oxidation on methionine and acetylation on protein N-terminus. As fixed modification, carbamidomethylation of cysteine was specified. MaxQuant data were filtered for re- verse identifications, with false discovery rate (FDR) set as 1%. DIA d d t i SCIE N C E A D V A NCES \| RESEA R CH A RT ICL E 8, eabj8360 (2022) 30 March 2022 SCIE N C E A D V A NCES \| RESEA R CH A RT ICL E Bioscience) using a DNA terminal transferase for 1 hour at 37°C (M0315, NEB). Beads were then successively washed with low-salt, high-salt, and LiCl buffer, and the chromatin was eluted for 15 min at 37°C in the SICAP elution buffer [7.5% SDS and 200 mM dithio- threitol (DTT)]. Chromatin was then resuspended in SICAP buffer [0.5% NP-40, 1% Triton X-100, 5 mM EDTA, 50 mM tris-HCl (pH 7.4), and 150 mM NaCl] and incubated with streptavidin beads (NEB) for 1 hour at room temperature. The beads were then exten- sively washed with the following solutions: three times with the SDS buffer [1% SDS, 1 mM EDTA, 10 mM tris-HCl (pH 7.4), and 200 mM NaCl], then once with the BW2x buffer [0.1% Triton X-100, 1 mM EDTA, 10 mM tris-HCl (pH 7.4), and 2 M NaCl], then twice with 20% isopropanol, and, lastly, four times with 40% acetonitrile. Dry beads were then frozen at −80°C and sent for MS analysis. Bioscience) using a DNA terminal transferase for 1 hour at 37°C (M0315, NEB). Beads were then successively washed with low-salt, high-salt, and LiCl buffer, and the chromatin was eluted for 15 min at 37°C in the SICAP elution buffer [7.5% SDS and 200 mM dithio- threitol (DTT)]. Chromatin was then resuspended in SICAP buffer [0.5% NP-40, 1% Triton X-100, 5 mM EDTA, 50 mM tris-HCl (pH 7.4), and 150 mM NaCl] and incubated with streptavidin beads (NEB) for 1 hour at room temperature. The beads were then exten- sively washed with the following solutions: three times with the SDS buffer [1% SDS, 1 mM EDTA, 10 mM tris-HCl (pH 7.4), and 200 mM NaCl], then once with the BW2x buffer [0.1% Triton X-100, 1 mM EDTA, 10 mM tris-HCl (pH 7.4), and 2 M NaCl], then twice with 20% isopropanol, and, lastly, four times with 40% acetonitrile. Dry beads were then frozen at −80°C and sent for MS analysis. subunit, suggesting that co-occurring mutations are not advanta- geous for cancer cells (40). Instead, NIPBL is very rarely mutated in cancer. On the other hand, NIPBL is a hallmark of the Cornelia de Lange syndrome, a disease exhibiting growth anomalies, facial dysmorphism, and cognitive retardation (53, 54). Chromatin immunoprecipitation–selective isolation of chromatin-associated proteins The procedure basically follows the initial publication with some adjustments (25). A total of 25 million mammary breast carcinoma cells were cultured for each condition and treated with corticoste- rone (600 M) or EtOH for 1 hour. Cells were then fixed for 13 min with 1% paraformaldehyde, washed three times with phosphate- buffered saline (PBS), and collected. After 1 hour of incubation with lysis buffer [0.5% SDS, 10 mM EDTA, and 50 mM tris-HCl (pH 8.1)], cells were sonicated to reach a 500-bp shredded chromatin (Bioruptor, Diagenode). Chromatin was then diluted and incubated overnight at 4°C with GR antibody preconjugated beads (sc-393232, Santa Cruz Biotechnology; MA1-510, Thermo Fisher Scientific). On-beads chromatin was then tagged with ddUTP-biotin (NU-1619-biox, Jena SCIE N C E A D V A NCES \| RESEA R CH A RT ICL E Boxplots represent minimum to maximum values, and error bars represent SE (n = 4 independent biological replicates). P values derived from unpaired t test. (D) Model of GR-NIPBL synergy to promote cohesin chromatin binding, activation of the loop extrusion, and long-range gene regulation. contradict either of these hypotheses, our results point to a mecha- nism wherein TFs initiate the binding of NIPBL-cohesin to chromatin, mediating the loop extrusion mechanisms that drive the enhancer near the promoter (Fig. 8D). Even if most of the interactions bound by the GR are already present before hormone activation (22), our analysis showed dex-induced strengthening of both chromatin loops and architectural stripes (Fig. 3). Our data demonstrate that GR and NIPBL promote the formation architectural stripes, reinforcing our hypothesis that TFs associate with the cohesin complex initiating DNA extrusion to promote long-range gene regulation. as for NIPBL, these factors do not recognize specific sites. We demonstrate here that the process is initiated, at enhancers, by TFs. as for NIPBL, these factors do not recognize specific sites. We demonstrate here that the process is initiated, at enhancers, by TFs. The association between TFs and the cohesin complex occurs in a positive feedback mechanism, increasing the concentration of TFs in the environment of specific sites, thus amplifying transcriptional output (Figs. 4 to 6). This concept is particularly evident through the SMT experiments, which demonstrate a significant loss of the confinement population of GR upon depletion of the cohesin com- plex (Fig. 7). The association between TFs and the cohesin complex occurs in a positive feedback mechanism, increasing the concentration of TFs in the environment of specific sites, thus amplifying transcriptional output (Figs. 4 to 6). This concept is particularly evident through the SMT experiments, which demonstrate a significant loss of the confinement population of GR upon depletion of the cohesin com- plex (Fig. 7). An important discussion in the community concerns whether an active regulatory enhancer must build a specific contact with the promoter or simply migrate into the neighborhood of the promoter (6, 51, 52). Although the data presented here neither support nor From the clinical perspective, NIPBL and cohesin subunits are frequently mutated in fast-growing cancers such as AML (40). How- ever, patients do not harbor mutations in more than one cohesin 12 of 18 Rinaldi et al., Sci. Adv. MATERIALS AND METHODS Cell culture, auxin treatment, and siRNA transfection Cells of 3134 mammary breast carcinoma were grown in Dulbecco’s modified Eagle’s medium (DMEM) and 10% fetal bovine serum (FBS) supplemented with sodium pyruvate, l-glutamine, and non- essential amino acids. HCT116 RAD21mAID cells were grown in McCoy medium supplemented with 10% charcoal-stripped FBS (CSS) and l-glutamine. Complete RAD21 depletion was achieved by the addition of 500 M indole-3-acetic acid (IAA; from Millipore/ Sigma-Aldrich). AML cells were grown in RPMI medium supple- mented with 20% FBS, pyruvate, l-glutamine, and essential amino acids. Before each dexamethasone (100 nM) treatment experiment, all cells were grown for 48 hours in CSS. To achieve an effective knockdown, 5 million 3134 cells were transfected during log-phase growth, by electroporation (140 V, 10 ms, three pulses) with 10 g of each siRNA against NIPBL, SMC1, or RAD21. Smartpool siRNAs were purchased from Dharmacon. Cells were treated with vehicle or dexamethasone after 48 and 72 hours after electroporation. After vehicle or dexamethasone treatment, nascent RNA was extracted using the Macherey-Nagel Kit following the manufacturer’s instruc- tions. Reverse transcription was carried out using 1 g of RNA using the Bio-Rad complementary DNA synthesis kit. qPCR was performed using Bio-Rad SYBR-Green Master Mix. g High-throughput HCR Proximity ligation assay Downloaded from https://www.science.org on April 11, 2022 DIA and data processing After generating a GR ChIP-SICAP spectral library using a combina- tion of DDA .raw files from method test runs (n = 6) and high-pH fractionations (n = 14), the remaining 80% of trypsin-digested pep- tides from each ChIP-SICAP sample were analyzed using a DIA method exactly as described in our previous works (55). DIA raw Rinaldi et al., Sci. Adv. 8, eabj8360 (2022) 30 March 2022 SCIE N C E A D V A NCES \| RESEA R CH A RT ICL E y Subcellular fractionation About 2 million 3134 cells were detached by Accutase digestion, then centrifuged (5 min, 300g, 4°C), and lastly washed three times with ice-cold PBS. Cells were then incubated on ice for 10 min with buffer A [15 mM tris-HCl (pH 8.0), 15 mM NaCl, 60 mM KCl, 1 mM EDTA, 0.5 mM EGTA, and 0.02% NP-40] to extract the cytoplasmic frac- tion by centrifugation at 1300g for 5 min at 4°C (supernatant). The purified nuclei (pellet) were then washed twice with buffer A and rotated for 1 hour at 4°C with buffer B (3 mM EDTA, 0.2 mM EGTA, and 1 mM DTT). Following that, the samples were centrifugated (5 min, 1400g, 4°C), and the chromatin fraction was collected (insoluble). The pellet of chromatin was then washed twice with buffer B, and the proteins were extracted with 50 mM tris (pH 8), 1 mM EDTA, 0.05% SDS, and 250 U of benzonase in the presence of 2 mM MgCl2 for 1 hour. After centrifugation (10 min, 15,000g, 4°C), the supernatant (chromatin fraction) was lastly collected. Equal amounts of protein from the different fractions were then processed for the Western blot procedure. SCIE N C E A D V A NCES \| RESEA R CH A RT ICL E assay was performed following the manufacturer’s instructions (DUO92101, Sigma-Aldrich). Briefly, after treatment, cells were fixed with 4% paraformaldehyde for 20 min and then washed extensively with PBS. Next, permeabilization was achieved with a PBS solu- tion with 0.5% Triton X-100 for 20 min, and cells were blocked with PBS with 3% bovine serum albumin and 0.05% Triton X-100 solution for an hour at room temperature. A second blocking was performed with the manufacturer’s solution for another hour at 37°C. After antibody incubation (see the “Antibodies” section; room temperature for 45 min), probes were added and incubated for an hour at 37°C, washed with buffer A, ligated (30 min at 37°C), and washed again with buffer A. Last, signal amplification was accomplished for 100 min at 37°C and washed with buffer B before getting stained with DAPI. The images were then acquired on a CV7000 microscope (Yokogawa) and analyzed on the Columbus platform. S b ll l f i i files were analyzed using Skyline v4.2.0.18305 (MacCoss Lab soft- ware, University of Washington) following the authors’ guidelines and settings. Library ion match tolerance was set to 5 mDa, and MS/ MS filtering was set to centroid with a 10-ppm mass accuracy. Retention time (RT) filtering was set to use only the scan within 15 min of the predicted RT and to extract the area under the curve relative to the five most intense product ions for each peptide. mProphet peak scoring algorithm was trained against the decoy peptide library and used to identify correctly integrated target peptide with a Q value of <0.01 (i.e., 1% FDR). Data matrix was exported in .csv format, and subsequent analysis of data was performed in Excel. In brief, after normalization using the median of MS/MS intensities within the runs, differential expression analysis was performed us- ing only proteins identified in at least 50% of the samples and with a fold change of ≥2 among the different GR treatment conditions. Missing values were filled by randomly picking a number in the 1% percentile of the distribution of each condition of each replicate. Significant GR interactors at chromatin level are listed in table S1. g High-throughput HCR g g p Appropriate siRNA oligos (0.25 pmol) were spotted at the bottom of each well of a 384-well imaging plate (6057300, Greiner) using an Echo525 (Beckman Coulter) acoustic liquid handler. The siRNA oligos were air-dried, and the plates were then sealed and stored at −20°C. The day of the transfection, plates were thawed and spinned down at 500g for 1 min. For each well, dried oligo siRNAs were re- suspended in 20 l of Opti-MEM (Thermo Fisher Scientific) con- taining 50 nl of RNAiMax (Thermo Fisher Scientific) for 30 min at room temperature. A total of 500 mammary breast carcinoma cells were added to the complexed siRNA/transfection mix in 20 l of culture media containing 2× serum, for a total volume of 40 l, and cultured for 72 hours at 37°C. Cells were then processed for HCR according to a slightly modified version of the original protocol (56). Briefly, after the specified treatment (+10 l), imaging plates were fixed by adding 50 l of 8% paraformaldehyde in PBS directly to the cells, incubated at room temperature for 20 min, and then washed for three times with PBS using a Bluewasher plate washer (Blue Cat Bio). Last, cells were permeabilized with 70% EtOH at −20°C for a minimum of 8 hours. Cells were rehydrated with 5× SSC Tween 0.1% buffer (5× SSCT) and preincubated with the hybridization buffer (Molecular Instruments) for 30 min at 37°C. Custom-design probes targeting intronic regions of the specified target genes Tsc22d3, Arl4d, Ccl2, and Cxcl5 (Molecular Instruments) were then added at a final concentration of 2 nM in a 10-l volume using a Mosquito liquid handler (SPT-Labtech) and incubated overnight at 37°C. Probes were later washed for 15 min at 37°C with the Probes Wash buffer (Molecular Instruments) with increasing proportions of 5× SSCT (25, 50, and 75%) then washed twice with 100% 5× SSCT (first at 37°C and second at room temperature). Later, probes were preincubated with the Amplifier buffer (Molecular Instruments) and then incubated with 75 nM proper hairpin amplifiers for 45 min at room temperature. Wells were next washed three times for 20 min each with 5× SSCT at room temperature. Following that, cells were stained with 4′,6-diamidino-2-phenylindole (DAPI) for nuclear localization, and images were acquired on a CV7000 high- throughput microscope (Yokogawa). Images analysis was lastly per- formed on the Columbus platform then with a custom R script. Micro-C Duplicate biological replicates were carried out for each Micro-C experiment, following the published protocol (30, 31). Briefly, after evaluating the best conditions for MNase treatment (nuclei prepa- ration using Igepal at 0.03% and 200 U of MNase enzyme for 10 min at 37°C) in our mouse breast adenocarcinoma cells, we performed all Micro-C protocol as described in detail in (30, 31) using 2.5 million 3134 mouse breast adenocarcinoma cells for each biological repli- cate. Briefly, after stopping the MNase reaction for 10 min at 65°C, the DNA ends were dephosphorylated using rSAP for 45 min at 37°C. Later, the 5′ overhangs were generated by 3′ resection by adding Klenow fragment polymerase and PNK in a nucleotide-free solution for 15 min at 37°C. The DNA overhangs were filled with biotinylated nucleotides for 45 min at room temperature using biotin-ATP, biotin-CTP, 2′-deoxyguanosine 5′-triphosphate, and 3′-deoxythymidine 5′-triphosphate. After stopping the reaction and isolating the nuclei by centrifugation, we incubated the isolated nuclei for 3 hours at room temperature with 12,500 U of NEB T4 ligase. After proximal ligation, nonligated biotinylated ends were removed by incubating the isolated nuclei with 200 U of exonucle- ase III for 5 min at 37°C. Samples were decross-linked for 7 hours at 65°C with shaking, and DNA was isolated by phenol-chloroform extraction. The 300- to 400-bp-sized (dinucleosome band) Micro-C g g ChIP and ATAC-seq analysis Biological duplicates were sequenced using Illumina NextSeq 500 single reads, whereas ATAC-seq was sequenced pair-ended. The reads were trimmed in silico to remove adapter sequences, low-quality reads, and 50-bp length using Trimmomatic 0.30 software and aligned to mm10 reference genome using Bowtie2 alignment tool. Mito- chondrial reads were filtered for the subsequent analyses. All peak calling of ATAC-seq data was performed using MACS2 v.2.1.1 with callpeak-format BAMPE parameters. The heatmaps were generated using an in-house R script. HiC balanced files were generated by cooltools (https://github. com/mirnylab/cooltools) and visualized by Juicebox (61). Loop calling and the detection of differential loops between dex-treated and EtOH cells (siCTRL and siNIPBL) were performed by diff_ mustache.py function in Mustache (60) with default parameter op- tions (loop call FDR = 0.2, differential loop detection FDR = 0.1) except sparsityThreshold (-st) as 0.7. Data were aligned to the mouse reference mm10 genome by STAR (58). ChIP-seq was analyzed mostly using homer (59) and bedtools. Cohesin quantification at previously identified GR sites was performed by homer using annotatePeaks.pl -ghist. siSMC1a and siNIPBL GR peaks were identified using homer findpeaks algo- rithm using style for TF and style histone for H3k27ac. Input DNA was used as control sequence. Genomic results such as cohesin en- richment after dexamethasone treatment are listed in table S2. Immunoprecipitation About 20 million cells were treated with EtOH or dexamethasone (100 nM) and fixed for 12 min with 1% paraformaldehyde. After three washes with PBS, cells were harvested and incubated with the lysis buffer (see ChIP-SICAP procedure) on ice for an hour. After sonication, to obtain an average DNA length of 500 bp, the lysate was diluted and incubated overnight at 4°C with GR antibody pre- incubated beads (sc-393232, Santa Cruz Biotechnology; MA1-510, Thermo Fisher Scientific). The next day, the beads were washed successively with low-salt, high-salt, LiCl, and lastly TE buffer. Then, the beads were incubated in 1× Laemmli buffer for 10 min at 95°C for elution/denaturation and loaded on a precast 3 to 8% tris-acetate gel (Thermo Fisher Scientific). Proteins were next transferred on a polyvinylidene difluoride membrane for Western blotting. ChIP-seq and ATAC-seq /www.science.org on April 11, 2022 Duplicate biological replicates were carried out for each ChIP-seq and ATAC-seq experiment. For GR ChIP-seq, 10 million to 20 million log-phase growth cells were treated with vehicle (EtOH) or treated with 100 nM dex (Sigma-Aldrich) for 1 hour. For ChIP, after cross-linking with paraformaldehyde and cell collection, the chro- matin was sonicated (Bioruptor, Diagenode) to an average DNA length of 200 to 700 bp. For immunoprecipitation, 1000 g of chro- matin was incubated with appropriate antibody coupled onto Dynabeads magnetic beads (Thermo Fisher Scientific) with rota- tion overnight at 4°C. Then, the chromatin-bead conjugates were washed with low-salt, high-salt, and LiCL buffer and eluted in 1% Proximity ligation assay Mammalian breast cancer cells were plated in 386-well plates (MGBB096-1-2-LG-L, Matriplate, Brooks), and proximity ligation 14 of 18 Rinaldi et al., Sci. Adv. 8, eabj8360 (2022) 30 March 2022 SCIE N C E A D V A NCES \| RESEA R CH A RT ICL E SCIE N C E A D V A NCES \| RESEA R CH A RT ICL E SDS and 100 M NACO3, proteinase K–treated, and the cross-linking was reversed at 65°C for 7 hours. DNA was extracted from the sam- ples with phenol-chloroform extraction and EtOH precipitation. ChIP-seq libraries were generated using a TruSeq ChIP sample prep kit (Illumina, IP-202-1012) according to the manufacturer’s instruc- tions. For NIPBL ChIP, we implemented a disuccinimidyl glutarate (DSG)/FA cross-link that has been shown to allow efficient detec- tion of weaker NIPBL-binding sites (57). Briefly, cells were sus- pended in PBS and treated for 45 min with 2 mM DSG. After three washes with PBS, cells were cross-linked with formaldehyde as described above. For ATAC-seq, the cells were detached from the flasks using 5 ml of Accutase (Thermo Fisher Scientific) by incubating for 5 min at room temperature. ATAC was performed according to Omni-ATAC protocol with double amount of transposase compared to the original protocol. Size selection was performed using SPRIselect (Beckman Coulter) to remove <150-bp and >1000-bp fragments accord- ing to the manufacturer’s instructions. Size selection was verified using the Agilent TapeStation System (Agilent Technologies). ChIP d ATAC l i library was purified by 2% agarose gel (run at 70 V) and purified using the Zimo DNA Clean and Concentrator Kit. Purified dinucleosome samples were quantified using the Qubit DNA HS Kit and balanced among samples. Then, purified DNA was incubated with 5 l of MyOne C1 streptavidin beads for 20 min at room temperature. After washes, Micro-C sequencing libraries were generated using the Kapa HyperPrep Kit from Roche. To reach high resolution, we sequenced around 3 billion to 4 billion reads for each biological sample using the Illumina NovaSeq sequencer. q q Micro-C analysis Micro-C datasets were analyzed following the published bioinformatic pipelines (30, 31). Briefly, raw data were analyzed through the dis- tiller pipeline (60) (https://github.com/mirnylab/distiller-nf), map- ping to the mouse reference assembly mm10 using bwa mem with the -SP flags. Pairtools package was then used to parse alignments and classify pairs to generate pair files compliant with the 4DN and deduplicated pair files with an option -max-mismatch=1 on either side. Pairs classified as uniquely mapped with high mapping quality scores (MAPQ > 30 for both) were used to obtain contact matrices in the cooler format at 500 bp and in balanced multiresolution cooler format files (500 bp, 1 kb, 2 kb, 5 kb, 10 kb, 20 kb, 50 kb, 100 kb, 250 kb, 500 kb, 1 mb, and 10 mb) using cooler with default options [low-coverage bins were excluded using the MADmax (maximum allowed median absolute deviation) filter on genomic coverage, de- scribed in (30) using a threshold of 5.0 MADs]. p p y Micro-C Stripenn analysis To identify the architectural stripes found in our Micro-C dataset, we used the mcool datasets to the compute function of the Stripenn algorithm (32) with default parameters (KR normalization, -p 0.2 and -m 0.95,0.96,0.97,0.98,0.99) set at 5000-bp resolution. To iden- tify the GR-bound stripes (n = 561), we used bedtools to intersect the Stripenn-identified architectural stripes with our GR ChIP-seq peaks. Then, we used the Stripenn score function to calculate the significance (P value) of the GR-bound architectural stripes in all our sample conditions, at 5000-bp resolution, using default parameters (32). from https://www.science.org on April 11, 2022 SCIE N C E A D V A NCES \| RESEA R CH A RT ICL E SCIE N C E A D V A NCES \| RESEA R CH A RT ICL E HiChIP of 83 Hz with 10-ms exposure time was used to study the mean square displacement. GR-HiChIP was performed following the previously published HiChIP protocol (28). Briefly, cells, after dexamethasone treatment, were detached using Accutase (Thermo Fisher Scientific) before cross- linking with formaldehyde for 10 min and quenched with 125 mM glycine. Here, 3134 cells were lysed in preparation for in situ con- tact generation. Isolated nuclei were permeabilized, and restric- tion digestion was carried out for 2 hours at 37°C with Mbo I (New England Biolabs). Restriction sites were filled with dNTPs using biotin-14-dATP (Jena Bioscience) for 1 hour at 37°C. The filled ends were then ligated together using a T4 ligase at room temperature for 4 hours before nuclei were lysed and sonicated (using Covaris), and then GR immunoprecipitation was carried out overnight using antibodies. The morning after, 30 l of beads was added to collect the chromatin-antibody complex; then, the ChIP DNA was collected and washed, and cross-links were reversed overnight using proteinase K. ChIP DNA was eluted, and samples were purified using the DNA Clean up Kit. The DNA was quantified using a Qubit before biotin ligation junction capture using streptavidin C-1 beads. Samples were washed and taken forward for Tn5 tagmentation. Tagmentation and PCR amplification were performed as described by Mumbach et al. (28). Libraries were size-selected to 200 to 700 bp and sequenced on the HiSeq using 2 × 150 bp. HiChIP fastq files were aligned using HiCPro (62), and thereafter, FitHiChIP (29) was used to identify significant interactions and interaction strength for each experimental condition. CoverageBias was used to normalize the data and call sig- nificant interaction. The genome browser WashU was used to visualize the HiChIP loops. GR-HiChIP significant interaction at q value of 0.01 for samples EtOH, siCTRL, and siNIPBL is listed in table S3. Viability assay The particle tracking was performed with the “TrackRecord” software developed in MATLAB (MathWorks Inc.). The procedure is summarized in a previous publication (63). The molecules were allowed to move a maximum of four pixels from one frame to the next for the 5-Hz acquisition and six pixels for 83-Hz acquisition; for both conditions, only tracks that were at least two frames long were kept. p Viability assay A total of 2000 cells treated with the indicated siRNA were plated into a 384-well plate (Greiner). Twenty-four hours later, we applied the Live/Dead Sytox staining procedure recommended by the manu- facturer (S1138, Thermo Fisher Scientific). Briefly, cells were stained for 15 min in regular growing medium then washed three times with PBS. The images were acquired on a CV7000 microscope (Yokagawa) and analyzed using the Columbus platform. y RNA was extracted using the Macherey-Nagel Kit, following the manufacturer’s instructions. RIN scores of all samples were above 7.6. RNA-seq libraries were constructed using the Illumina TruSeq Stranded Total RNA with Ribo-Zero Human/Mouse/Rat library kit according to the manufacturer’s instructions. We sequenced two biological replicates for each condition using Illumina NovaSeq6000 S4 with 150-bp paired-end reads. RTA was used for base calling, and Bcl2fastq was used for demultiplexing. Trimmomatic 0.39 was used to trim for adapters. RNA-seq alignment to human hg19 genome was performed by STAR (58) using default parameters with the following modifications: “--genomeDir hg19-150 --outSAMunmapped Within --outFilterType BySJout --outFilterMultimapNmax 20 --outFilterMismatchNmax 999 --outFilterMismatchNoverL- max 0.04 --alignIntronMin 20 --alignIntronMax 1000000 --align- MatesGapMax 1000000 --alignSJoverhangMin 8 --limitSjdbInsertNsj 2500000 --alignSJDBoverhangMin 1 --sjdbScore 1 --sjdbFileChrStartEnd hg19-150/sjdbList.out.tab --sjdbGTFfile gencode.v19.annotation. gtf --peOverlapNbasesMin 10 --alignEndsProtrude 10 ConcordantPair.” Raw count data for a total of 55,765 genes were obtained using htseq 0.11.4 using the default parameters and the option “--stranded=re- verse.” Low-count genes were removed by requiring more than 15 reads in at least 2 samples for each gene across the 12 samples, and the remaining 15,612 genes were used for the subsequent analyses. Differentially expressed genes were identified on the basis of the criteria of adjusted P < 0.05 and shrunken log2 fold change (LFC) > log2(1.5) using DESeq2 (65). Shrunken LFC was obtained using the adaptive shrinkage estimator. Wald test was used to detect p CTCF boundary prediction CTCF boundaries were predicted using the script published by Oti et al. (27) run on CTCF ChIP-seq peaks published previously in mouse breast adenocarcinoma 3134 cells (24). Briefly, we over- lapped the genome-wide CTCF motifs with the CTCF peaks to ob- tain the subset of peak motifs. We used homer to quantify CTCF peaks, and we considered for the follow-up analysis only the CTCF peaks above 40 normalized sequenced tag. Peak-contained motifs were considered as possible anchors for loop prediction. Two possi- ble anchors constituted a TAD loop when the two CTCF motifs were convergent. The probability and scores of the loop anchors were computed from both peak and motif scores by multiplying the ChIP-seq peak scores. We intersected the predicted TADs (with high probability) with GR ChIP-seq dataset to obtain a list of pre- dicted TADs containing a GR-bound site and a list of TADs with no GR-bound sites. 15 of 18 Rinaldi et al., Sci. Adv. 8, eabj8360 (2022) 30 March 2022 p SMT analysis An improved method that accounts for photobleaching effects was applied to the dwell time distribution analysis (64). Briefly, the dwell time distribution of histone H2B was measured at the focal plane under identical SMT acquisition conditions and then fitted to a triple-exponential model to calculate photobleaching parameters. The dwell time distribution is obtained by calculating the weighted ensemble average distribution of bound times for each diffusive state in different cells in the experiment, and then it is corrected by dividing the exponential component estimated in the H2B dwell time distribution analysis. After photobleaching correction, the dwell time distribution is fitted to a power-law distribution. Using a machine learning–based classification described previ- ously (36), we measured the nuclear mobility of chromatin factors. Specifically, we used short exposure times (10 and 12 ms) to mini- mize motion blur. Then, images were acquired, and trajectories were generated. Perturbation expectation maximation (pEM) together with BIC was used to classify the trajectories of the protein into the least number of diffusive modes. The posterior probability weighted mean-squared displacement (MSD) for each diffusive state was computed. 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Mirny, RNA polymerases as moving barriers to condensin loop extrusion. Proc. Natl. Acad. Sci. U.S.A. 116, 20489–20499 (2019). 19. H. B. Brandão, P. Paul, A. A. van den Berg, D. Z. Rudner, X. Wang, L. A. Mirny, RNA The following are the antibodies used in this study: NIPBL (Bethyl Laboratories, A301-779A; Thermo Fisher Scientific, MA1-72534), YY1 (Active Motif, 61779), H3K27ac (Active Motif, 39133), GR (Santa Cruz Biotechnology, sc-393232; Thermo Fisher Scientific, MA1-510), SMC1a (Bethyl Laboratories, A300-055A; Abcam, ab133643), SMC3 (Abcam, ab9263), RAD21 (Abcam, ab992), H2B (Abcam, ab61250), and tubulin (Abcam, ab6160). polymerases as moving barriers to condensin loop extrusion. Proc. Natl. Acad. Sci. U.S.A. 116, 20489–20499 (2019). 20. E. P. Nora, A. Goloborodko, A.-L. Valton, J. H. Gibcus, A. Uebersohn, N. Abdennur, J. Dekker, L. A. Mirny, B. G. Bruneau, Targeted degradation of CTCF decouples local insulation of chromosome domains from genomic compartmentalization. Cell 169, 930–944.e22 (2017). 21. F. L. Dily, E. Vidal, Y. Cuartero, J. Quilez, A. S. Nacht, G. P. Vicent, J. Carbonell-Caballero, P. Sharma, J. L. Villanueva-Cañas, R. Ferrari, L. I. De Llober, G. Verde, R. H. G. Wright, M. Beato, Hormone-control regions mediate steroid receptor-dependent genome organization. Genome Res. 29, 29–39 (2019). SCIE N C E A D V A NCES \| RESEA R CH A RT ICL E SCIE N C E A D V A NCES \| RESEA R CH A RT ICL E differentially expressed genes from the pairwise comparison be- tween two contrasting groups. Fold inductions of differentially ex- pressed genes are listed in table S4. The GEO accession number for the genomic data is GSE162617. 17. Y. Zhu, M. Denholtz, H. Lu, C. Murre, Calcium signaling instructs NIPBL recruitment at active enhancers and promoters via distinct mechanisms to reconstruct genome compartmentalization. Genes Dev. 35, 65–81 (2021). 18. H. Yan, I. Surovtsev, J. F. Williams, M. L. P. Bailey, M. C. King, S. G. J. Mochrie, Extrusion of chromatin loops by a composite loop extrusion factor. Phys. Rev. E 104, 024414 (2021). Single-molecule tracking Data acquisition of SMT experiments was described previously (36, 63, 64). Briefly, HCT116 RAD21mAID cells were plated into two-well Lab-Tek chamber slides (Thermo Fisher Scientific, Waltham, MA, USA) in Phenol Red–free DMEM medium containing 10% CSS (Hyclone, Logan, UT) and transfected with a Halo-GR plasmid. Later, cells were treated with 500 M IAA (from Millipore/Sigma-Aldrich) for 24 hours and, the next day, incubated with 0.25 nM cell-permeable Janelia Fluor 549 HALOTag ligand (JF549) for 20 min. Cells were lastly extensively washed with media and treated with 100 nM dexamethasone or EtOH for 20 min before acquisition. A custom-built microscope (Optical Microscopy Core facility, LRBGE, NCI) controlled by Micro-Manager software (Open Imag- ing Inc., San Francisco, CA) was used. It was equipped with a 150× 1.45 numerical aperture objective (Olympus Scientific Solutions, Waltham, MA), a 561-nm laser (iFLEX-Mustang, Excelitas Technolo- gies Corp., Waltham, MA), an acousto-optic tunable filter (AOTFnC- 400.650, AA Optoelectronic, Orsay, France), and HILO (highly inclined and laminated optical sheet) illumination microscope. Eight hundred frames of fluorescent images were collected on an EM-CCD camera (Evolve 512, Photometrics) at a rate of 5 Hz with 10-ms exposure time to resolve the confinement and slow bound state, while a rate 16 of 18 Rinaldi et al., Sci. Adv. 8, eabj8360 (2022) 30 March 2022 SCIE N C E A D V A NCES \| RESEA R CH A RT ICL E Genome Biol. 15, 550 (2014). 46. E. H. Finn, G. Pegoraro, H. B. Brandão, A.-L. Valton, M. E. Oomen, J. Dekker, L. Mirny, T. Misteli, Extensive heterogeneity and intrinsic variation in spatial genome organization. Cell 176, 1502–1515.e10 (2019). Acknowledgments: We thank M. Kanemaki for providing the HCT116 RAD21mAID cells. We thank J. Taub for providing the DS-AMKL cells. We thank the Center for Cancer Research (CCR) Genomics Core, CCR Sequencing Facility, Flow Cytometry, and High- Throughput Facility at NCI. We would like to thank T. Karpova and D. Ball at the Optical Microscopy Core at NIH. We thank P. Rocha, E. Arda, and L. Wang for technical support to perform Micro-C and HiChIP experiments. This work used the computational resources of the NIH HPC Biowulf cluster. We thank G. Vahedi and S. Yoon from Penn University of Medicine for technical support to run the Stripenn algorithm. We would like to thank the Danish National Research Foundation (DNRF grant no. 141 to ATLAS) and Novo Nordisk Foundation (NNF18OC0052768). We thank A. Lillich, NIH Library Editing Service, for manuscript editing assistance. The current affiliation of S.F. is the Kanagawa Cancer Center of Yokohama, Japan, while G.F. is at the University of Liège, Belgium. D.A.G. is currently employed at the Boston Consulting Group, Chicago, IL. Funding: S.F. was supported by the JSPS Research Fellowship for Japanese Biomedical and Behavioral Researchers at NIH. A.U. acknowledges support from NSF PHY 1806903 and NSF PHY 1915534. L.R. was supported by the EMBO long-term postdoctoral fellowship. This work was supported (in part) by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research. Author contributions: L.R., G.F., and G.L.H. conceived the study and wrote the manuscript with input of all the other coauthors. L.R. performed and analyzed genomic experiments such as ChIP-seq and HiChIP. L.R. and T.A.J. performed the Micro-C experiments, analyzed by L.R. and S.K. G.F. performed ChIP-SICAP, HCR, IP, PLA, and IF, helped by D.A.S. G.F., M.P., and B.B. analyzed ChIP-SICAP data, while HCR was analyzed by G.F. and G.P. L.R. and G.F. performed SMT, analyzed by D.A.G. and G.F. with valuable advice provided by A.U. 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Copyright © 2022 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution License 4.0 (CC BY).
https://openalex.org/W2800287677	https://repository.ubn.ru.nl/bitstream/handle/2066/193604/1/193604.pdf	English	null	Dutch guideline for clinical foetal-neonatal and paediatric post-mortem radiology, including a review of literature	European journal of pediatrics	2,018	cc-by	9,437	Dutch guideline for clinical foetal-neonatal and paediatric post-mortem radiology, including a review of literature Sonnemans, L.J.P.; Vester, M.E.; Kolsteren, E.E.M.; Erwich, J.; Nikkels, P.G.; Kint, P.A.M.; Rijn, R.R. van; Klein, W.M. 2018, Article / Letter to editor (European Journal of Pediatrics, 177, 6, (2018), pp. 791-803) Doi link to publisher: https://doi.org/10.1007/s00431-018-3135-9 Version of the following full text: Publisher’s version Downloaded from: http://hdl.handle.net/2066/193604 Download date: 2024-10-24 Dutch guideline for clinical foetal-neonatal and paediatric post-mortem radiology, including a review of literature Sonnemans, L.J.P.; Vester, M.E.; Kolsteren, E.E.M.; Erwich, J.; Nikkels, P.G.; Kint, P.A.M.; Rijn, R.R. van; Klein, W.M. 2018, Article / Letter to editor (European Journal of Pediatrics, 177, 6, (2018), pp. 791-803) Doi link to publisher: https://doi.org/10.1007/s00431-018-3135-9 Dutch guideline for clinical foetal-neonatal and paediatric post-mortem radiology, including a review of literature L. J. P. Sonnemans1 & M. E. M. Vester2,3,4 & E. E. M. Kolsteren5 & J. J. H. M. Erwich6 & P. G. J. Nikkels7 & P. A. M. Kint8 & R. R. van Rijn2,3,4 & W. M. Klein1,9 & On behalf of the Dutch post-mortem imaging guideline group L. J. P. Sonnemans1 & M. E. M. Vester2,3,4 & E. E. M. Kolsteren5 & J. J. H. M. Erwich6 & P. G. J. Nikkels7 & P. A. M. Kint8 & R. R. van Rijn2,3,4 & W. M. Klein1,9 & On behalf of the Dutch post-mortem imaging guideline group Received: 22 November 2017 /Revised: 5 February 2018 /Accepted: 26 March 2018 /Published online: 19 April 2018 # The Author(s) 2018 Abstract Clinical post-mortem radiology is a relatively new field of expertise and not common practice in most hospitals yet. With the declining numbers of autopsies and increasing demand for quality control of clinical care, post-mortem radiology can offer a solution, or at least be complementary. A working group consisting of radiologists, pathologists and other clinical medical specialists reviewed and evaluated the literature on the diagnostic value of post-mortem conventional radiography (CR), ultra- sonography, computed tomography (PMCT), magnetic resonance imaging (PMMRI), and minimally invasive autopsy (MIA). Evidence tables were built and subsequently a Dutch national evidence-based guideline for post-mortem radiology was devel- oped. We present this evaluation of the radiological modalities in a clinical post-mortem setting, including MIA, as well as the recently published Dutch guidelines for post-mortem radiology in foetuses, neonates, and children. In general, for post-mortem radiology modalities, PMMRI is the modality of choice in foetuses, neonates, and infants, whereas PMCT is advised in older children. There is a limited role for post-mortem CR and ultrasonography. In most cases, conventional autopsy will remain the diagnostic method of choice. g Conclusion: Based on a literature review and clinical expertise, an evidence-based guideline was developed for post-mortem radiology of foetal, neonatal, and paediatric patients. L. J. P. Sonnemans and M. E. M. Vester shared first authorship and contributed equally to this work. Communicated by Piet Leroy * M. E. M. Vester m.e.vester@amc.uva.nl L. J. P. Sonnemans lianne.sonnemans@radboudumc.nl E. E. M. Kolsteren e.kolsteren@kennisinstituut.nl J. J. H. M. Erwich j.j.h.m.erwich@umcg.nl P. G. J. Nikkels P.G.J.Nikkels@umcutrecht.nl P. A. M. Kint pkint@amphia.nl R. R. van Rijn r.r.vanrijn@amc.uva.nl W. M. Klein willemijn.klein@radboudumc.nl 1 Department of Radiology, Radboud University Medical Center, Nijmegen, The Netherlands 2 Department of Radiology, Academic Medical Center, Amsterdam, The Netherlands 3 Department of Forensic Medicine, Netherlands Forensic Institute, The Hague, The Netherlands 4 Amsterdam Centre for Forensic Science and Medicine, Amsterdam, The Netherlands 5 Knowledge Institute of Medical Specialists, Utrecht, The Netherlands 6 Department of Obstetrics and Gynaecology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands 7 Department of Pathology, University Medical Center Utrecht, Utrecht, The Netherlands 8 Department of Radiology, Amphia Hospital, Breda, The Netherlands 9 Department of Radiology, Maastricht University Medical Center, Maastricht, The Netherlands 1 Department of Radiology, Radboud University Medical Center, Nijmegen, The Netherlands 1 Department of Radiology, Radboud University Medical Center, Nijmegen, The Netherlands * M. E. M. REVIEW Dutch guideline for clinical foetal-neonatal and paediatric post-mortem radiology, including a review of literature L. J. P. Sonnemans1 & M. E. M. Vester2,3,4 & E. E. M. Kolsteren5 & J. J. H. M. Erwich6 & P. G. J. Nikkels7 & P. A. M. Kint8 & R. R. van Rijn2,3,4 & W. M. Klein1,9 & On behalf of the Dutch post-mortem imaging guideline group REVIEW REVIEW Note: Note: To cite this publication please use the final published version (if applicable). To cite this publication please use the final published version (if applicable). European Journal of Pediatrics (2018) 177:791–803 https://doi.org/10.1007/s00431-018-3135-9 Materials and methods The guideline was developed under the guidance of the Dutch knowledge institute of medical specialists. An im- portant objective of the Dutch knowledge institute is to preserve and pool knowledge and expertise about the de- sign and execution of quality assurance projects in the realm of specialist medical care. Medline and Embase were searched for studies comparing clinical post-mortem radi- ology to autopsy in foetal, neonatal, and paediatric patients from January 2000 up to January 2016, when the guideline committee started her work (Appendix 1, a further detailed search strategy is available upon request). Language selec- tion was restricted to studies published in Dutch and English. The study selection and analysis was performed separately for the group of foetal and neonatal cases (de- ceased within 28 days post-partum) and for the group of paediatric cases (aged 1 month to 18 years). Studies were initially screened on title and abstract (JE, RR), and here- after analysed on full text (EK, JE, RR). Case reports and forensic articles were excluded. Outcomes in sensitivity and specificity were mandatory. Reference lists of included studies were screened for additional relevant studies. What is Known: • Post-mortem investigations serve as a quality check for the provided health care and are important for reliable epidemiological registration. • Post-mortem radiology, sometimes combined with minimally invasive techniques, is considered as an adjunct or alternative to autopsy. What is New: • We present the Dutch guidelines for post-mortem radiology in foetuses, neonates and children. • Autopsy remains the reference standard, however minimal invasive autopsy with a skeletal survey, post-mortem computed tomography, or post-mortem magnetic resonance imaging can be complementary thereof. Keywords Post-mortem . Paediatric . Neonatal . Foetal . Radiology . Autopsy Keywords Post-mortem . Paediatric . Neonatal . Foetal . Radiology . Autopsy the basis for the evidence-based Dutch guideline for clinical foetal, neonatal, and paediatric post-mortem radiology [17]. Abbreviations CR Conventional radiography GRADE Grading Recommendations Assessment, Development and Evaluation MaRIAS Magnetic Resonance Imaging Autopsy Study MIA Minimally invasive autopsy PMCT Post-mortem computed tomography PMMRI Post-mortem magnetic resonance imaging Abstract Vester m.e.vester@amc.uva.nl 2 Department of Radiology, Academic Medical Center, Amsterdam, The Netherlands 2 Department of Radiology, Academic Medical Center, Amsterdam, The Netherlands L. J. P. Sonnemans lianne.sonnemans@radboudumc.nl E. E. M. Kolsteren e.kolsteren@kennisinstituut.nl J. J. H. M. Erwich j.j.h.m.erwich@umcg.nl P. G. J. Nikkels P.G.J.Nikkels@umcutrecht.nl P. A. M. Kint pkint@amphia.nl R. R. van Rijn r.r.vanrijn@amc.uva.nl W. M. Klein willemijn.klein@radboudumc.nl 3 Department of Forensic Medicine, Netherlands Forensic Institute, The Hague, The Netherlands 4 Amsterdam Centre for Forensic Science and Medicine, Amsterdam, The Netherlands 6 Department of Obstetrics and Gynaecology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands 7 Department of Pathology, University Medical Center Utrecht, Utrecht, The Netherlands 7 Department of Pathology, University Medical Center Utrecht, Utrecht, The Netherlands 8 Department of Radiology, Amphia Hospital, Breda, The Netherlands 9 Department of Radiology, Maastricht University Medical Center, Maastricht, The Netherlands Eur J Pediatr (2018) 177:791–803 792 Introduction Paediatric post-mortem radiology, in addition to autopsy, is becoming widely accepted as an important component of cause of death determination [1–5]. The trend in declining clinical autopsy rates in adults [6–9] is also evident in the foetal and paediatric population, though higher autopsy rates of approximately 50% remain [3, 10–13]. This decline is in spite of evidence that clinical error rates persist: approximately 25% discrepancy between clinical ante-mortem diagnosis and autopsy cause of death diagnosis [3, 11, 14, 15]. If an alternative, less- or non-invasive diagnostic method could adequately determine the cause of death, current objec- tions to conventional autopsy (e.g. its invasiveness) could be met. Consequently, this might increase quality control and subsequently improve clinical care. Post-mortem radiology might be such an alternative diagnostic method. It can be helpful for diagnosing anatomic abnormalities, identification of syndromes, or to narrow down the differential diagnosis of genetic disorders. Consequently, it can also be useful for identifying potential siblings at risk, counselling for future pregnancies, and helping the parents in their process of grief [7, 16]. Methodological quality assessment of included studies was performed (EK) according to the AMSTAR checklist, PRISMA checklist, or QUADAS II, depending on the type of article [18–20]. The joint evidence of included articles was scored (EK) according to the Grading Recommendations Assessment, Development and Evaluation (GRADE) tool [21]. GRADE divides the quality (or certainty) of evidence and conclusions into four categories: high, medium, low or very low. A high GRADE level of evidence means that the conclusion is unlikely to change with future research, whereas in a very low GRADE level of evidence the conclusion is very precarious. In addition to the level of evidence in literature, expertise from the Dutch post-mortem imaging guideline group members was taken into account, along with prefer- ences of bereaved relatives, costs, availability of devices, Post-mortem radiology is evolving into a subspecialty, reflected by the large increase of publications and the broad spectrum of used techniques [1]. Nevertheless, a guideline on the indications and contraindications for the use of post- mortem conventional radiography (CR), ultrasonography, computed tomography (PMCT), magnetic resonance imaging (PMMRI), and minimally invasive autopsy (MIA), was not yet available. This article provides the literature review that is 793 Eur J Pediatr (2018) 177:791–803 Fig. 2 Paediatric study selection. * Several papers had multiple reasons for full text exclusion. Study identification The literature search resulted in 268 eligible articles for foe- tuses and neonates and 415 articles for paediatric studies. After title, abstract, and full-text selection 14 foetal-neonatal articles and 9 paediatric studies remained (Figs. 1 and 2). Studies on CR, PMCT, PMMRI, and MIA were included, other post-mortem imaging methods (e.g. post-mortem ultra- sound) did not meet the inclusion criteria. Fig. 2 Paediatric study selection. * Several papers had multiple reasons for full text exclusion. Maximum one reason per article was scored, according to the order presented Introduction Maximum one reason per article was scored, according to the order presented and organisational issues when formulating the guideline recommendations. and organisational issues when formulating the guideline recommendations. Foetal-neonatal Both in foetal and neonatal patients, as well as in paediatric patients, the GRADE evidence for post-mortem CR, PMCT, and MIA was graded as low because few studies, with few patients included, have been performed. More studies were pub- lished on PMMRI, yet the evidence for PMMRI was also graded as low in both groups, because almost all results were based on the Magnetic Resonance Imaging Autopsy Study (MaRIAS). This study was performed in a specialised setting with well- trained specialists and a relatively low number of patients. A clinical, foetal post-mortem skeletal survey consists mainly of a whole-body radiograph; a ‘babygram’ (Fig. 3). A study of 377 foetal post-mortem skeletal surveys showed a 100% sensitivity and 97% specificity for Fig. 3 Example of a diagnostic babygram. This pregnancy was terminated at 22 weeks of gestation because of micromelia on prenatal 2nd trimester ultrasound, suspected to be a skeletal dysplasia. The babygram showed skeletal abnormalities with shortened ribs, metaphyseal flaring (1) and shortened and bowed long bones (2). Histology revealed abnormalities in the liver, kidneys, lungs, bone and cartilage compatible with ciliopathy with major skeletal involvement. Jeune syndrome is the most likely diagnosis Example of a diagnostic babygram. This pregnancy was ed at 22 weeks of gestation because of micromelia on prenatal mester ultrasound, suspected to be a skeletal dysplasia. The am showed skeletal abnormalities with shortened ribs Post-mortem conventional radiography (CR) One article was included from the literature review on post- mortem CR in foetal and neonatal patients. No articles on post-mortem CR of paediatric patients met the inclusion criteria. Fig. 1 Foetal-neonatal study selection. * Several papers had multiple reasons for full text exclusion. Maximum one reason per article was scored, according to the order presented Fig. 3 Example of a diagnostic babygram. This pregnancy was terminated at 22 weeks of gestation because of micromelia on prenatal 2nd trimester ultrasound, suspected to be a skeletal dysplasia. The babygram showed skeletal abnormalities with shortened ribs, metaphyseal flaring (1) and shortened and bowed long bones (2). Histology revealed abnormalities in the liver, kidneys, lungs, bone and cartilage compatible with ciliopathy with major skeletal involvement. Jeune syndrome is the most likely diagnosis Fig. 1 Foetal-neonatal study selection. * Several papers had multiple reasons for full text exclusion. Maximum one reason per article was scored, according to the order presented Eur J Pediatr (2018) 177:791–803 794 Table 1 Table of evidence of diagnostic performance of PMCT in foetuses, neonates and paediatric patients Author (ref) Year Study design Anatomical system Outcome parameter Foetuses < 24 weeks of gestation Foetuses > 24 weeks of gestation Neonates and children Sensitivity (95%CI) Specificity (95%CI) Sensitivity (95%CI) Specificity (95%CI) Sensitivity (95%CI) Specificity (95%CI) Arthurs [23] 2016 Prospective (MaRIAS) General Main diagnosis or cause of death 28 (13–51) 100 (44–100) 50 (22–79) 100 (61–100) 50 (29–71) 100 (74–100) Any lesion (cardiac, thoracic, neurologic, abdominal or skeletal) 67 (35–88) 91 (81–96) 62 (36–82) 93 (80–97) 58 (42–72) 95 (89–98) Cardiac Cardiac lesions NA 100 (61–100) 100 (21–100) 100 (65–100) 50 (22–79) 100 (83–100) Non-cardiac thoracic Non-cardiac thoracic lesions 0 (0–79) 100 (51–100) 100 (34–100) 80 (38–96) 60 (36–80) 85 (58–96) Neurologic Neurologic lesions 100 (44–100) 67 (44–84) 50 (22–79) 100 (57–100) 58 (32–81) 100 (82–100) Abdominal Abdominal lesions 0 (0–79) 100 (65–100) 100 (21–100) 67 (30–90) 50 (10–91) 93 (77–98) Musculo-skeletal Skeletal lesions 75 (30–95) 100 (89–100) 0 (0–79) 100 (82–100) 100 (21–100) 96 (82–99) Arthurs [24] 2015 Prospective Non-cardiac thoracic Abnormal lung areas 100 (52–100)a 63% (31–86)a detection of skeletal abnormalities compared to diagnosis based on autopsy, genetics, and prenatal investigations [22]. However, the number of diagnostic abnormalities in this population was limited. Post-mortem computed tomography (PMCT) One study was included on the sensitivity and specificity of PMCT for both foetal-neonatal and paediatric patients, and an additional article on paediatric patients (Table 1). Foetal-neonatal PMCT and PMMRI were both compared to autopsy, and to each other in 53 foetuses, finding 40% of the PMCT’s non- diagnostic in foetuses below 24 weeks of gestation (n = 35) compared to 11% of PMMRI’s, with twice as many correct diagnoses on PMMRI compared to PMCT (10 vs. 5, p < 0.005) [23]. In foetuses above 24 weeks of gestation (n = 18), 22% of PMCT’s were non-diagnostic, compared to 0% of PMMRI’s (p < 0.005). In cases where radiology was diag- nostic, both PMCTand PMMRI showed a 50% sensitivity and 100% specificity for main diagnosis or cause of death. Also, no significant differences were observed for identification of pathological lesions in individual organ systems, irrespective of contribution to death. Post-mortem conventional radiography (CR) The authors concluded that there is no indication for a foetal post-mortem skeletal sur- vey in cases without previous suspicion of skeletal abnor- malities on prenatal ultrasound or during post-mortem ex- ternal inspection. If a foetal ‘babygram’ is obtained, it should preferably be done using a high resolution ‘cabinet radiography’ system. If this is not available, the use of a mammography system is advised. Paediatric Sensitivity of PMCT for cause of death determination depends on the type of pathology and age of the child [23, 24]. The same study as for foetuses and neonates, included 29 children with an average age of 6.9 months (range 1 day–16 years) [23]. In this small group, both PMCT and PMMRI showed a 50% sensitivity and 100% specificity for the main diagnosis or cause of death. The overall concordance was slightly lower for PMCT than PMMR (59.4% vs. 62.8%). In another study with 12 children under the age of 1 year, PMCT’s of the lungs were non-diagnostic in 75% prior to post-mortem ventilation, com- pared to 0% of PMCT’s with ventilation [24]. A 100% sensi- tivity and 63% specificity were found for the detection of abnormal lung areas with ventilated PMCT. Therefore, venti- lated PMCT could be used to improve identification of abnor- mal areas of the lungs. Eur J Pediatr (2018) 177:791–803 795 Foetal-neonatal MaRIAS reported a 100% sensitivity and 98% specificity for major and minor structural heart defects in neonates and chil- dren with 1.5 T PMMRI [25]. A substantial lower sensitivity of 62% was observed for any cardiac pathology, both struc- tural and non-structural. Identification of non-cardiac, thoracic abnormalities was difficult, especially in case of pneumonia [27]. Sensitivities of 100% and specificities of 98–100% were reported for the detection of brain malformations and minor and major intracranial haemorrhages [28]. In contrast to foe- tuses, PMMRI showed a high sensitivity (93%) for ischaemic brain injury in neonates and children. Just as in foetuses, PMMRI showed a moderate sensitivity (71%) and high spec- ificity (87%) for abdominal abnormalities [29]. The sensitivity for skeletal abnormalities was poor (31%) [30]. Before the MaRIAS study, a systematic review, investigating the diagnostic accuracy of PMMRI, included five studies on foetuses [33]. In four of those five studies, a complete autopsy was used as the reference standard. The included studies were of moderate quality as the groups were small and the popula- tion heterogeneity large. There was a pooled sensitivity of 69% (95%CI 56–80) and a pooled specificity of 95% (95%CI 88–98) for detection of clinically significant abnormalities. The MaRIAS study reported high sensitivities (82–100%) and high specificities (93–97%) for both major and minor cardiac pathology, as well as for structural and non-structural heart disease in foetuses below and above 24 weeks of gesta- tion [25]. Votino et al. (2012) compared high-field PMMRI (9.4 T) to lower-field PMMRI (1.5 T and 3.0 T) and stereo- microscopic autopsy (MIA) [26]. In contrast to lower-field PMMRI, the heart situs, four-chamber view and outflow tracts could be visualised in all foetuses with 9.4 T, irrespective of gestational age. High-field PMMRI identified seven out of eight cases with major congenital heart disease. In foetuses below and above 24 weeks of gestation, MaRIAS reported low sensitivities of 30 and 38% and high specificities of 96 and 88% respectively for the detection of non-cardiac, thorac- ic abnormalities with 1.5 T [27]. Based on these results and the reasonable negative predictive values of approximately 85%, PMMRI appeared to be more useful in the exclusion of tho- racic abnormalities, rather than in its identification. Detection of pulmonary tract infection and diffuse alveolar haemorrhage was difficult, whereas PMMRI was most sensitive for detec- tion of anatomical abnormalities, including pleural effusions and lung hypoplasia. Minimal invasive autopsy (MIA) One article included from the literature search reported on MIA in both foetal-neonatal and paediatric patients (MaRIAS) [31]. This study compared the diagnostic accuracy of MIA to conventional autopsy. MIA consisted of PMMRI, combined with other post-mortem radiology, genetic and met- abolic tests (ante-mortem and post-mortem blood sampling), a review of the clinical history, external examination, and ex- amination of placental tissue, if available. No foetal-neonatal or paediatric studies combining PMMRI or PMCT with tissue biopsies or angiography met the inclusion criteria. Post-mortem magnetic resonance imaging (PMMRI) provided clinically important information in 23 out of 43 foe- tuses in whom neuropathological examination was non- diagnostic due to maceration. Seven articles were included on the diagnostic performance of PMMRI in foetuses and neonates, along with five articles on paediatric patients (Table 2). The majority of these studies reported on the Magnetic Resonance Imaging Autopsy Study (MaRIAS) (sub)population [23, 25, 27–30]. MaRIAS is a large, 3.5 year, double-blind prospective study in 277 foetuses (185 foetuses of 24 weeks gestation or less and 92 foetuses of 24 weeks gestation or more) and 123 children (42 neonates, 53 infants up to 1 year of age, and 28 children above 1 year of age), which compared the diagnostic accuracy of 1.5 T PMMRI to conventional autopsy [31, 32]. PMMRI showed moderate sensitivities of 77 and 65% for abdominal abnormalities in foetuses below and above 24 weeks gestation, respectively [29]. Diagnostic accuracy was variable per organ system, with the highest sensitivity for renal abnormalities (18/21 = 86%) and the lowest for in- testinal abnormalities (2/7 = 29%). In addition, MaRIAS re- ported moderate and very low sensitivities for detection of musculoskeletal abnormalities in foetuses below and over 24 weeks of gestation, respectively 69 and 17% [30]. Foetal-neonatal Both a high sensitivity (100%, 95%CI 97–100) and high spec- ificity (98%, 95%CI 88–100) were reported for the detection of major pathological abnormalities or cause of death in foe- tuses below 24 weeks of gestation [31]. In foetuses above 24 weeks of gestation, sensitivity and specificity were also high (respectively 96%, 95%CI 86–99, and 95%, 95%CI 84–99). Moreover, MIA had a higher sensitivity and specific- ity compared to PMMRI alone. In both groups of foetuses, the Based on MaRIAS, very high sensitivities (80–100%) and specificities (87–100%) were found for the detection of brain malformations (Fig. 4) and minor and major intracranial bleedings [28]. A lower sensitivity of 30% was found for the detection of hypoxic-ischaemic brain injury in foetuses above 24 weeks of gestation. Foetal-neonatal Furthermore, cerebral PMMRI 796 Eur J Pediatr (2018) 177:791–803 Table 2 Table of evidence of diagnostic performance of PMMRI in foetuses, neonates and paediatric patients Author (ref) Year Study design Anatomical system Outcome parameter Foetuses < 24 weeks of gestation Foetuses > 24 weeks of gestation Neonates and children Sensitivity (95%CI) Specificity (95%CI) Sensitivity (95%CI) Specificity (95%CI) Sensitivity (95%CI) Specificity (95%CI) Arthurs [23] 2016 Prospective (MaRIAS) General Main diagnosis or cause of death 40 (23–59) 100 (61–100) 50 (24–76) 100 (68–100) 50 (29–71) 100 (74–100) Any lesion (cardiac, thoracic, neurologic, abdominal or skeletal) 58 (39–76) 88 (81–93) 79 (57–92) 92 (83–96) 70 (55–82) 84 (76–90) Cardio-vascular Cardiac lesions 100 (44–100) 86 (69–94) 100 (44–100) 100 (80–100) 63 (31–86) 95 (77–99) Non-cardiac thoracic Non-cardiac thoracic lesions 20 (4–62) 96 (78–99) 67 (21–94) 87 (62–96) 47 (26–69) 50 (25–75) Neurological Neurologic lesions 83 (44–97) 52 (32–72) 75 (41–93) 70 (40–89) 100 (76–100) 77 (53–90) Abdominal Abdominal lesions 40 (12–77) 100 (86–100) 100 (51–100) 93 (69–99) 100 (34–100) 82 (63–92) Musculo-skeletal Skeletal lesions 60 (23–88) 100 (88–100) 0 (0–79) 100 (82–100) 100 (21–100) 96 (82–99) Taylor [25] 2014 Prospective (MaRIAS) Cardio-vascular Structural and non-structural heart diseases 82 (59–94) 96 (91–98) 83 (44–97) 94 (87–97) 62 (41–79) 98 (93–100) Structural heart defects (major and minor) 83 (61–94) 97 (92–99) 100 (57–100) 93 (85–98) 100 (77–100) 98 (94–100) Major structural heart defects 87 (62–96) 99 (95–100) 100 (51–100) 99 (94–100) 100 (68–100) 100 (97–100) Votino [26] 2012 Prospective (High-field MRI, 9.4 T) Cardio-vascular Abnormalities of the four-chamber view 67 (30–92) 80 (52–95) Outflow-tract-abnormalities 75 (20–96) 100 (83–100) Abnormalities of the aortic arch 100a 100a Abnormalities of the systemic veins 100a 100a Arthurs [27] 2014 Prospective (MaRIAS) Non-cardiac thoracic Non-cardiac thoracic abnormalities 30 (17–47) 96 (91–98) 38 (19–61) 88 (79–94) 45 (33–58) 61 (48–72) Arthurs [28] 2015 Prospective (MaRIAS) Neurological Ischaemic brain injury NA 100 (97–100) 30 (11–60) 90 (81–95) 93 (70–99) 95 (89–98) Major intracranial bleed 100 (21–100) 99 (95–97) 100 (21–100) 100 (96–100) 100 (82–100) 99 (95–100) Minor intracranial bleed 100 (21–100) 87 (80–91) 80 (38–96) 99 (93–100) 100 (44–100) 98 (94–100) Brain malformations 86 (69–94) 90 (83–94) 90 (60–98) 96 (87–99) 100 (57–100) 100 (97–100) Overall brain pathology 87 (71–95) 69 (60–77) 71 (53–84) 77 (64–86) 98 (90–100) 81 (70–89) Arthurs [29] 2015 Prospective (MaRIAS) Abdominal Abdominal abnormalities 77 (61–88) 95 (90–98) 65 (41–83) 89 (80–95) 71 (47–87) 87 (79–92) Arthurs [30] 2014 Prospective (MaRIAS) Musculo-skeletal Skeletal abnormalities 69 (50–84) 100 (97–100) 17 (3–56) 98 (92–99) 31 (13–58) 96 (91–99) 797 Eur J Pediatr (2018) 177:791–803 ventricle (3) was found on PMMRI along with the additional findings of a left choroid plexus cyst (4) and polymicrogyria (5). Foetal-neonatal Furthermore, autopsy diagnosed a choroid plexus papilloma in the left lateral ventricle, but the additional finding of polymicrogyria (5) on PMMRI revealed Aicardi syndrome as the most likely diagnosis. a Axial. b Sagittal Fig. 4 a, b Example of abnormalities of the central nervous system diagnosed at PMMRI in a female foetus. This pregnancy was terminated at 23 weeks of gestation because of corpus callosum agenesis (1), an interhemispheric cyst (2) and fossa posterior anomalies on prenatal 2nd trimester ultrasound, which were confirmed by PMMRI and/or conventional autopsy. A non-cystic dilatation of the fourth ventricle (3) was found on PMMRI along with the additional findings of a left choroid plexus cyst (4) and polymicrogyria (5). Furthermore, autopsy diagnosed a choroid plexus papilloma in the left lateral ventricle, but the additional finding of polymicrogyria (5) on PMMRI revealed Aicardi syndrome as the most likely diagnosis. a Axial. b Sagittal Fig. 4 a, b Example of abnormalities of the central nervous system diagnosed at PMMRI in a female foetus. This pregnancy was terminated at 23 weeks of gestation because of corpus callosum agenesis (1), an interhemispheric cyst (2) and fossa posterior anomalies on prenatal 2nd trimester ultrasound, which were confirmed by PMMRI and/or conventional autopsy. A non-cystic dilatation of the fourth Discussion respectively 94% (95%CI 84–98) and 27% (95%CI 14–44) were reported for the detection of non-infectious and in- fectious pathologies, with specificities of respectively 96% (95%CI 89–99) and 100% (95%CI 96–100). Pneumonia and myocarditis were the main undetected abnormalities. This study showed an increase in the diagnostic accuracy of post-mortem radiology when PMMRI was extended with additional (minimal-invasive, genetic and metabolic) tests or examination of placental tissue. Like in the foetal patient group, MIA showed better results than PMMRI alone. Autopsy is traditionally considered as the gold standard for post-mortem diagnoses and quality assessment of provided health care. However, the declining autopsy rates of the last decennia result in decreasing expertise, especially in foetal- neonatal and paediatric cases where mortality rates are low. Although autopsy remains the preferred diagnostic method in foetal, neonatal, and paediatric death, post-mortem radiology, after consent, can be used in adjunct to autopsy or as an alter- native in cases without consent for conventional autopsy. In general, PMMRI is advised in foetuses, neonates, and young children, as PMMRI has a higher soft-tissue contrast com- pared to PMCT. The small body size enables high-resolution whole-body imaging in a reasonable amount of time. The limited value of PMCT in young children is illustrated in a study of 54 children (median age 1.0 years old, range 2 days– 17.9 years) who died of an assumed natural cause, where PMCT could establish the cause of death in mere 12.9% [34]. In older children, just as in adults, PMCT is the preferred modality because of the lack of evidence of superiority of PMMRI over PMCT, its high availability, lower costs, and reduced scan time compared to PMMRI. With the limited Paediatric 6 Flowchart for post-mortem radiology in paediatric deaths* * The Dutch ‘Nader Onderzoek naar de DoodsOorzaak van Kinderen’ (i e Fig. 6 Flowchart for post-mortem radiology in paediatric deaths. adapted from the Dutch guideline for clinical foetal, neonatal, and paedi- atric post-mortem radiology [17]. PMMRI: post-mortem magnetic reso- nance imaging. PMCT: post-mortem computed tomography. NODOK: The Dutch ‘Nader Onderzoek naar de DoodsOorzaak van Kinderen’ (i.e. ‘further examination of cause of death in children’) procedure is a step- wise approach to investigate the cause of death in children with an as- sumed natural unexpected and unexplained death [34] Paediatric sensitivity and specificity for detection of non-infectious pa- thologies were above 95%. Sensitivity for infectious patholo- gies was with 80% (95%CI 38–96) lower in foetuses above 24 weeks of gestation than in foetuses below 24 weeks of gestation (100%, 95%CI 92–100). A 69% sensitivity (95%CI 58–78) and 93% specificity (95%CI 81–98) were found for major pathological abnor- malities or cause of death in children [31]. Sensitivities of Fig. 5 Flowchart for post-mortem radiology in foetal and neonatal deaths. adapted from the Dutch guideline for clinical foetal, neonatal, and paediatric post-mortem radiology [17]. GA: gestational age. US: ultrasonography. The ‘routine 2nd trimester ultrasound’ is a standard pre- natal US in all growing foetuses. The ‘US for foetal death determination’ is a second, separate antenatal US by the gynaecologist in order to confirm death. PMMRI: post-mortem magnetic resonance imaging. CNS: central nervous system. NODOK:: The Dutch ‘Nader Onderzoek naar de DoodsOorzaak van Kinderen’ (i.e. ‘further examination of cause of death in children’) procedure is a stepwise approach to investigate the cause of death in children with an assumed natural unexpected and un- explained death [34] Fig. 5 Flowchart for post-mortem radiology in foetal and neonatal deaths. adapted from the Dutch guideline for clinical foetal, neonatal, and paediatric post-mortem radiology [17]. GA: gestational age. US: ultrasonography. The ‘routine 2nd trimester ultrasound’ is a standard pre- natal US in all growing foetuses. The ‘US for foetal death determination’ is a second, separate antenatal US by the gynaecologist in order to confirm death. PMMRI: post-mortem magnetic resonance imaging. CNS: central nervous system. NODOK:: The Dutch ‘Nader Onderzoek naar de DoodsOorzaak van Kinderen’ (i.e. ‘further examination of cause of death in children’) procedure is a stepwise approach to investigate the cause of death in children with an assumed natural unexpected and un- explained death [34] Eur J Pediatr (2018) 177:791–803 798 Fig. 6 Flowchart for post-mortem radiology in paediatric deaths. adapted from the Dutch guideline for clinical foetal, neonatal, and paedi- atric post-mortem radiology [17]. PMMRI: post-mortem magnetic reso- nance imaging. PMCT: post-mortem computed tomography. NODOK: The Dutch ‘Nader Onderzoek naar de DoodsOorzaak van Kinderen’ (i.e. ‘further examination of cause of death in children’) procedure is a step- wise approach to investigate the cause of death in children with an as- sumed natural unexpected and unexplained death [34] Fig. Eur J Pediatr (2018) 177:791–803 799 survey or conventional radiographs in natural causes of death. In a study in 542 perinatal deaths (from 16 weeks gestation to 1 week after birth), the diagnostic value was very limited: 30% had abnormal radiographs, of which only 0.9% were of diag- nostic importance for establishing the cause of death [40]. amount of studies in children, it is not possible to be more specific about the age range where PMCT and PMMRI have equal diagnostic performances. The Dutch guideline for pae- diatric post-mortem radiology describes PMCT as a possible adjunct to PMMRI and autopsy, in children of 2 to 5 years of age (Fig. 7). Furthermore, either PMCT or PMMRI is advised in children of 5 years or older, depending on the type of pa- thology expected. Given the limited amount of evidence, we would like to underline that, especially in infants and children, post-mortem imaging should be seen as an adjunct to the autopsy and not as a replacement. The cut-off age levels were the results of combined expert opinion, this as there is insuf- ficient evidence to define a set cut-off age level. Although ultrasound did not meet the inclusion criteria for the guideline it is a technique that could be considered in selected cases where parents do not approve the use of PMCT or PMMRI [41–43]. Due to open sutures and absence of inhaled air, the brain and lungs can be examined by ultra- sonography in cases of foetal demise [44]. In 88 foetuses of 11–40 weeks of gestation sensitivities of 91, 88, and 87% and specificities of respectively 90, 92, and 95% were reported with ultrasound for respectively brain, thoracic and abdominal anomalies [45]. No eligible paediatric studies on post-mortem CR were included. Nevertheless, in deceased children up to 4 years of age, a skeletal survey (consisting of 20–30 images) is advised to detect fractures, potentially caused by non-accidental injury [35–39]. In deceased children of 5 years or older, with possi- ble child abuse, conventional radiographs of the areas of in- terest are advised on a low-threshold basis. This is despite a lack of evidence for the supplementary value of a skeletal To meet the demand for less invasive alternatives to autop- sy [46, 47], as well as a high diagnostic performance, it is likely that a combination of imaging and minimal invasive tissue acquisition will be increasingly used in future. Fig. 8 a, b Example of the difference in resolution between 1.5 (a) and 7 (b) Tesla PMMRI in a foetus of 18 weeks and 2 days of gestation. The 7 T image shows development of polymicrogyria (arrow) of the left temporal cor- tex, which is not detectable at the 1.5 T images Dutch post-mortem imaging guideline The Dutch guideline working group developed an evidence and practice-based flowchart for post-mortem radiology in non-forensic foetal and neonatal deaths (Fig. 5), and pae- diatric deaths (Fig. 6). It must be emphasised that, based on the literature, due to the low GRADE level of evidence, post-mortem radiology without clinical autopsy should be considered as insufficient for best-practice post-mortem diagnosis. PMCTand PMMRI showed a volvulus of the ileum around its mesentery (whirl sign) (arrow). Ischemic haemorrhagic volvulus of the ileum was confirmed by autopsy (b) as the cause of death Fig. 7 a, b Example of a PMCT (a) of a 4-year-old child with an unex- pected and unexplained but assumed natural cause of death. Cardiopulmonary resuscitation was performed but not successful. PMCTand PMMRI showed a volvulus of the ileum around its mesentery (whirl sign) (arrow). Ischemic haemorrhagic volvulus of the ileum was confirmed by autopsy (b) as the cause of death Fig. 7 a, b Example of a PMCT (a) of a 4-year-old child with an unex- pected and unexplained but assumed natural cause of death. Cardiopulmonary resuscitation was performed but not successful. Fig. 9 a, b Examples of normal post-mortem findings. (a) Opacification dorsal in the lung lobes due to septal oedema and pleural fluid (arrow). (b) Distension of bowel lumen due to post-mortem gas formation, and portal venous (1) and ventricular gas (2) Eur J Pediatr (2018) 177:791–803 Other minimal invasive techniques such as genetic and metabolic testing as well as virology and microbiology sampling can a, b Examples of normal mortem findings. (a) fication dorsal in the lung due to septal oedema and l fluid (arrow). (b) nsion of bowel lumen due to mortem gas formation, and venous (1) and ventricular ) Fig. 9 a, b Examples of normal post-mortem findings. (a) Opacification dorsal in the lung lobes due to septal oedema and pleural fluid (arrow). (b) Distension of bowel lumen due to post-mortem gas formation, and portal venous (1) and ventricular gas (2) 800 Eur J Pediatr (2018) 177:791–803 J.J.E. screened studies on title, abstract, and full text for the Dutch guideline development, and developed the Dutch guideline for clinical postmortem radiology. be added on indication. The more post-mortem radiology is expanded with minimally invasive investigations, the higher the diagnostic yield will be; the border area of a minimal invasive radiological test and a restricted autopsy demands for close collaboration between these two specialities. Furthermore, the diagnostic performance of post-mortem ra- diology will increase by improvements of diagnostic tech- niques such a high-field PMMRI (Fig. 8), post-mortem angi- ography, and post-mortem ventilation [24, 48, 49]. Non- or minimally invasive autopsy evokes much less objections from parents compared to conventional autopsy, resulting in overall increasing post-mortem investigation rates [46, 47]. Hence, post-mortem radiology can increase post-mortem investiga- tion rates, and subsequently improve family counselling and quality control of clinical diagnosis. P.K. developed the Dutch guideline for clinical postmortem radiology. P.K. developed the Dutch guideline for clinical postmortem radiology. P.N. developed the Dutch guideline for clinical postmortem radiology. P.N. developed the Dutch guideline for clinical postmortem radiology. R.R. screened studies on title, abstract, and full text for the Dutch guideline development, and developed the Dutch guideline for clinical postmortem radiology. W.K. chaired the Dutch post-mortem imaging guideline group and developed the Dutch guideline for clinical postmortem radiology. All authors contributed to the interpretation of the data and revision of the manuscript for important intellectual content. Funding The development of the Dutch postmortem imaging guideline was funded by the Quality Foundation of the Dutch Medical Specialists (SKMS). Guideline working group This guideline was developed and sponsored by the Radiological Society of the Netherlands (NVvR), using gov- ernmental funding from the Stichting Kwaliteitsgelden Medisch Specialisten in the Netherlands (SKMS, Quality foundation of the Dutch Federation of Medical Specialists). The early preparative phase started July 2015 and the guide- line will officially be authorized by the Radiological Society of the Netherlands at the end of 2017. The working group had nine in-person meetings (between September 2015 and September 2017) and otherwise communicated by phone and email. Decisions were made by consensus. At the start of guideline development, all working group members com- pleted conflict of interest forms. Acknowledgements M.M.J. Ploegmakers (Knowledge Institute of Medical Specialists, Utrecht, The Netherlands), M. Wessels (Knowledge Institute of Medical Specialists, Utrecht, The Netherlands), I.M.B. Russel (Department of Paediatrics, University Medical Center Utrecht, the Netherlands), M. ten Horn (Patientfederation, Utrecht, the Netherlands), R. Kranenburg (Patientfederation, Utrecht, the Netherlands), D. van Meersbergen (The Royal Dutch Medical Association (KNMG), the Netherlands). Compliance with ethical standards As post-mortem radiology is a relatively new subspecialty, images should be evaluated by an experienced radiologist. This should preferably be a paediatric radiologist who is fa- miliar with normal post-mortem changes, which to the un- trained eye can mimic pathologic abnormalities (Fig. 9) [50, 51]. Therefore, it is advised for non-specialised centres to ask assistance from experienced radiologists. Conflict of interest The authors declare that they have no conflict of interest. Appendix 1. Materials and methods To conclude, post-mortem radiology without clinical au- topsy is yet considered as insufficient to establish the cause of death, due to the low GRADE level of evidence. Autopsy is therefore still regarded as the reference standard [23]. Post- mortem radiology, especially as part of a MIA procedure, is considered a useful adjunct or valuable alternative in cases where autopsy is not performed. In general, neonatologists or paediatricians will be the referring physicians and as such they will be the ones obtaining parental informed consent. Therefore, it is imperative that they are aware of the advan- tages and limitations of post-mortem imaging. A multidisci- plinary approach including clinicians, radiologists, and pa- thologists seems most beneficial. At present, PMMRI is the imaging modality of choice in foetuses, neonates, and young children, whereas PMCT is preferred in in older children. Text adjusted from Acta Orthopedica (Besselaar et al [52]; PubMed PMID: 28266239). Text adjusted from Acta Orthopedica (Besselaar et al [52]; PubMed PMID: 28266239). Methodology bodyofevidenceacrossallrelevant outcomes,valuesandprefer- ences, and resource use. The strength of a recommendation re- flects the extent to which the guideline panel was confident that desirableeffectsoftheinterventionoutweighundesirableeffects, or vice versa, across the range of patients for whom the recom- mendation is intended. The strength of a recommendation is de- terminedbyweightingallrelevantargumentstogether,theweight ofthebodyofevidence from thesystematicliterature analysis,as well as the weight of all complementary arguments. Guideline panels must use judgment in integrating these factors to make a strong or weak recommendation. Thus, a low quality of the body of evidence from the systematic literature analysis does not ex- clude a strong recommendation, and weak recommendations may follow from high quality evidence [56]. The guideline was developed in agreement with the criteria set by the advisory committee on guideline development of the Federation of Medical Specialists in the Netherlands (Medisch Specialistische Richtlijnen 2.0; OMS [53], which are based on theAGREEII instrument (Brouwers [54]; www.agreetrust.org). Theguidelinewasdevelopedusinganevidence-basedapproach endorsing GRADE methodology, and meeting all criteria of AGREE-II. Grading of Recommendations Assessment, Development and Evaluation (GRADE) is a systematic ap- proach for synthesizing evidence and grading of recommenda- tions offering transparency at each stage of the guideline devel- opment [55, 56]. The guideline development process involves a number of phases, a preparative phase, development phase, commentary phase, and authorization phase. After authorization, the guide- line has to be disseminated and implemented, and uptake and use have to be evaluated. Finally, the guideline has to be kept up-to-date. Each phase involves a number of practical steps (see Schünemann [57]). After reaching consensus in the working group, the concept guideline was subjected to peer review by all relevant stakeholders. Amendments were made and agreed upon by the working group, and the final text was presented to the Dutch societies of medical specialists and other organiza- tions that participated in the working group for approval and formal authorization. The guideline will be published and be freely accessible in the Dutch guideline database (Richtlijnendatabase, www.richtlijnendatabase.nl). The Dutch guideline database has a modular structure, with each clinical question as a separate entry, thus allowing for modular updates. A methodologist together with the chairman of the working group drafted a concept list of key issues which was exten- sively discussed in the working group. The selected (high priority) issues were translated into carefully formulated clin- ical questions, defining patient/problem, intervention, and pri- oritizing the outcomes relevant for decision-making. Target group and aims This guideline was developed for Dutch radiologists con- cerned with postmortem radiology, and other medical special- ists involved in postmortem diagnostics. The main purpose of the guideline is to provide best possible care to fetuses or neonates, children and their relatives in a postmortem radiol- ogy setting, by informing optimal treatment decisions, and reduce unwarranted variation in the delivery of postmortem diagnostic care. Authors’ contributions L.S. reported on the literature review and Dutch guideline for postmortem radiology/wrote the submitted manuscript, and provided tables and figures for the manuscript. M.V. reported on the literature review and Dutch guideline for post- mortem radiology/wrote the submitted manuscript, and provided tables and figures for the manuscript. E.K. analysed appropriate articles on full text, performed methodolog- ical quality assessment of included studies, and assessed the joint evi- dence of included articles according to the GRADE tool. Eur J Pediatr (2018) 177:791–803 801 Methodology Particular attention was paid to relevant outcomes for relatives of fetuses or children undergoing postmortem radiology and defining minimal clinically important differences. Therefore, a focus group was organized in cooperation with the Federation of Patient Organizations in the Netherlands. Dutch post-mortem imaging guideline group Apart from the named authors, the following persons are the collaborators in the Dutch post-mortem imaging guideline group: W.L.J.M. Duijst (GGD IJsselland Zwolle, the Netherlands, and Maastricht University, the Netherlands), P.A.M. Hofman (Department of Radiology, Maastricht University Medical Center, the Netherlands), J.J.F. Kroll (Department of Radiology, Maastricht University Medical Center, the Netherlands), N.S. Renken (Department of Radiology, Reinier de Graaf Gasthuis, Delft, the Netherlands), Y.O. Rosier (NVMBR Utrecht, the Netherlands), C.I.E. Scheeren (Department of Intensive Care, Zuyderland Heerlen, the Netherlands), S.J. Stomp (GGD, Amsterdam, the Netherlands), P. van der Valk (Department of Pathology, Free University Medical Center, Amsterdam, the Netherlands). Theliteraturewassystematicallysearchedusingthedatabases MEDLINE (Ovid) and Embase (a detailed search strategy is available upon request). Selection of the relevant literature was based on predefined inclusion and exclusion criteria and was carried out by members of the working group (JE, RR) in collab- oration with the methodologist (EK). For each of the clinical questions, the evidence was summarized by the guideline meth- odologist using the GRADE approach: a systematic review was performed for each of the relevant outcomes and the quality of evidencewasassessedinoneoffourgrades(high,moderate,low, very low) by analyzing limitations in study design or execution (risk of bias), inconsistency of results, indirectness of evidence, imprecision, and publication bias. The evidence synthesis was complemented by a working group member (JE or RR) consid- ering any additional arguments relevant to the clinical question, including relatives values and preferences, and resource use (costs, organization of care issues). Evidence synthesis, comple- mentary arguments, and concept recommendations were exten- sivelydiscussedintheworkinggroupandfinalrecommendations were formulated. Final recommendations are based on the bal- ance of desirable and undesirable outcomes, the quality of the 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 appro- priate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. 1. Baglivo M, Winklhofer S, Hatch GM, Ampanozi G, Thali MJ, Ruder TD (2013) The rise of forensic and post-mortem References Burton JL, Underwood J (2007) Clinical, educational, and epide- miological value of autopsy. Lancet 369(9571):1471–1480. https:// doi.org/10.1016/s0140-6736(07)60376-6 8. 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https://openalex.org/W4206453370	https://www.frontiersin.org/articles/10.3389/fmed.2021.799412/pdf	English	null	Highly Engaged Video-Watching Pattern in Asynchronous Online Pharmacology Course in Pre-clinical 4th-Year Medical Students Was Associated With a Good Self-Expectation, Understanding, and Performance	Frontiers in medicine	2,022	cc-by	10,379	ORIGINAL RESEARCH published: 21 January 2022 doi: 10.3389/fmed.2021.799412 Highly Engaged Video-Watching Pattern in Asynchronous Online Pharmacology Course in Pre-clinical 4th-Year Medical Students Was Associated With a Good Self-Expectation, Understanding, and Performance Jann-Yuan Wang 1,2, Chia-Hsien Yang 3, Wei-Chih Liao 1,2, Kai-Chien Yang 1,4, I-Wen Chang 5, Bor-Ching Sheu 1,6* and Yen-Hsuan Ni 1,7 1 Division of Curriculum Integration, Center of Faculty Development, National Taiwan University College of Medicine, Taipei, Taiwan, 2 Department of Internal Medicine, National Taiwan University College of Medicine, Taipei, Taiwan, 3 Ofﬁce of International Affairs and Global Master of Business Administration Program, National Taiwan University College of Management, Taipei, Taiwan, 4 Department and Graduate Institute of Pharmacology, National Taiwan University College of Medicine, Taipei, Taiwan, 5 School of Medicine, National Taiwan University College of Medicine, Taipei, Taiwan, 6 Department of Obstetrics and Gynecology, National Taiwan University College of Medicine, Taipei, Taiwan, 7 Department of Pediatrics, National Taiwan University College of Medicine, Taipei, Taiwan Edited by: Ray Samuriwo, Cardiff University, United Kingdom Reviewed by: Maria Bloksgaard, University of Southern Denmark, Denmark Hosam Eldeen Elsadig Gasmalla, Al-Neelain University, Sudan Reviewed by: Maria Bloksgaard, University of Southern Denmark, Denmark Hosam Eldeen Elsadig Gasmalla, Al-Neelain University, Sudan Background: Online video-based learning is more common in higher education. Investigating students’ viewing behaviors while watching online video lectures is essential for instructors to understand their learning status so that the course content, structure, and media selection can be improved continuously. The current study identiﬁed the engagement level of the learners based on their online video-watching behaviors, and tested the correlation between the engagement level and learning outcome. Correspondence: Bor-Ching Sheu bcsheu@ntu.edu.tw Correspondence: Bor-Ching Sheu bcsheu@ntu.edu.tw Methods: The action logs of watching online video lectures in 2020 Spring Pharmacology of the 4th-year medical students of the 6-year course and their feedbacks by questionnaires after each exam during the semester were provided anonymously. The data were analyzed and visualized for an efﬁcient way to comprehend and interpret. To deﬁne the student’s engagement level in his or her video-based learning journey, three viewing criteria, “Completion,” “Pausing,” and “Repeated watching” were identiﬁed. We evaluated the association between the engagement level and the students’ learning outcomes, including their learning satisfaction, knowledge acquisition progresses based on assessment results, and the grades measured by the instructors. Specialty section: This article was submitted to Healthcare Professions Education, a section of the journal Frontiers in Medicine Received: 21 October 2021 Accepted: 16 December 2021 Published: 21 January 2022 Received: 21 October 2021 Accepted: 16 December 2021 Published: 21 January 2022 Edited by: Ray Samuriwo, Cardiff University, United Kingdom Citation: Wang J-Y, Yang C-H, Liao W-C, Yang K-C, Chang I-W, Sheu B-C and Ni Y-H (2022) Highly Engaged Video-Watching Pattern in Asynchronous Online Pharmacology Course in Pre-clinical 4th-Year Medical Students Was Associated With a Good Self-Expectation, Understanding, and Performance. Front. Med. 8:799412. doi: 10.3389/fmed.2021.799412 Results: The graphs and the charts demonstrate whether the students allocated enough time to ﬁnish the video lectures (completion), paused for a while, then resumed the video (pausing), or replayed the speciﬁc sections of video content (repeated watching). The engagement level with video lectures, evaluated by pre-deﬁned thresholds for “Completion,” “Pausing,” and “Repeated watching” had a positive correlation with the learning outcomes. January 2022 \| Volume 8 \| Article 799412 Frontiers in Medicine \| www.frontiersin.org 1 Wang et al. Video-Watch Pattern and Learning Outcome Conclusions: We suggested that an engagement dashboard containing real-time visualized information on students’ online video-watching behaviors can be developed to help instructors to monitor students’ learning progress and improve teaching in a timely fashion. It can also help each student to re-feel the stimulation of peers, prompt self-monitoring, improve their learning attitudes and disciplines for better learning outcomes. This innovative way of assessing student’s engagement during online video- based learning can also be used for quality assurance purposes. Keywords: engagement, learning analytics, learning outcome, medical education, online video-based learning, video-watching pattern Hypothesis We hypothesized that the engagement level of online video watching can be measured by combinations of learning analytics and may correlate with learning outcome. Assessment of the online video learning process is even more crucial for instructors, given that it may serve as a means of understanding students’ involvement and engagement with the course materials (20). Such information generated by well- designed learning analytics may beneﬁt instructors in several ways, including (1) understanding students’ involvements and performances, (2) improving instruction and assessment in real- time, (3) modifying online teaching materials to accommodate students’ needs, (4) adjusting teaching styles to raise students’ interests and facilitate persistence, and (5) tracking students’ INTRODUCTION Research in learning analytics has revealed that providing a real-time dashboard to check online learning progress and participation level compared with peers can support students’ self-management and facilitate their learning attitudes to improve their comprehensions and learning achievements (19). Therefore, tracing and analyzing the online learning behaviors presents an eﬀective solution for students to manage their learnings better, which reduces the instructor’s workloads in class management. Therefore, we analyzed the video viewing logs, delimited completion, pausing, and repeated watching as the engagement criteria, and tested the feasibility of deﬁning three levels of engagement with video lectures. The correlation between engagement level and learning outcome regarding the learner’s satisfaction and performance was also investigated. Frontiers in Medicine \| www.frontiersin.org INTRODUCTION usage of diﬀerent parts of a learning website to have a better understanding with regards to their learning processes, eﬀectiveness, and suitability (21–23). In other words, the ability to understand students’ online video-based learning activities and evaluate teaching performance is indispensable and helpful for instructors to design online courses and support their students (24). Higher education has increasingly developed and oﬀered online courses as part of their academic curriculum in recent two decades (1–3). It reaches a wide range of audiences and improves teaching and learning environments (4, 5). In an asynchronous online course, the time-independent delivery mode respects student’s autonomy and has beneﬁted students with diﬀerent paces and learning strategies (6, 7). Although students can learn at their own pace freely, they are required to be self-disciplined for eﬀective learning (8). To brace for digital education, we started to discuss and re- design some courses for online video-based learning starting from 2019. However, in 2020 Spring, the required Pharmacology course, initially taught in the classroom with more than 150 pre- clinical medical students (4th year of the 6-year course), was changed to be online video lectures under the COVID-19 crisis. Though the COVID-19 outbreak pushed us moving faster, it also revealed opportunities for teaching innovations. As online courses are more and more common, students often report diﬃculties in attention and commitments to their online courses (9). There is an increasing concern with regards to students’ lack of persistence and engagement, reﬂected by low activity (10, 11) and high dropout rate compared (12–15) with traditional in-person courses. Previous studies demonstrated that lack of persistence, reﬂecting low engagement and poor self-management, is an essential factor leading to attrition among students in online courses and suboptimal academic achievements (11, 16, 17). Therefore, instructors need to track online learners’ engagement status to ensure teaching eﬀectiveness. However, as we adapted online video-based learning to the COVID-19 situation, we experienced challenges in understanding the students’ real-time learning statuses without in-person interactions like we usually do in the classrooms. Although we still had measurements to grade the students’ learning results, we were considering a better solution to know their learning progresses so that we would have references to enhance the course content, media selection, and the lecturing style in the future. g Unlike in-person or hybrid courses, online learning does not involve physical interaction between students and instructors (18). Research Context The research context for this study was a Pharmacology course oﬀered for the pre-clinical senior medical students (4th year of the six-year course) at NTUCM, a top medical school in Taiwan. The course was mandatory for medical students and not open to other majors as an elective course. The course content was delivered by 20 video lectures online asynchronously (9 and 11 video lectures in the ﬁrst and second half of the course, respectively), delivered by 10 teachers. Each video was around 60 min and uploaded weekly to the National Taiwan University COurses OnLine (NTU COOL) system with the related course materials. The Curriculum Committee and the course instructors deﬁne the learning route that drives the students to reach proﬁciency in the semester. The students could arrange their online learning on their own. To brace for online learning, NTU initiated a learning management platform, the NTU COOL, in 2017, which went live oﬃcially in 2019, with the following main features: 1. Fundamental LMS (Learning Management System) Functions: NTU COOL has all necessary functions to support learning in NTU, including the curriculum organization and delivery, course material management, discussion board, learner assessment, and peer evaluation. 1. Fundamental LMS (Learning Management System) Functions: NTU COOL has all necessary functions to support learning in NTU, including the curriculum organization and delivery, course material management, discussion board, learner assessment, and peer evaluation. 2. Video-based Learning: It allows users to upload their videos or to import videos from YouTube. Students can play videos at diﬀerent speeds and jump to speciﬁc segments accordingly to their learning needs. When the network bandwidth is limited, the video resolution can be adjusted accordingly. Both instructors and learners can post comments and reply to each other on speciﬁc parts of videos. Since the students learned from the videos during the outbreak, the instructors could use the reserved in-person class times to foster learning. Before the original in-class times, the teaching assistant would collect the students’ questions, feedback, and comments posted on the NTU COOL system. According to the students’ learning needs, the teaching assistant discussed with the related instructors the online workshop itinerary (independent beyond the online video-learning courses) for question clariﬁcations or further discussions. If nothing were posted, the teaching assistant would consult with the associated instructors whether the students are in fear of appearing foolish, having gaps in knowledge, or hard to form the question. Learning Management System g g y Currently, NTU provides two platforms to manage course delivery, CEIBA and NTU COOL. CEIBA links up with educational administration systems to assist faculty members in creating courses websites, by which instructors can upload courses handouts, announce assignments, and release academic grades. It also provides other functions such as forums, etc., to support the teaching and learning of the courses. Although CEIBA fulﬁlls the fundamental teaching requirements, it is a legacy system without supporting online video-learning. Study Aim This study aimed to investigate whether the undergraduate senior medical students’ online video-watching engagement levels, measured by their viewing action logs, were associated with their online Pharmacology learning outcome. A real-time engagement dashboard can be further developed to facilitate online video- based learning and teaching. January 2022 \| Volume 8 \| Article 799412 2 Video-Watch Pattern and Learning Outcome Wang et al. RESEARCH CONTEXT AND LEARNING MANAGEMENT SYSTEM Though the movement of Pharmacology course from in-person to online video lectures seemed to be a forced shift resulted from the COVID-19 pandemic, the video lectures have been used as part of the teaching materials to improve learning eﬀectiveness before the COVID-19 crisis, based on the advice from the Curriculum Committee at our institution, the National Taiwan University College of Medicine (NTUCM) and the suggestions from students, and alumni. In recent years, many high educational institutions started developing web-based learning in either a hybrid or a purely online mode before the COVID-19 outbreak. The online learning strategy enables educational institutions to implement a learner- centered approach to teaching where learners are given space and ﬂexibility to indulge in constructive learning activities (5, 25, 26). It can lessen teacher’s workload, increase the ﬂexibility and diversity of course designs, support students to manage their own pace of learning, and foster students’ autonomy. Frontiers in Medicine \| www.frontiersin.org Research Questions The students were required to take four online exams and ﬁll out the learning satisfaction questionnaire after each exam. The teaching assistants maintained the syllabus, announced updates, or posted reminders on the course bulletin board over the Collaborative Enhanced Instruction By Asynchronous (CEIBA) course system to ensure all the students were on the same page. 1. How can we deﬁne completion, pausing, and repeated watching by analyzing the online Pharmacology video viewing logs? 2. How can we use completion, pausing, and repeated watching to determine the engagement level? 3. Does the highly engaged learning lead to a better learning outcome in the online pharmacology video lectures? Pausing and Repeated Watching During online video learning, in order to understand better what has been taught in the video, learners may either pause the lecture to take a break, to think and reﬂect, to write a synthesizing note on the content, or to collaborate with a peer, or to hover over the ﬁlmstrip to locate a speciﬁc piece of information to navigate and repeat explanations until they are fully understood (40). In a study investigating the underlying meaning of activities during online video learning with ViDeX, Seo and his colleagues found that during exam week, students “Search” more and “Reﬂect” less, probably in an attempt to be more eﬃcient and mindful in their time in seeking video segments that they perceive are valuable for the exam (40). The way of time utilization is another sign of strategic engagement in online video learning, implying that students selectively pick parts that they consider vitally important to re-watch (41, 42). When the COVID-19 outbreak happened, in-person classes of more than 60 students at NTU had to be distance learning, according to the guidance announced by the Central Epidemic Command Center (CECC) of Taiwan Centers for Disease Control (TCDC). As of more than 150 students, the face-to- face Pharmacology class was soon replaced by the online video lectures and virtual workshops for question clariﬁcations or group discussions since April 6th, 2020 (2nd semester). Study Regularity and Timing Study regularity has also been acknowledged as a strong indicator of self-regulated learning in asynchronous online courses, representing learners’ earnestness and engagement in this learning environment. These self-regulated learners work hard, regularly allocate their time and, overcome obstacles on the way forward (43, 44). In a study analyzing log data from 284 undergraduate students in an asynchronous online statistics course, Kim et al. noted that self-regulated learners were more likely to study regularly and demonstrated consistent commitment to their learnings, leading to signiﬁcantly higher ﬁnal grades (35). Asynchronous online video learning is a self-regulated learning process involving systematic eﬀorts, including planning, conducting, regulating, and evaluating to attain learning goals (27, 28) to transform the learner’s mental abilities into academic skills (29). Since 1960, educational videos have been used as learning aids in medical education (30). Though online video learning has become a feasible and popular learning module that can be applied to clinical medicine (31), its success in medical education requires learners’ self-regulated learning and active engagement (32). In general, online video-based learnings are more autonomous than traditional face-to-face or blended courses (33). Therefore, it is vital to identify prominent characteristics or strategies leading to successful learning in asynchronous online video-earning environments (34). Moreover, understanding the video-watching behaviors of students over time is crucial to provide timely instructional support for improving both teaching and learning. Previous studies have identiﬁed and validated several attributes of asynchronous online video learning, detailed in the following. In the previously mentioned study done by Seo and his colleagues (40), the authors found that the video viewing behavior in exam weeks (the one week before students take the exam) was diﬀerent from that in non-exam weeks. Students “Clarify” less during exam weeks probably because the activity of “Clarify,” such as rewinding and reducing playback speed, increases engagement time with the video. Usually, this activity implies that learners intend to understand information comprehensively or to clarify video segments. Speciﬁcally, content learning no later than two weeks before exams signiﬁcantly increases the probability of being a self- regulated learner and implies that the learner engages in content learning with suﬃcient time. In contrast, students who studied right before or during the exam week are likely to have a learning pattern of cramming (29). Research Context Based on the previous teaching experiences or feedback from the alumni, the associated instructors would guide the teaching assistant to conduct an oral quiz to reﬂect students’ learning or oﬀer Supplementary Materials to enhance students’ understanding in the workshop. Otherwise, the associated instructors might suspend online workshops one time, regardless of whether they considered students had engaged and learned. The learning outcome would then be evaluated by mid-term and ﬁnal exams. 3. Interaction Environment: In addition to the bulletin board or discussion board, instructors can set up diﬀerent forums by subject or group to facilitate teaching eﬀectiveness. Recently, a new feature, named “Symphony,” was launched for users to exchange ideas. Teachers can release text ﬁles (PDF) onto the system, and students can mark on the languages to comment or clarify their questions. 4. Tracking and Report: NTU COOL visualizes some learning data of students, including their video-viewing time, activity participation records, and learning progresses. Those graphs are valuable tools for both instructors and students to understand their learning situations. Since NTU COOL is more advanced and certiﬁed by ISO 27001, the Oﬃce of Academic Aﬀairs has decided to stop the service January 2022 \| Volume 8 \| Article 799412 3 Video-Watch Pattern and Learning Outcome Wang et al. of CEIBA on August 1, 2022, after several years of operation in parallel to ease user anxiety and reduce replacement risk. of CEIBA on August 1, 2022, after several years of operation in parallel to ease user anxiety and reduce replacement risk. one of the fundamental dimensions of engagement and has been validated to positively impact students’ achievements, ﬁnal grades, and retentions in the online courses (37, 38). Speciﬁcally, imperative attributes for time spent may include the total number of online sessions completed (4, 22), hours viewing main content (22, 39), time spent in each week (35, 40), and time spent on learning tools (27). Detailed introduction and usage of the NTU COOL system are available on the website (https://www.dlc.ntu.edu.tw/en/ coolsupport/). A series of screenshots have been added in the online Supplementary File to illustrate the NTU COOL learning platform (Supplementary Figure 4 in the Supplementary File). The videos were Microsoft PowerPoint slideshow with the instructor’s audio narration. These were uploaded to the integrated organ system-based online scaﬀolding learning modules in the NTU COOL system for students’ convenience to access and view. Frontiers in Medicine \| www.frontiersin.org Deﬁnition of Parameters A total of 11 parameters (Table 1) were used to measure the students’ online video-watching behaviors in this study, including ﬁve for time investment, one for pausing, one for repeated watching, and four for regularity and timing. Time Investment Asynchronous online courses require students to study on their own without the instructor’s direct supervision. Therefore, time spent on video content of asynchronous online courses is a fundamental issue in understanding course topics and successful learning (35). Having suﬃcient time is particularly important in asynchronous online courses because learning time depends entirely on students’ perceptions of the importance of the study and students’ decisions and abilities to secure and allocate time for learning activities (36). Time spent is thus regarded as Learning analytics is a signiﬁcant asset for discovering crucial information and knowledge from an education setting to assess learning eﬀectiveness and improve teaching (45, 46). In this study, the learners’ anonymous video-viewing data and course feedback were collected and analyzed. The parameters were Frontiers in Medicine \| www.frontiersin.org January 2022 \| Volume 8 \| Article 799412 4 Video-Watch Pattern and Learning Outcome Wang et al. developed to generate various attributes of viewing behaviors, which were visualized and used to deﬁne engagement levels with online Pharmacology video lectures. four senior tutors and one teaching assistant from the Center of Faculty Development (CFD) of NTUCM in 2010 for assessing the quality and eﬃcacy of teaching, rather than for research purpose. The questionnaires have been used for years and revised several times. Five key questions in the questionnaires assessing students’ learning expectation, satisfaction, and eﬃcacy by using 5-point Likert scale system (strongly disagree, disagree, neutral, agree, strongly agree) (47) were analyzed in the study (Supplementary Table 1 in online Supplementary File). METHODS First, we analyzed the students’ video viewing logs to identify the parameters of time investment, the situation of pausing and repeated watching, learning regularity, and watching timing. The associated parameters determined three key viewing behaviors (completion, pausing, and repeated watching) and evaluated their video usage’s time investment and regularity. Secondly, we deﬁned their online video learning engagement level (high, intermediate, or low) based on the composition of the three key viewing behaviors. Lastly, the student’s learning satisfaction, self- assessment of learning eﬀectiveness, and grades were compared among students with diﬀerent engagement levels. Participants a) The ﬁnished rate, “the watched video length (excluding re-watch) divided by the video length” (unit: %), was the percent of the video the audience watched. Initially, a total of 155 pre-clinical medical students (4th year of 6-year course) were enrolled in the course. Three of the 155 students were repeating the course for a second time, and one student neither ﬁnished the questionnaires after the exams nor provided his feedback during the whole semester. Therefore, we used the data from a total of 149 students for further analyses. The 149 participants were all 4th-year medical students of the 6-year course. Among them, 111 (74.5%) were male. The students were informed that completion of questionnaires was compulsory but would not be accounted for grading. b) The watch rate, “the watch time (including re-watch) divided by the video length” (unit: %), was performed to understand how much more or less time the students were watched compared to the total video length. c) The engaged-view rate, the percentage of videos viewed in high engagement level (unit: %), was to understand the high engagement situation of the class as a whole. The deﬁnitions of engagement levels are stated in Section 4.6. d) The initial watch rate, “sum of each initial video-watching time divided by the total length of all videos” (unit: %), was to know the students’ learning activation status. The “initial” referred to the ﬁrst time seeing a video. Frontiers in Medicine \| www.frontiersin.org Data Collection and Processing Students’ video-viewing data (action logs), grades, course feedback, and questionnaires data as well as their personal information were stored in the Digital Learning Center, National Taiwan University (NTM-DLC). After the Institutional Review Board (IRB) approved our research proposal, the de-identiﬁed data (anonymous to the users) in CSV (comma-separated values) ﬁle was provided by the Digital Learning Center, National Taiwan University (NTU-DLC) after the course was completed. For the convenience of analysis, the ﬁrst two and the last two exam scores were averaged separately to be used as their exam results for the ﬁrst and the second half- semester, respectively. e) The initial learning progress, “the sum of each initial video- watching time divided by the total video-watching time” (unit: %), was developed to understand the students’ initial learning progress compared to their total watching time. The “initial” referred to the ﬁrst time seeing a video. GURE 1 \| Examples of high engagement (upper panel), intermediate engagement (middle panel) and low engagement (lower panel) with video-based learning FIGURE 1 \| Examples of high engagement (upper panel), intermediate engagement (middle panel) and low engagement (lower panel) with video-based learning (X: play rate). FIGURE 1 \| Examples of high engagement (upper panel), intermediate engagement (middle panel) and low engagement (lower panel) with video-based learning (X: play rate) FIGURE 1 \| Examples of high engagement (upper panel), intermediate engagement (middle panel) and low engagement (lower panel) with video-based learning (X: play rate) FIGURE 1 \| Examples of high engagement (upper panel), intermediate engagement (middle panel) and low engagement (lower p (X: play rate). Frontiers in Medicine \| www.frontiersin.org 2. Pausing: 2. Pausing: The pause rate, “total paused duration divided by total video length” (unit: %), was computed to understand how long a student paused the videos during online video-watching. JYW processed the raw data of action logs of each participant using Microsoft Access. The action logs were visualized to express watching speed, rewinding, repetition, and pausing. Furthermore, three online video-viewing behaviors, completeness, pausing, and repeated watching (Figure 1) were identiﬁed (23). 3. Repeated watching: The re-watch rate, “the sum of video seconds watched for three or more times divided by total video length” (unit: %), gave us an overview regarding the proportion of the video length being repeatedly watched. In this study, a threshold for a parameter of video-watching behavior was initially selected by the best dichotomization for correlation with grades. In cases that two cutoﬀpoints performed equally well, the threshold was further determined by the consensus of JYW, CHY, and IWC. 4. Regularity and timing: The following measurements were determined for each student to investigate their learning regularity and timing. a) Learning days between two exams: number of days in watching videos between two exams (unit: day). The questionnaires were initially created by a committee, which consisted of the program directors from course-related departments, two pre-clinical senior medical students, and b) When to start learning right after an exam: how many days after an exam to start watching the video (unit: day). January 2022 \| Volume 8 \| Article 799412 5 Wang et al. Video-Watch Pattern and Learning Outcome \| Examples of high engagement (upper panel), intermediate engagement (middle panel) and low engagement (lower panel) with video-based learning e). Deﬁnition of Engagement Level c) The view rate before exam week: the number of exam videos viewed before exam week divided by the total number of exam videos (unit: %) was performed to learn their exam preparation activation status before exam week. Video learning engagement is related to students’ time investment and interactions with the video content. Therefore, we used the ﬁnished rate, the pausing rate, and the re-watch rate to identify three key watching behaviors, “Completion,” “Pausing,” and “Repeated Watching.” d) The initial ﬁnished rate before exam week: “the sum of the initial video watch length (excluding re-watch) between two exams and before exam week divided by the total length of the watched videos before exam week” (unit: %), was used to understand their exam preparation progress. “Completion” was deﬁned as the ﬁnished rate >90%, “Pausing” was deﬁned as the pausing rate ≥10%, and “Repeated watching” was deﬁned as repeated watching rate >3%. A Frontiers in Medicine \| www.frontiersin.org January 2022 \| Volume 8 \| Article 799412 6 Video-Watch Pattern and Learning Outcome Wang et al. TABLE 1 \| Learning analytics metrics glossary and deﬁnition. Terms Equation/Deﬁnition Measurement in the scenario Finished rate (%) Total video length been watched Total video length 270/540 = 50.0% Watch rate (%) Total video−watching time Total video length 360/540 = 66.7% Initial watch rate (%) Sum of each initial video−watching time Total video length 180/540 = 33.3% Pause rate (%) Total paused duration Total video length 10.8/540 = 2.0% Re-watch rate (%) Video length been watched≥3times Total video length 5.4/540 = 1.0% Engaged-view rate (%) No. of videos been watched in highly engaged mode Total No. of videos 1/9 = 11.1% Initial learning progress (%) Sum of each initial video−watching time Total video−watching time 180/360 = 50.0% Learning days between two exams (day) No. of days in watching videos between two exams 8/50 = 16.0% When to start learning right after an exam (day) How many days after an exam to start watching the video 15 View rate before exam week (%) No. of videos been initially watched before exam week Total No. of videos between 2 exams 3/9 = 33.3% Initial ﬁnished rate before exam week (%) Video length been initially watched before exam week Total length of videos been initially watched before exam week 45/180 = 25.0% The table below explains the terms we used in this study. Deﬁnition of Engagement Level A student started watching the video on the 15th day of the semester. In eight days, he spent 360 min watching 5 videos. Among these 5 watched videos, only one was viewed at a high engagement level. In addition, the watched length of these 5 videos is 270 min and the sum of the ﬁrst watch time in these 5 videos is 180 min. In the 360-min watch time, a total of 10.8 min were paused. During the 270-min watched video length, there were 5.4-min re-watched more than three times. Before exam week, 3 videos were viewed. The sum of the ﬁrst watch time for those 3 videos is 45 min. The table below is his video-watching measurements. (Abbreviation: No., number). The table below explains the terms we used in this study. Following is the scenario to provide a case example. There are nine 60-min videos in the ﬁrst half of the course. A student started watching the video on the 15th day of the semester. In eight days, he spent 360 min watching 5 videos. Among these 5 watched videos, only one was viewed at a high engagement level. In addition, the watched length of these 5 videos is 270 min and the sum of the ﬁrst watch time in these 5 videos is 180 min. In the 360-min watch time, a total of 10.8 min were paused. During the 270-min watched video length, there were 5.4-min re-watched more than three times. Before exam week, 3 videos were viewed. The sum of the ﬁrst watch time for those 3 videos is 45 min. The table below is his video-watching measurements. (Abbreviation: No., number). half (median: 99.2 vs. 60.5 vs. 0%, p < 0.001) and the second half (median: 92.8 vs. 56.4 vs. 0%, p < 0.001) of the online course (Figure 2, left panel). Similarly, the pause rate was signiﬁcantly diﬀerent among the 3 groups of participants in the ﬁrst half (median: 42.8 vs. 24.4 vs. 0%, p < 0.001) and the second half (median: 57.4 vs. 25.4 vs. 0%, p < 0.001) of the course (Figure 2, middle panel). The re-watch rate was also diﬀerent among the 3 groups in the ﬁrst half (median: 2.2 vs. 0.5 vs. 0%, p < 0.001) and the second half (median: 4.2 vs. 0.6 vs. 0%, p < 0.001) of the course (Figure 2, right panel). Deﬁnition of Engagement Level video-watching status was considered highly engaged in the presence of 2 or 3 attributes, intermediately engaged in the presence of only one attribute, and low engaged in the absence of all 3 attributes. Statistical Analysis Quantitative data were expressed in percentages where appropriate. Non-parametric tests were employed to compare group diﬀerences. Categorical variables were compared using the chi-squared test, whereas Mann–Whitney U and the Kruskal–Wallis tests were employed to compare the diﬀerence in continuous variables for 2 and 3 groups, respectively. The Pearson correlation coeﬃcient assessed the correlation between two variables. A two-sided p < 0.05 was considered statistically signiﬁcant. Statistical analyses were performed using SPSS version 21.0 (SPSS Inc., Chicago, IL, USA). Other characteristics of online video-watching among the 3 groups with diﬀerent levels of engagement were shown in Figure 3. Statistical analyses revealed that the watching rate (Figure 3, left upper panel), the engaged-view rate (Figure 3, upper center panel), the initial watch rate (Figure 3, right upper panel), and the initial learning progress (Figure 3, left middle panel) (all p < 0.001) of highly engaged participants were higher than the other two groups. Online Video-Watching Behaviors in Online Video-Watching Behaviors in Different Engagement Status Deﬁnition of Engagement Level Following is the scenario to provide a case example. There are nine 60-min videos in the ﬁrst half of the course. A student started watching the video on the 15th day of the semester. In eight days, he spent 360 min watching 5 videos. Among these 5 watched videos, only one was viewed at a high engagement level. In addition, the watched length of these 5 videos is 270 min and the sum of the ﬁrst watch time in these 5 videos is 180 min. In the 360-min watch time, a total of 10.8 min were paused. During the 270-min watched video length, there were 5.4-min re-watched more than three times. Before exam week, 3 videos were viewed. The sum of the ﬁrst watch time for those 3 videos is 45 min. The table below is his video-watching measurements. (Abbreviation: No., number). TABLE 1 \| Learning analytics metrics glossary and deﬁnition. Measurement in the scenario Finished rate (%) Watch rate (%) Initial watch rate (%) Pause rate (%) Re-watch rate (%) Engaged-view rate (%) Initial learning progress (%) Learning days between two exams (day) When to start learning right after an exam (day) View rate before exam week (%) Initial ﬁnished rate before exam week (%) The table below explains the terms we used in this study. Following is the scenario to provide a case example. There are nine 60-min videos in the ﬁrst half of the course. A student started watching the video on the 15th day of the semester. In eight days, he spent 360 min watching 5 videos. Among these 5 watched videos, only one was viewed at a high engagement level. In addition, the watched length of these 5 videos is 270 min and the sum of the ﬁrst watch time in these 5 videos is 180 min. In the 360-min watch time, a total of 10.8 min were paused. During the 270-min watched video length, there were 5.4-min re-watched more than three times. Before exam week, 3 videos were viewed. The sum of the ﬁrst watch time for those 3 videos is 45 min. The table below is his video-watching measurements. (Abbreviation: No., number). The table below explains the terms we used in this study. Following is the scenario to provide a case example. There are nine 60-min videos in the ﬁrst half of the course. RESULTS The highly engaged group spent more learning days between two exams (Figure 3, center panel) during the whole semester, began to watch the videos earlier than the other two groups in the second half (p = 0.003) of the course, but had no diﬀerence in the ﬁrst half (p = 0.107) of the course (Figure 3, right middle panel). The highly engaged group also had a higher view rate before exam week (Figure 3, left lower panel) and a better initial ﬁnished rate before exam week (Figure 3, lower center panel) (all p < 0.001). Among the 149 students, 46 (30.9%), 42 (28.2%), and 61 (40.9%) students were considered as highly engaged, intermediately engaged, and low engaged in the ﬁrst half of the course; while during the second half of the course, 30 (20.1%), 48 (32.2%), and 71 (47.7%), respectively. The engagement status in the ﬁrst and the second half of the online Pharmacology course was signiﬁcantly correlated (Pearson correlation coeﬃcient = 0.557) (Table 2). The illustrations of high, intermediate, and low engagement with online video-watching were shown in Figure 1. Unlike the latter two, the high engagement composed 2 or 3 viewing behaviors of completion, pausing, and re-watching. Engagement Status and Learning Outcome Statistical analysis showed that the exam grades were signiﬁcantly diﬀerent (p < 0.001 in both the ﬁrst and second half of the course) among the 3 groups with diﬀerent levels of engagement. Not surprisingly, the high-engaged group had the best exam result (Figure 3, right lower panel). Online Video-Watching Behaviors in Different Engagement Status Different Engagement Status The ﬁnished rate was signiﬁcantly diﬀerent among participants with high, intermediate, and low levels of engagement in the ﬁrst Results of survey questionnaires were shown in Supplementary Table 2 in online Supplementary File. For January 2022 \| Volume 8 \| Article 799412 Frontiers in Medicine \| www.frontiersin.org 7 Video-Watch Pattern and Learning Outcome Wang et al. TABLE 2 \| Correlations between the engagement status in ﬁrst and second half of Pharmacology course (Pearson correlation coefﬁcient = 0.557). Engagement status in second half of Pharmacology course Low Intermediate High Engagement status in ﬁrst half of Pharmacology course Low 47 (31.3%) 11 (7.3%) 4 (2.7%) Intermediate 18 (12.0%) 21 (14.0%) 3 (2.0%) High 7 (4.7%) 16 (10.7%) 23 (15.3%) FIGURE 2 \| The differences in the ﬁnished rate, the pause rate, and the re-watch rate among the students with different engagement levels (p < 0.001 for all). represents extreme value; ◦represents potential outlier. TABLE 2 \| Correlations between the engagement status in ﬁrst and second half of Pharmacology course (Pearson correlation coefﬁcient = 0.557). FIGURE 2 \| The differences in the ﬁnished rate, the pause rate, and the re-watch rate among the students with different engagement levels (p < 0.001 for all). represents extreme value; ◦represents potential outlier. FIGURE 2 \| The differences in the ﬁnished rate, the pause rate, and the re-watch rate among the students with different engagement levels (p < 0.001 for all). represents extreme value; ◦represents potential outlier FIGURE 2 \| The differences in the ﬁnished rate, the pause rate, and the re-watch rate among the students with different engagem represents extreme value; ◦represents potential outlier. in the ﬁnished rate, the pause rate, and the re-watch rate among the students with different engagement levels (p < 0.001 for all). represents potential outlier. lower center panel) and initial ﬁnished rate before exam week (Figure 4, right lower panel) (all p < 0.001). each of the ﬁve questions, <5% of students reported either disagree or strongly disagree. In the ﬁrst half of the course, the high engagement was associated with better learning outcomes, including a higher self- learning satisfaction and a better understanding of the underlying concepts. In addition, the students who learned in high- engaged mode expressed that they could connect Pharmacology with other classes more eﬃciently and used what they had learned from Pharmacology to construct the understandings of other subjects more widely (Table 3). DISCUSSION The study demonstrated three signiﬁcant ﬁndings. First, the visualization plot (Figure 1) we constructed from action logs provides a very intuitive presentation on the student’s engagement status in online video learning. Furthermore, using these three essential viewing behaviors, “Completion,” “Pausing,” and “Repeated Watching” can measure the students’ engagement levels with the online video lectures eﬀectively. Second, learning analytics revealed that the students with diﬀerent levels of engagement have completely distinct online video-watching behaviors. Third, the engagement level with online video lectures is positively correlated with self-learning satisfaction, concept understanding, integrations among various subjects, and grades. Online Video-Watching Behaviors in Different Engagement Status Though the ﬁndings were similar in the second half of the course, the diﬀerences failed to reach statistical signiﬁcance except for understanding underlying concepts. Frontiers in Medicine \| www.frontiersin.org Changing of Engagement Level and Learning Outcome As the course progressed, 18 (12.1%) participants enhanced their engagement, 90 (60.4%) remained at the same level, and 41 (27.5%) became less engaged (Table 4). Compared students’ grade rankings between the ﬁrst and the second half of the semester, 78, 47, and 42% of each track showed the class ranking improvement, respectively (p = 0.030). When a teacher is teaching in a physical classroom, there are plenty of visible clues to manage the class. The teacher can look for participation patterns, body language, and other non-verbal cues that give some sense of what students might be thinking. The observation and interaction are invaluable because it helps inform the teacher what the next steps are. With online learning, especially asynchronous online video lectures, this rich, analog information stream from a traditional in-person classroom has turned into a multichannel digital data stream in the virtual classroom. The data is very diﬀerent, and teachers are much less experienced at interpreting it. The changing of engagement levels implied their learning behavior adjustments. Those who became more engaged showed a better ﬁnished rate (Figure 4, left upper panel), pause rate (Figure 4, upper center panel), re-watch rate (Figure 4, right upper panel), watch rate (Figure 4, left middle panel), engaged- view rate (Figure 4, center), initial watch rate (Figure 4, right middle panel), and view rate before exam week (Figure 4, January 2022 \| Volume 8 \| Article 799412 8 Wang et al. Video-Watch Pattern and Learning Outcome FIGURE 3 \| Online video-watching characteristics among the 3 groups with different engagement levels (p < 0.001 for all unless otherwise mentioned) (Abbreviation: No., number). represents extreme value; ◦represents potential outlier. FIGURE 3 \| Online video-watching characteristics among the 3 groups with different engagement levels (p < 0.001 for all unless otherwise mentioned) (Abbreviation: No., number). represents extreme value; ◦represents potential outlier. In a study enrolling psychology students at an Australian university, 67 participants provided 102 reasons in justifying their modality preferences about completing the class face- to-face rather than face-to-screen (48). The two themes that emerged were “more engagement” and “immediate feedback.” When an instructor teaches an online course, a great deal of time is required to learn about the learners and their needs (49). Frontiers in Medicine \| www.frontiersin.org LIMITATIONS AND FURTHER WORK However, they cannot capture whether a student is actively paying attention to the video or just playing it in the background while multitasking (53). On the contrary, pausing, rewinding, or repeating videos may represent the purpose of driving attention to certain parts of the learning material (54). • Students were required to provide feedback and response to the questionnaires in the Pharmacology course, which may introduce bias because the answers we obtain in a mandatory questionnaire can diﬀer from optional ones. • Students were required to provide feedback and response to the questionnaires in the Pharmacology course, which may introduce bias because the answers we obtain in a mandatory questionnaire can diﬀer from optional ones. By simultaneously incorporating three fundamental elements (completion, pausing, and repeated watching) of active viewing behavior, the visualization plot (Figure 1) we constructed can provide a very intuitive interpretation of the student’s engagement level in online video learning. The instructor can quickly realize whether the learners have allocated enough time for self-learning (completion), generated curiosity and questions about the video content (repeated watching), and tried to resolve their questions (pausing). Furthermore, an engagement dashboard containing real-time visualizations that provide insight into how students are engaging with their online video lectures can be designed for instructors to monitor their learning status. With the intuitional presentation on the dashboard, instructors can quickly identify at-risk learners and provide timely assistance to get them back to track (55). Indeed, ways to measure students’ engagement in online video lectures and blended learning courses remains an unmet need. • It is important not to judge students as active or passive learners based on the predicted level of engagement and video- watching behaviors alone (58) because watching itself can be considered an active process. On the other hand, we cannot know precisely that this “watching” occurs while the videos are playing. • Those 149 4th-year medical students are from a top medical school in Taiwan. They should have always been serious about learning and studying hard since early in their school stage. However, they may not represent medical students in other generations. Furthermore, the ﬁndings in this study should be validated in western countries and college students other than medicine. The learning pattern in the Pharmacology course may not be the same as that in other courses. Changing of Engagement Level and Learning Outcome Since there is no facial expressions nor body language that can alert the teachers, teachers need constantly look for other information that can help them understand the impact of their decisions and continually ask the learners to provide feedback to know where the students are, how they feel, do they appear engaged. Teachers do need data to help them answer these questions. Therefore, teachers must encourage students to express themselves in writing as much as possible so teachers can sense whether everyone is on the same page. However, writing may not suit everyone and may be diﬃcult for some. For example, Asian students tend to be passive learners and seldom ask questions or participate in class discussions (50–52). Available studies on video-based learning lack suﬃcient understanding of how students’ behaviors are related to their engagements with video content. Many studies measured engagement based on the duration of students’ viewing patterns (53) or whether they navigate away from a video before completion (42). Notably, these measures focus on whether students accessed and covered diﬀerent parts of the video. January 2022 \| Volume 8 \| Article 799412 9 Wang et al. Video-Watch Pattern and Learning Outcome LIMITATIONS AND FURTHER WORK The threshold for “Completion,” “Pausing,” “Repeated watching” in determining the level of engagement may be diﬀerent from various participants. Though we showed that the student’s engagement level was positively correlated with the learning outcome, the measurement of engagement level did not directly assess the educational objectives and learning goals, as presented in the six hierarchical levels of Bloom’s Taxonomy (56, 57). In this study, more than 50% of the students had a low engagement level (either low in both the ﬁrst and second half of the course, or a low-intermediate combination) and more than 50% with low engagement level answered “no” to the question “Does pharmacology meet your learning expectations.” Therefore, the teacher should worry that the engagement level may not be a good measure of learning, in particular if it can stand alone. In fact, we should not use a single dimension to measure learning eﬀectiveness. Given that this study was designed to deﬁne engagement level by video-watching patterns and correlate engagement level with general assessment of learning outcomes, further studies on the correlation between engagement level and the achievement of diﬀerent learning goals should be performed. • The questionnaires used in this study was for the routine assessment of the quality and eﬃcacy of teaching. They were not developed in a scientiﬁcally rigorous way. Validation of the questionnaires should be done. • Self-eﬃcacy, deﬁned as the conﬁdence to carry out the courses of action necessary to accomplish desired goals, plays an important role in inﬂuencing achievement outcomes through its dynamic interplay with environmental and behavioral determinants (59, 60). Though we recognize the importance of motivating the students to increase their self-eﬃcacy especially for online learning, the current study is a purely observation design. We are currently planning to build an engagement dashboard on the NTU COOL platform. We look forward to seeing whether the learning behaviors are changed and learning outcomes are improved by the engagement dashboard. Frontiers in Medicine \| www.frontiersin.org ngag n = 7 %) %) %) %) CONCLUSION With the visualization plot and algorithm which we constructed to measure the engagement status of online video learning, the teachers could realize the extent of students’ involvement and engagement, identify parts of the video that are diﬃcult to understand. Therefore, the teachers can re-design video content, supplement video clips, or critically discuss the problematic parts in the next oﬃce hours. Because students in online video classes often feel more disconnected from their peers and lecturers (35), the engagement dashboard containing their engagement status and learning behaviors compared with the others can also help learners re-feel the stimulation of peers, prompt self-monitoring, and increase understanding of the learning content (55). In addition, this innovative way of assessing student’s engagement during online video-based learning can be used for quality assurance purposes. Since online video-based learning is more common in higher education, learning analytics is essential for instructors to understand students’ engagement situations and evaluate teaching eﬀectiveness for continuous improvements. This study developed the pre-deﬁned thresholds and algorithms on how the pre-clinical medical students’ video viewing logs of online Pharmacology course can be analyzed and visualized more intuitively to present their learning situations. Furthermore, the high, intermediate, and low engagement level with online video-based lectures was deﬁned by the composition of diﬀerent learning behaviors of completion, pausing, and repeated watching. The study results showed that the highly engaged students had a better learning outcome, higher learning satisfaction and were more beneﬁcial in knowledge construction and integration. Frontiers in Medicine \| www.frontiersin.org January 2022 \| Volume 8 \| Article 799412 10 Second half of the online Pharmacology course ngagement = 71) Intermediate engagement (n = 48) High engagement (n = 30) P Yes No Yes No Yes %) 29 (41%) 25 (52%) 23 (48%) 12 (40%) 18 (60%) 0.209 %) 37 (52%) 9 (19%) 39 (81%) 7 (23%) 23 (77%) 0.002 %) 55 (77%) 11 (23%) 37 (77%) 2 (7%) 28 (93%) 0.140 %) 56 (79%) 11 (23%) 37 (77%) 2 (7%) 28 (93%) 0.159 %) 55 (77%) 10 (21%) 38 (79%) 2 (7%) 28 (93%) 0.159 59 59 Video-Watch Pattern and Learning Outcome Wang et al. ranking between ﬁrst and second halves of the Pharmacology course, stratiﬁed by the change in engagement status of online 030 for inter-group difference by chi-square test). ETHICS STATEMENT The studies involving human participants were reviewed and approved by Research Ethics Committee (REC) of National Taiwan University Hospital (NTUH), Taipei, Taiwan. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements. FUNDING The engagement dashboard containing students’ engagement status and learning behaviors compared with the others can also be customized and provided for each student to re-feel the stimulation of peers, prompt self-monitoring, improve their learning attitudes and disciplines for better learning outcomes. The study was supported by the High Education Sprout Project of the Ministry of Education (NTU-109L3901; NTU-110L3901). The funders had no role in the study design, data collection and analysis, decision to publish, or manuscript preparation. ACKNOWLEDGMENTS The datasets used and analyzed during the current study are available from the corresponding author upon reasonable request. We thank the student participants for providing feedback generously and the Center of Faculty Development, NTUCM, and Center for Teaching and Learning Development, Digital Learning Center, National Taiwan University, for their technical assistance. REFERENCES 10. Clay MN, Rowland S, Packard A. Improving undergraduate online retention through gated advisement and redundant communication. J Coll Stud Retent Res Theory Pract. (2008) 10:93–102. doi: 10.2190/CS.10.1.g 1. Cohen A, Soﬀer T. Academic instruction in a digital world: the virtual TAU case. Proc Soc Behav Sci. (2015) 177:9–16. doi: 10.1016/j.sbspro.2015. 02.322 Res Theory Pract. (2008) 10:93–102. doi: 10.2190/CS.10.1.g 11. Otter RR, Seipel S, GraeﬀT, Alexander B, Boraiko C, Gray J, et al. Comparing student and faculty perceptions of online and traditional courses. Int Higher Educ. (2013) 19:27–35. doi: 10.1016/j.iheduc.2013.08.001 2. Lee L. Autonomous Learning Through Task-Based Instruction in Fully Online Language Courses. 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Change in engagement status of online video-watching No. of students No. (%) of students with improve in grades ranking No. (%) of students without improve in grades ranking More engaged 18 14 (78%) 4 (22%) Stationary 90 42 (47%) 48 (53%) Less engaged 41 17 (42%) 24 (58%) FIGURE 4 \| Engagement level changes and watching behavior adjustments (p < 0.001 for all unless otherwise mentioned). represents extreme value; ◦represents potential outlier. FIGURE 4 \| Engagement level changes and watching behavior adjustments (p < 0.001 for all unless otherwise mentioned). represents extreme value; ◦represents potential outlier. Frontiers in Medicine \| www.frontiersin.org January 2022 \| Volume 8 \| Article 799412 12 Video-Watch Pattern and Learning Outcome Wang et al. AUTHOR CONTRIBUTIONS Unlike in-person or hybrid courses, instructors of online video-based learning cannot interact with students in real-time. 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https://openalex.org/W4210290811	https://czasopisma.kul.pl/index.php/recl/article/download/12240/12252	English	null	Issues of awards given as a part of social arbitration in a collective dispute. De lege lata and de lege ferenda remarks	Review of European and Comparative Law	2,022	cc-by	11,224	Maciej Jarota Dr., Assistant Professor, Faculty of Law, Canon Law and Administration, The John Paul II Catholic University of Lublin, correspondence address: Al. Racławickie 14, 20-950 Lublin, Poland; e-mail: maciejjarota@kul.lublin.pl https://orcid.org/0000-0001-6568-1626 Keywords: social arbitration, collective disputes, trade union, strike, arbitration award Abstract: Social arbitration as the third method of resolution of collective disputes can be used to resolve a dispute in an amica- ble manner. Thanks to this method, parties to the collective dis- pute can end their conflict thanks to the arbitration award with no need to go on strike. The author analyses the legal nature of arbitration awards and presents consequences of the related labour law legislation. The conclusion is as follows: current legal regulations are in need of change, especially when it comes to the execution, amendment and supplementation of an award is- sues as a part of social arbitration with the involvement of trade unions, employers or their organisations. Review of European and Comparative Law \| 2022 Vol. 48, No. 1, 7–28 https://doi.org/10.31743/recl.12240 Received: 11 February 2021 \| Accepted: 14 April 2021 \| Published: 10 March 2022 Review of European and Comparative Law \| 2022 Vol. 48, No. 1, 7–28 https://doi.org/10.31743/recl.12240 Received: 11 February 2021 \| Accepted: 14 April 2021 \| Published: 10 March 2022 This is an open access article under the CC BY license ISSN 2545-384X (Online) 1 Act on the resolution of collective disputes of 23 May 1991, Journal od Laws 2020, item 123, hereinafter: “RCD” or “Act”. 2 Article 4 (1) RCD. 3 Article 17 (2) and Article 15 RCD. 4 Article 16 (2) and Article 16 (6) RCD. 1. Introduction Collective labour disputes constitute a material element of the economic system of a state. It remains a valid question whether amicable methods of dispute resolution can be used effectively to avoid using strikes as a last resort. Social arbitration is one such amicable form. To evaluate the impor- tance of this institution and its possible application objectively, one cannot ignore the nature of an arbitration award. However, it has to be explained first what collective disputes are and who they apply to. i While analysing collective disputes, one should note that a collective dispute is a legal concept separate from the concept of an individual em- ployee dispute. The principal difference between an industrial dispute and This is an open access article under the CC BY license ISSN 2545-384X (Online) tps://creativecommons.org/licenses/by/4.0/d cle under the CC BY license ISSN 2545-384X (Online) Maciej Jarota an individual dispute is the scope of the subject and object of the dispute. According to art. 2 of the Act of 23 May 1991 on the resolution of collective disputes1, parties to the dispute can be employers, their organisations and employees who can only be represented by their trade union. The party to the dispute will therefore not be the individual employee, but the employees represented by an entity acting for the collective interests of the employees. In fact, a trade union is the one who can initiate and carry out a collective dispute. Additionally, pursuant to art. 1 of the said Act, a dispute can relate to working, remuneration or social benefit terms as well as to union rights and freedoms of employees or other groups having the right to form trade unions. It is not permitted to conduct a collective dispute to support indi- vidual employee claims if their resolution is possible by way of proceedings before the agency resolving disputes relating to employee claims2. h The legislator provided for three amicable methods of collective dis- pute resolution. The first method is by bargaining. If it is ineffective as un- derstood in art. 10 of the Act one shall proceed to mediation. The lack of an agreement at the stage of the mediation proceedings authorises the trade union to initiate a strike action3. However, a trade organisation that does not wish to take advantage of this right can also initiate social arbitration pursuant to art. 16 RCD. 8 Review of European and Comparative Law \| 2022 Vol. 48, No. 1 5 Ordinance of the Council of Ministers of 16th August 1991, Journal of Laws 1991, No. 73, item 324, hereinafter: “PSA”. 1. Introduction The essence of social arbitration is to authori- tatively resolve a dispute with the involvement of the social arbitration college in a competent District Court (one employer dispute) or Supreme Court (multi-employer dispute). Such a college can issue an award ending this stage of the collective dispute4. h The paper analyses two ways to conclude arbitration proceedings, i.e. conclusion of a collective agreement and issuance of an arbitration award. For this form of dispute resolution to be successful, it is important to actually be able to end the dispute by opting for one of these two var- iants of conflict resolution. They should give the parties a feeling of a sta- ble settlement of the dispute that is consistent with the applicable laws. 8 Review of European and Comparative Law \| 2022 Vol. 48, No. 1 Issues of awards given as a part of social arbitration in a collective dispute The institution of social arbitration must inspire confidence in both parties to the dispute. Both the employee and the employer side should be cer- tain that the award or agreement will be enforced without any problems whatsoever. Only clear and comprehensive regulation of the arbitration will make it possible to resort to arbitration proceedings to end a collective dispute rather than go on a strike. For this reason, the analysis covers the le- gal nature of an arbitration award in the context of practical problems with applying social arbitration in Polish legal reality. h y g g y The aim of this study is to determine whether legal regulations concern- ing the methods for ending social arbitration are comprehensive and clear or whether they need to be changed, and if yes, to what extent. The analy- sis uses the dogmatic-legal and legal-comparative methods. The countries under analysis share one common feature: they have all implemented leg- islation to resolve collective disputes. Research into legal mechanisms in the states concerned leads to the conclusion that these mechanisms are close to one another in terms of their objective: in each state, the legislator strives to ensure that the arbitration proceedings are effective in discourag- ing non-amicable forms of collective dispute resolution. Legal regulations of the countries in question are influenced by labour law standards adopted by international and European organisations. 9 Review of European and Comparative Law \| 2022 Vol. 48, No. 1 2. Ways to end social arbitration 2.1. Collective agreements as the second method of collective dispu in arbitration proceedings in addition to arbitration awards 2.1. Collective agreements as the second method of collective dispute resolution in arbitration proceedings in addition to arbitration awards An award issued by a social arbitration college is not the only way to resolve a dispute at this stage. Just like with bargaining or mediation, parties to arbi- tration proceedings can enter into agreements resolving the dispute. Accord- ing to § 9 of the Regulation of the Council of Ministers of 16 August 1991 on the procedure before social arbitration colleges5 after the opening of a col- lege session encourages the parties to reach an agreement. Review of European and Comparative Law \| 2022 Vol. 48, No. 1 9 Maciej Jarota Such an agreement as the source of the appropriate content of the labour law, determining rights and obligations6 has the legal nature characteristic for agreements based on RCD7 resulting from bargaining and mediation8. It should determine: parties to the dispute, precise claims of the trade un- ions and normative collective provisions if they apply to the general sit- uation of the employees. It can also contain individual provisions if they influence the contents of the employment relationship9. According to the Supreme Court, collective agreements are considered contractual provisions applying exclusively to entities covered with their contents. However, they are not treated as generally applicable legal reg- ulations10. According to the representative theory, parties to a collective agreement execute it not only on their behalf but also on behalf of mem- bers they represent or individuals other than their members. It means that it is possible to make direct demands regarding the fulfilment of duties by individual employees and employers towards each other11. Agreements are based on the principle of the freedom of agreements by way of the submis- sion of declarations of will in the course of social arbitration12. The doctrine Such an agreement as the source of the appropriate content of the labour law, determining rights and obligations6 has the legal nature characteristic for agreements based on RCD7 resulting from bargaining and mediation8. It should determine: parties to the dispute, precise claims of the trade un- ions and normative collective provisions if they apply to the general sit- uation of the employees. It can also contain individual provisions if they influence the contents of the employment relationship9. l According to the Supreme Court, collective agreements are considered contractual provisions applying exclusively to entities covered with their contents. However, they are not treated as generally applicable legal reg- ulations10. 6 See Krzysztof Wojciech Baran, “Porozumienia zawierane w sporach zbiorowych jako źródła prawa pracy,” Monitor Prawa Pracy, no. 9 (2008): 455; Janusz Żołyński, “Postępowan- ie arbitrażowe jako metoda rozwiązywania sporu zbiorowego,” Monitor Prawa Pracy, no. 10 (2011): 517. 7 Grzegorz Goździewicz, “Charakter porozumień zbiorowych w polskim prawie pracy,” Work and Social Security, no. 3 (1998): 23; Łukasz Pisarczyk, “Pokojowe (ireniczne) metody ro- związywania sporów zbiorowych,” in System prawa pracy. Zbiorowe prawo pracy, vol. V, ed. Krzysztof Wojciech Baran (Warszawa: Wolters Kluwer S.A., 2014), 643–644; see Baran, “Porozumienia,” 453–54. The labour law doctrine includes doubts regarding the distinc- tion of agreements being specific sources of the labour law, see Ludwik Florek, “Charakter prawny porozumień zbiorowych,” in Prawo pracy. Refleksje i poszukiwania. Księga jubile- uszowa Profesora Jerzego Wratnego, ed. Gertruda Uścińska (Warszawa: IPISS, 2013), 114–17. 8 L d ik Fl k U t i i (W W lt Kl P l k 12 Compare Paweł Nowik, Metoda negocjacji układowych w zakresie kształtowania wyna- grodzenia za pracę pracowników administracji publicznej. Doświadczenia polskie i europejsk- ie (Lublin: Wydawnictwo KUL, 2014), 90–1. 9 Pisarczyk, “Pokojowe,” 650–52. 13 See Pisarczyk, “Pokojowe,” 650–51; see also the topic of a collective agreement in Florek, Ustawa, 224–27. 14 Andrzej Marian Świątkowski, “Ustawa o rozwiązywaniu sporów zbiorowych,” in Zbiorowe prawo pracy, eds. Jerzy Wratny, Krzysztof Walczak (Warszawa: C.H. Beck, 2009), 334. 15 E.g. according to the information obtained as a part of access to the public information, the award rejecting the related request of the applicant was issued by the social arbitration college at the Regional Court in Piotrkow Trybunalski in 2012, Decision of the College for Social Arbitration at the Regional Court in Piotrków Trybunalski, Judgment of 2012, Ref. No. KAS - z 1/12, unreported. Data referring to specific awards under social arbitration that were referred to in the article were collected on the basis of enquiries emailed to all Regional Courts in Poland. 16 Artur Rycak, “Praktyka arbitrażu społecznego w zbiorowych sporach pracy w Polsce,” in Ar- 2.1. Collective agreements as the second method of collective dispute resolution in arbitration proceedings in addition to arbitration awards According to the representative theory, parties to a collective agreement execute it not only on their behalf but also on behalf of mem- bers they represent or individuals other than their members. It means that it is possible to make direct demands regarding the fulfilment of duties by individual employees and employers towards each other11. Agreements are based on the principle of the freedom of agreements by way of the submis- sion of declarations of will in the course of social arbitration12. The doctrine 7 Grzegorz Goździewicz, “Charakter porozumień zbiorowych w polskim prawie pracy,” Work and Social Security, no. 3 (1998): 23; Łukasz Pisarczyk, “Pokojowe (ireniczne) metody ro- związywania sporów zbiorowych,” in System prawa pracy. Zbiorowe prawo pracy, vol. V, ed. Krzysztof Wojciech Baran (Warszawa: Wolters Kluwer S.A., 2014), 643–644; see Baran, “Porozumienia,” 453–54. The labour law doctrine includes doubts regarding the distinc- tion of agreements being specific sources of the labour law, see Ludwik Florek, “Charakter prawny porozumień zbiorowych,” in Prawo pracy. Refleksje i poszukiwania. Księga jubile- uszowa Profesora Jerzego Wratnego, ed. Gertruda Uścińska (Warszawa: IPISS, 2013), 114–17. 9 Pisarczyk, “Pokojowe,” 650–52. 10 Polish Supreme Court, Judgment of 22 February 2008, Ref. No. II BP 36/07, Journal of Laws 2009, no. 11–12, item 138. 11 Wacław Szubert, Układy zbiorowe pracy (Warszawa: Państwowe Wydawnictwo Naukowe, 1960), 10–11. 12 Compare Paweł Nowik, Metoda negocjacji układowych w zakresie kształtowania wyna- grodzenia za pracę pracowników administracji publicznej. Doświadczenia polskie i europejsk- ie (Lublin: Wydawnictwo KUL, 2014), 90–1. 10 Review of European and Comparative Law \| 2022 Vol. 48, No. 1 10 Issues of awards given as a part of social arbitration in a collective dispute notices elements of a civil law agreement contained in such agreements that, however, do not rule out the possibility that above-mentioned acts compiled at the arbitration stage in a collective dispute can be seen as sources of the la- bour law based on the act13. Agreements signed by parties to the dispute in the course of social arbitration entail an automatic change of collective interests of employees the satisfaction of which the trade union demanded into individual subjective rights just like agreements executed at the bar- gaining and mediation stage14. 16 Artur Rycak, “Praktyka arbitrażu społecznego w zbiorowych sporach pracy w Polsce,” in Ar- bitraż i mediacja w polskim prawie pracy, ed. Grzegorz Goździewicz (Lublin: Wydawnictwo KUL, 2005), 142. 17 It has to be stated that both RCD and PSA do not introduce the possibility to reject a re- quest, only to return it due to formal defects not remedied on time, see: Walery Masewicz, Zatarg zbiorowy pracy (Poznań: Polski Dom Wydawniczy “Ławica”, 1994), 97–98. Some representatives of the labour law science indicate that the college issues substantive awards considering demands of the trade union in part or in whole or dismissing them in part of in while, see: Bogusław Cudowski, Spory zbiorowe w polskim prawie pracy (Białystok: Tem- ida, 1998), 114. However, if arbitration is not admissible due to the fact that there was no bargaining or mediation carried out or when proceedings before the college demonstrate that the dispute is not a collective dispute the request will have to be rejected for purposive reasons. The postulate that the trade union’s request can be rejected by the issue of an award by the social arbitration college is justified. Checking whether a dispute is a collective dis- pute lies within the competences of the college, see Rycak, “Praktyka,” 142. The labour law doctrine also contains the view according to which a request can be destroyed due to the in- admissibility of arbitration proceedings due to its return by the court president, see: Walery Masewicz, Ustawa o związkach zawodowych Ustawa o rozwiązywaniu sporów zbiorowych (Warszawa: Wydawnictwo Prawnicze PWN, 1998), 180. 20 Decision of the College for Social Arbitration at the Supreme Court of January 26, 2006, Ref. No. III KAS 1/05, Journal of Laws 2007, no. 3–4, item 59. 19 Rycak, “Praktyka,” 142. 2.2. Arbitration awards – decisions of social arbitration colleges If an agreement resolving a collective dispute is not reached in the arbitration proceedings an arbitration award will be issued. In practice, one can distin- guish substantive decisions considering the demand made by a trade union and dismissing collective claims made with regard to payment, working and social benefit conditions15. When a trade union makes a claim regarding union rights and freedoms, a decision can be made to dismiss that claim, i.e. considering the trade union’s position ungrounded or to grant the appli- cation in whole or in part16. h A social arbitration college also issues typically formal awards. These type of awards includes a decision to refuse to hear the arbitration, de facto to reject the application to the inadmissibility of social arbitration as under- stood in RCD regulations, even though no legal basis for such a conclusion Review of European and Comparative Law \| 2022 Vol. 48, No. 1 11 Maciej Jarota of arbitration proceedings can be found in PSA or even less in RCD17. Addi- tionally, a decision to discontinue proceedings exists in legal transactions18. y p g g In the issued award, the social arbitration college also refers the case to a competent social arbitration college for the ruling, e.g. the college at the Supreme Court19. Decisions of the social arbitration college at the Supreme Court adopted the rule according to which “employer dis- putes can be transformed during the proceedings before a social arbitration college into a single multi-employer dispute if parties to these disputes are willing and the subject matter of the dispute applies to workers employed in at least two workplaces”20. In the analysed case being the basis for the issue of the above-mentioned award, the college primarily examined its compe- tence to hear the dispute. It was necessary due to the fact that the dispute was conducted as eight separate disputes in the bargaining and media- tion phase. Findings made by the college led to the conclusion according to which trade unions representing all workers covered with disputes in the bargaining and mediation phase jointly applied for the submission of 17 It has to be stated that both RCD and PSA do not introduce the possibility to reject a re- quest, only to return it due to formal defects not remedied on time, see: Walery Masewicz, Zatarg zbiorowy pracy (Poznań: Polski Dom Wydawniczy “Ławica”, 1994), 97–98. 12 Review of European and Comparative Law \| 2022 Vol. 48, No. 1 2.2. Arbitration awards – decisions of social arbitration colleges Some representatives of the labour law science indicate that the college issues substantive awards considering demands of the trade union in part or in whole or dismissing them in part of in while, see: Bogusław Cudowski, Spory zbiorowe w polskim prawie pracy (Białystok: Tem- ida, 1998), 114. However, if arbitration is not admissible due to the fact that there was no bargaining or mediation carried out or when proceedings before the college demonstrate that the dispute is not a collective dispute the request will have to be rejected for purposive reasons. The postulate that the trade union’s request can be rejected by the issue of an award by the social arbitration college is justified. Checking whether a dispute is a collective dis- pute lies within the competences of the college, see Rycak, “Praktyka,” 142. The labour law doctrine also contains the view according to which a request can be destroyed due to the in- admissibility of arbitration proceedings due to its return by the court president, see: Walery Masewicz, Ustawa o związkach zawodowych Ustawa o rozwiązywaniu sporów zbiorowych (Warszawa: Wydawnictwo Prawnicze PWN, 1998), 180. 18 18 E.g. in 2005-2014, the social arbitration college at the Regional Court in Łodz dismissed ar- bitration proceedings twice: in one case, it was due to the trade union’s loss of its mandate to represent employees, in another case, it was due to the withdrawal of the request for social arbitration. 18 E.g. in 2005-2014, the social arbitration college at the Regional Court in Łodz dismissed ar- bitration proceedings twice: in one case, it was due to the trade union’s loss of its mandate to represent employees, in another case, it was due to the withdrawal of the request for social arbitration. 12 Review of European and Comparative Law \| 2022 Vol. 48, No. 1 12 Issues of awards given as a part of social arbitration in a collective dispute the dispute for resolution by the social arbitration college at the Supreme Court as a multi-employer dispute21. 27 In turn, the college at the Regional Court in Olsztyn examining the case, granted the trade union’s request in full by obliging to introduce the salary agreement of 21 February 2005 in the award not binding the parties, issued on 4 April 2006. Decision of the College for Social 26 Bogusław Cudowski, “Rola państwa w rozwiązywaniu sporów zbiorowych,” Państwo i Prawo, no. 10 (1994): 69. 24 See Grzegorz Goździewicz, “Mediacja i arbitraż w polskim prawie pracy,” in Arbitraż i me- diacja w polskim prawie pracy, ed. Grzegorz Goździewicz (Lublin: Wydawnictwo KUL, 2005), 24. 25 Zbigniew Hajn, Zbiorowe prawo pracy. Zarys systemu (Warszawa: Wolters Kluwer Pols- ka sp. z o.o., 2013), 188. 23 Janusz Żołyński, Ustawa o rozwiązywaniu sporów zbiorowych. Komentarz. Wzory pism (Warszawa: Wolters Kluwer Polska sp. z o.o., 2011), 92. 21 Ibid. 22 Świątkowski, “Ustawa,” 374. 23 Janusz Żołyński, Ustawa o rozwiązywaniu sporów zbiorowych. Komentarz. Wzory pism (Warszawa: Wolters Kluwer Polska sp. z o.o., 2011), 92. 24 See Grzegorz Goździewicz, “Mediacja i arbitraż w polskim prawie pracy,” in Arbitraż i me- diacja w polskim prawie pracy, ed. Grzegorz Goździewicz (Lublin: Wydawnictwo KUL, 2005), 24. 25 Zbigniew Hajn, Zbiorowe prawo pracy. Zarys systemu (Warszawa: Wolters Kluwer Pols- ka sp. z o.o., 2013), 188. 26 Bogusław Cudowski, “Rola państwa w rozwiązywaniu sporów zbiorowych,” Państwo i Prawo, no. 10 (1994): 69. 27 In turn, the college at the Regional Court in Olsztyn examining the case, granted the trade union’s request in full by obliging to introduce the salary agreement of 21 February 2005 in the award not binding the parties, issued on 4 April 2006. Decision of the College for Social ą , , 23 Janusz Żołyński, Ustawa o rozwiązywaniu sporów zbiorowych. Komentarz. Wzory pism (Warszawa: Wolters Kluwer Polska sp. z o.o., 2011), 92. 24 See Grzegorz Goździewicz, “Mediacja i arbitraż w polskim prawie pracy,” in Arbitraż i me- diacja w polskim prawie pracy, ed. Grzegorz Goździewicz (Lublin: Wydawnictwo KUL, 2005), 24. 3. Legal nature of arbitration awards In light of art. 16 clause 6 RCD, the arbitration award is binding for par- ties to the dispute unless they agree differently. If both the trade union in its application for social arbitration and the employer responding to that application fail to submit, pursuant to art. 16 clause 6 RCD, an appropriate statement of application for arbitration with the issue of an award non-bind- ing for the parties, the decision of the social arbitration college, in prin- ciple, will bind the parties22. The employer can express their position on the subject responding to the application of the trade union or in another letter before the date of the commission’s session but not later than upon the opening of that session23. Contrary to the situation in which both par- ties express their conclusive consent to the binding nature of the college’s decision, a trade union can decide to initiate a protest action in the form of a strike if the award is non-binding. Purposive considerations are in favour of this postulate24. The parties can autonomously choose the dispute reso- lution variant after the mediations end and apply non-amicable resolution methods. An award in arbitration proceedings whose binding power results from the will of the parties ends the proceedings related to the occurrence of a collective dispute with the employer25; as a consequence, the collective dis- pute is resolved26. The statement of the binding power27 shall be contained in igniew Hajn, Zbiorowe prawo pracy. Zarys systemu (Warszawa: Wolters Kluwer Pols- sp. z o.o., 2013), 188. gusław Cudowski, “Rola państwa w rozwiązywaniu sporów zbiorowych,” Państwo i Prawo, . 10 (1994): 69. Review of European and Comparative Law \| 2022 Vol. 48, No. 1 13 Maciej Jarota the award itself28; however, this situation does not always happen29. Accord- ing to § 11 clause 3 PSA, an arbitration award should also contain the name and content of the college, award issue date, definition of parties, indication of the subject matter of the dispute, the resolution30 and its justification, the statement whether the award binds the parties and signatures of mem- bers of the college31. If the award does not contain any of the above-mentioned elements, it seems that it is not possible to supplement them pursuant to art. 351 § 3 of the Act of 17 November 1964 – Code of Civil Procedure32 for two reasons. Arbitration at the District Court in Olsztyn, Judgment of 2006, Ref. No. Kas-z.1/06, un- reported. The above data come from the information obtained by email as an answer to enquiries addressed to Regional Courts. See more Maciej Jarota, “Arbitraż społeczny – fakultatywna czy obligatoryjna metoda rozwiązywania sporów zbiorowych? Przyczynek do dyskusji o wykorzystaniu postępowania arbitrażowego w zbiorowych stosunkach pracy,” ADR Arbitraż i Mediacja, no. 2 (2018): 48–49. 32 Act on the Code of Procedural Civil of 17 November 1964, Journal od Laws 2020, item 1575, as amended, hereinafter: “CPC”. 29 Rycak, “Praktyka,” 145. 28 Goździewicz, “Mediacja,” 23. 3. Legal nature of arbitration awards Firstly, provisions of the Code of Civil Procedure do not apply to arbitra- tion proceedings before social arbitration colleges – obviously with some exceptions. Civil procedure was referred to only in one place of the provi- sion regulating the arbitration procedure, however, it only had to do with evidentiary proceedings. In the light of § 8 clause 2 PSA, a college can take evidence in line with the provisions of the Code of Civil Procedure on evi- dence. Secondly, it is the social arbitration college rather than a public court that issues an award; this is why, in light of the linguistic interpretation of 29 Rycak, “Praktyka,” 145. 30 In practice, the awards of the social arbitration college sometimes lacks the dispute resolu- tion, which cannot be considered positive at this stage of the dispute. E.g. in the operative part of the award of 7 May 2015 regarding the proceedings requested by the trade union against the employer, the social arbitration college at the Regional Court in Warsaw limited itself to the indication that the parties failed to reach an agreement. Decision of the Col- lege for Social Arbitration at the District Court in Warszawa, Judgment of 2015, Ref. No. XXI Kas-z 2/14, unreported. The operative part of the decision did not refer to the dispute resolution while item 5 of the award states that the award is not binding for the parties. See more Maciej Jarota, “Arbitraż społeczny – fakultatywna czy obligatoryjna metoda rozwiązy- wania sporów zbiorowych? Przyczynek do dyskusji o wykorzystaniu postępowania arbi- trażowego w zbiorowych stosunkach pracy,” ADR Arbitraż i Mediacja, no. 2 (2018): 48–49. 31 Świątkowski, “Ustawa,” 374. 32 Act on the Code of Procedural Civil of 17 November 1964, Journal od Laws 2020, item 1575, as amended, hereinafter: “CPC”. 14 Review of European and Comparative Law \| 2022 Vol. 48, No. 1 14 Issues of awards given as a part of social arbitration in a collective dispute art. 351 § 3 CPC in connection with art. 351 § 1 CPC, it is not possible to supplement an award issued pursuant to § 11 PSA. It is worth mentioning that, in the past, according to art. 33 Ordinance of the President of the Republic of Poland 27th October 1933, Journal of Laws 1933, No. 82, item 604. 34 Marek Włodarczyk, “Historia porozumień normatywnych na szczeblu zakładu pracy w Polsce,” in Studia z prawa pracy. Księga pamiątkowa ku czci Docenta Jerzego Logi, ed. Zbigniew Góral (Łódź: Wydawnictwo Uniwersytetu Łódzkiego 2007), 210. 3. Legal nature of arbitration awards 9 of the Reg- ulation of the President of the Republic of Poland of 27 October 1933 on extraordinary disputes committees for the resolution of collective disputes between employers and employees in the industry and commerce33, the le- gal construct in force in Poland provided that the committee’s award had the economically prevailing importance in the work branch covered with the award, the Council of Ministers could issue a regulation if requested by the minister of social care to give binding legal effect to the award in the entire area for which the award was issued or in a part of the area where it attained prevailing importance. Such an award, as understood in the said regulation, would apply directly to all employees and employers. Said le- gal regulations have overcome the rule of committee awards contained in the pre-WWII Law of Obligations, typical for collective labour agreements (c.l.a.), according to which they are only binding for those parties who have executed them34. To summarize this part of the discussion, a concern may be expressed about the impossibility to supplement an existing award, if necessary. Sim- ilarly, the Polish legislation does not provide for rectification of an award should there be an obvious error in the dispute resolution. In the Polish legal reality, there are no legal regulations that would precisely define the manner in which the panel should act if it is necessary to amend the award from the proceedings. 15 Review of European and Comparative Law \| 2022 Vol. 48, No. 1 35 Żołyński, Ustawa, 95. In 1960ies, doubts existed regarding the enforceability of collective labour agreements as far as the enforcement of resulting obligations is concerned. Free mar- ket economies adopted the inadmissibility of the formulation of individual employee claims against a participant in the collective labour agreement. A particular significance of rights vested in the employee organisation representing employees rather than individual rights was stressed, see Szubert, Układy, 229. 35 Żołyński, Ustawa, 95. In 1960ies, doubts existed regarding the enforceability of collective labour agreements as far as the enforcement of resulting obligations is concerned. Free mar- ket economies adopted the inadmissibility of the formulation of individual employee claims against a participant in the collective labour agreement. A particular significance of rights vested in the employee organisation representing employees rather than individual rights was stressed, see Szubert, Układy, 229. 36 See Świątkowski, “Ustawa,” 376; see also Pisarczyk, “Pokojowe,” 676. 37 See Świątkowski, “Ustawa,” 376; see also a different position: Żołyński, Ustawa, 94. 38 See Pisarczyk, “Pokojowe,” 680; Krzysztof Wojciech Baran, Zbiorowe prawo pracy. Komen- tarz (Warszawa: Wolters Kluwer Polska sp. z o.o., 2010), 442; Cudowski, Spory, 115; see also a different position: Żołyński, Ustawa, 94. 39 Goździewicz, “Mediacja,” 25. 40 Pisarczyk, “Pokojowe,” 680. y 36 See Świątkowski, “Ustawa,” 376; see also Pisarczyk, “Pokojowe,” 676.f 37 See Świątkowski, “Ustawa,” 376; see also a different position: Żołyński, Ustawa, 94. 38 See Pisarczyk, “Pokojowe,” 680; Krzysztof Wojciech Baran, Zbiorowe prawo pracy. Komen- tarz (Warszawa: Wolters Kluwer Polska sp. z o.o., 2010), 442; Cudowski, Spory, 115; see also a different position: Żołyński, Ustawa, 94. 4. Execution of arbitration awards While analysing the issues of arbitration awards, it is worthwhile to con- sider whether parties to the employment relationship are entitled to the claim for the execution of an award issued by a social arbitration col- lege on the same terms as those applying to the formulation of demands Review of European and Comparative Law \| 2022 Vol. 48, No. 1 15 Maciej Jarota referring to the execution of provisions of the collective agreement executed in the course of social arbitration. The view that it is possible for employees and employer to make direct civil law claims based on an arbitration award just as is the case with the collective agreement35 is debatable36. j g As soon as demands made by the trade union are transformed into in- dividual rights pursuant to the collective agreement executed at the arbitra- tion stage, individual employees acquire the right to claim the satisfaction of individual rights guaranteed to them. In turn, the issue of an arbitration award not being a source of labour law does not entail the legal transfor- mation of employee interests to be satisfied by the employer on the basis of the decision of the social arbitration college into rights of individuals with an employment relationship. The conclusion of this analysis is that the issue of an award does not result in an automatic transformation of employees’ collective interests into their rights37. Reference publications present the prevailing view that, to enforce the employer’s observance of the arbitration award referring to employee interests, it is only possible for the trade union to exert pressure by organizing a strike or another non-am- icable method of resolution of collective disputes even with no renewed procedure for the initiation of a collective dispute38. h The Polish model of an amicable resolution of collective disputes does not provide for sanction for the failure to comply with an award issued in the arbitration proceedings39 even though the failure to comply can be con- sidered a violation of art. 26 clause 1 clause 2 RCD40. The enforceability of 16 Review of European and Comparative Law \| 2022 Vol. 48, No. 1 16 Issues of awards given as a part of social arbitration in a collective dispute arbitration awards is not subject to enforcement proceedings41. This fact is demonstrated in the linguistic interpretation of art. 41 Żołyński, Ustawa, 93. 42 Baran, Zbiorowe, 442. 4. Execution of arbitration awards 777 CPC, in particular, the lack of an indication of awards issued in the social arbitration mode in its contents and the absence of a legal standard in RCD that would estab- lish the admissibility of enforcement of such awards by way of enforcement proceedings42.i Such deficiencies may raise doubts whether a trade union is actually able to effectively enforce pay and work conditions, social benefits, or trade union rights and freedoms established by an arbitration award. Hence, the trade union organisation, unlike the employer, will essentially go for a strike method rather than arbitration proceedings. Mediation failing, the trade union party will count on a strike as a viable method to achieve its demands from a collective dispute stage. The mere fact that an arbitra- tion award will become non-binding if one of the parties submits a rele- vant declaration hardly encourages the use of the social arbitration method. Since parties to a collective dispute are not bound by the arbitration award, decisions of the social arbitration committee are treated as non-mandato- ry for the trade union and the employer. On the other hand, the inability to effectively enforce a binding arbitration award means that arbitration proceedings actually lose their sense as a constructive method of resolving a controversy. 5. Arbitration awards in selected European states vs. Polish legal realities 41 Żołyński, Ustawa, 93. 42 Baran, Zbiorowe, 442. 5. Arbitration awards in selected European states vs. Polish legal realities In light of the unique nature of an arbitration award and a limited possibility of its enforcement in Polish labour law, it is worthwhile to analyse the said institution from the perspective of selected examples of European states. Legal regulations of individual European states define the legal nature of arbitration awards in an inconsistent manner. In some European states, ar- bitrators issue awards. In the Russian Federation, an arbitration award is binding for the parties. They are obliged to execute it in pain of the fine of 2000-4000 roubles. If the employer fails to comply with the award the trade Review of European and Comparative Law \| 2022 Vol. 48, No. 1 Review of European and Comparative Law \| 2022 Vol. 48, No. 1 17 17 Maciej Jarota union can initiate a protest action in the form of a strike43. In France, the ar- bitrator’s decision can be appealed against to the Supreme Court of Arbitra- tion consisting of the equal number of judges from the Council of State and judges from the Court of Cassation44. j g In light of art. 20 clause 4 of the Latvian Act of 26 September 2002 on employment relationships, compliance with an arbitration award is volun- tary in Latvia. If parties compile a written agreement to determine the bind- ing power of the arbitration award that arbitration award shall have legal effects typical for a collective agreement45. In Great Britain, an award issued in the course of an optional arbitration is binding if the parties decide dur- ing the pending procedure that they would comply with the award irre- spective of its contents46. In Slovakia, awards issued by an arbitrator regarding the execution of collective agreements can be appealed against to a District Court that re- peals the arbitrator’s award if it is in conflict with the legal regulations or with the collective agreement. In the same country, an arbitrator’s decision regarding the conclusion of a collective labour agreement is final with no appeal possible, unlike decisions referring to disputes regarding the execu- tion of duties under collective agreements. If a court annuls the arbitrator’s award the dispute shall be referred to the same arbitrator for reassessment. 43 Elena Gerasimowa, “The Resolution Of Collective Labour Disputes,” in Labour Law in Rus- sia: Recent Developments and New Challenges, eds. Vladimir Lebedev and Elena Radevich (Newcastle: Cambridge Scholars Publishing, 2014), 274. 44 Michel Despax, Jaques Rojot, and Jean Pierre Laborde, Labour Law in France (Alphen aan den Rijn: Kluwer Law International, 2011), 347. 45 In Greece, like in Latvia, the arbitrator’s award entails legal effects typical for a collective agreement, see Theodore Konaris, Labour law in Hellas (Alphen aan den Rijn: Kluwer Law International, 2002), 239. 46 Steven Hardy, Labour Law in Great Britain (Alphen aan den Rijn: Kluwer Law Internation- al, 2002), 44–45. 47 Helena Barancová and Andrea Olšowská, Labour Law in Slovak Republic (Alphen aan den Rijn: Kluwer Law International, 2002), 191. Review of European and Comparative Law \| 2022 Vol. 48, No. 1 47 Helena Barancová and Andrea Olšowská, Labour Law in Slovak Republic (Alphen aan den Rijn: Kluwer Law International, 2002), 191. 43 Elena Gerasimowa, “The Resolution Of Collective Labour Disputes,” in Labour Law in Rus- sia: Recent Developments and New Challenges, eds. Vladimir Lebedev and Elena Radevich (Newcastle: Cambridge Scholars Publishing, 2014), 274. Michel Despax, Jaques Rojot, and Jean Pierre Laborde, Labour Law in France (Alphe aan den Rijn: Kluwer Law International, 2011), 347.f 48 Manuel Alonso Olea and Fermĺn Rodríguez - Sañudo, Labour Law in Spain (Alphen aan de Rijn: Kluwer Law International, 2002), 156. 49 Manfred Weiss and Marlene Schmidt, Labour Law in Germany (Alphen aan den Rijn: Klu- wer Law International, 2008), 236. 50 Ole Hasselbalch, Labour Law in Denmark (Alphen aan den Rijn: Kluwer Law International, 2011), 300–01. 51 Polish Supreme Court, Resolution of 23 May 1986, Ref. No. III PZP 9/86, Journal of Laws 1987, no. 4, item 77; Polish Supreme Court, Resolution of 10 December 1986, Ref. No. III PZP 72/86, Journal of Laws 1987, no. 4, item 47. 52 Act on the Labour Code of 26 June 1974 Journal od Laws 2020 item 1320 5. Arbitration awards in selected European states vs. Polish legal realities The lack of consent to the participation of the same person as the arbitrator results in the nomination of an arbitrator on the request of any of the parties by the Minister of Labour, Social Affairs and Family of the Slovak Republic47. yf y p In Spain, art. 21 clause 3 V ASEC provides that a binding arbi- tration award is enforceable immediately. Each award is submitted to 8 Review of European and Comparative Law \| 2022 Vol. 48, No. 1 18 Issues of awards given as a part of social arbitration in a collective dispute the SIMA office. After that, the award is forwarded to an appropriate agen- cy for publication if required under the law. The award has the same legal effects as a collective labour agreement48. f In Germany, parties to a dispute can appeal from the decision of an arbitration committee to a labour court within 2 weeks of the award announcement. The appeal can be upheld in the event of a law violation. The German labour law doctrine indicates that parties rarely decide to un- dermine the resolution made at the arbitration stage in collective disputes in this manner49. In Denmark, an award issued at the arbitration stage is final even though, if material rules of the procedure influencing the res- olution of the case are violated it is possible to consider the award invalid before the labour court50. Polish legal regulations do not assume the two-tiered procedure in arbitration proceedings51. As already mentioned, parties in Slovakia and Germany have the possibility to appeal against an arbitration award to a labour court. Analysed legal solutions applied in above-mentioned coun- tries make the appellate review of an arbitration award possible, which is particularly desirable for a discretionary ruling by an arbitration agency in a specific case, in a manner not limited with statutory criteria. The aspect of determination of an entity competent to consider the means of challenge of arbitration awards is also extremely important. The labour court seems to be competent to assess an arbitration award because judges who are labour law practitioners are able to guarantee reliability and independence while analysing arbitration proceedings in a collective dispute. It should be remembered that, according to the rule set out in art. 52 Act on the Labour Code of 26 June 1974, Journal od Laws 2020, item 1320. 54 In the previous legal regime applicable in 1980ies, the Public Prosecutor General argued that purposive considerations (the need to amend awards) warrant the application of rem- edies provided for in civil procedure regulations but the Supreme Court did not share this position in its decision of 10 December 1986, III PZP 72/86, see Polish Supreme Court, Resolution of 23 May 1986, Ref. No. III PZP 72/86, unreported. The labour law doctrine also indicated that, even though the nature of arbitration proceedings is different than the nature of litigation, this circumstance does not entail the right to conclude that it is not possible to appeal against an arbitration award on the basis of autonomous findings of the parties, see Andrzej Marian Świątkowski, “Spory zbiorowe (I),” Praca i Zabezpieczenie Społeczne, no. 8 (1987): 13–17. p 55 See Cudowski, Spory, 112; Żołyński, Ustawa, 93; Żołyński, Ustawa, 438.i Http://www.mpips.gov.pl/gfx/mpips/userfiles/File/Departament%20Prawa%20Pracy/kod- eksy%20pracy/ZKP_04.08..pdf., accessed May 9, 2016, hereinafter: CLC. The CLC draft was submitted to the President of the Council of Ministers on 5.12.2006 even though its contents refer to “April 2007”, while the draft description found at the website provides the information that the draft originated in April 2008. The Labour Law Codification Commission had been preparing the draft for a few years on the basis of the Ordinance of the Council of Ministers of 20th August 2002 on the establishment of a labour law codifica- tion commission, Journal of Laws 2002, No. 139 item 1167, as amended. The Commission initially worked under the leadership of Tadeusz Zieliński. As of 5.12.2003, the Commis- sion consisted of: Michał Seweryński, a professor at the Łódz University (the chairman); Ludwik Florek, a professor at the Warsaw University (deputy chairman); Grzegorz Goździewicz, a professor at the M. Kopernik University in Toruń; Zbigniew Hajn, a profes- sor at the Łódz University, a judge at the Supreme Court; Andrzej Kijowski, a professor at the A. Mickiewicz University in Poznań, a judge of the Supreme Court; Walerian Sanetra, a professor at the Białystok University, President of the Supreme Court; Barbara Wagner, a professor at the Jagiellonian University, judge of the Supreme Court; Jan Wojtyła, a pro- fessor at the K. Adamiecki University of Economics in Katowice; Jerzy Wratny, a professor Review of European and Comparative Law \| 2022 Vol. 48, No. 1 53 Żołyński, Ustawa, 93. 5. Arbitration awards in selected European states vs. Polish legal realities 262 § 2 item 1 of the Act of 26 June 1974 – Labour Code52 public courts cannot Review of European and Comparative Law \| 2022 Vol. 48, No. 1 19 Maciej Jarota interfere with disputes regarding the establishment of new payment and working terms or the application of labour law standards to the nomina- tion of college members at the social arbitration stage53. The lack of a two- tier system in social arbitration in Poland provokes doubts54. In particular, the literature rightly indicates that the delegation of three, by definition, in- dependent members of the college from the employer side and three mem- bers from the trade union to form the college is only apparent; in fact, a dis- pute is resolved by a professional judge. Individuals indicated by the trade union or by the employer do not vote against their principals55. ht y p y g p p The statement of reasons for the draft collective labour code of 2008 pre- pared by the Labour Law Codification Commission56 provides that social p y y y Http://www.mpips.gov.pl/gfx/mpips/userfiles/File/Departament%20Prawa%20Pracy/kod- eksy%20pracy/ZKP_04.08..pdf., accessed May 9, 2016, hereinafter: CLC. The CLC draft was submitted to the President of the Council of Ministers on 5.12.2006 even though its contents refer to “April 2007”, while the draft description found at the website provides the information that the draft originated in April 2008. The Labour Law Codification Commission had been preparing the draft for a few years on the basis of the Ordinance of the Council of Ministers of 20th August 2002 on the establishment of a labour law codifica- tion commission, Journal of Laws 2002, No. 139 item 1167, as amended. The Commission initially worked under the leadership of Tadeusz Zieliński. As of 5.12.2003, the Commis- sion consisted of: Michał Seweryński, a professor at the Łódz University (the chairman); Ludwik Florek, a professor at the Warsaw University (deputy chairman); Grzegorz Goździewicz, a professor at the M. Kopernik University in Toruń; Zbigniew Hajn, a profes- sor at the Łódz University, a judge at the Supreme Court; Andrzej Kijowski, a professor at the A. Mickiewicz University in Poznań, a judge of the Supreme Court; Walerian Sanetra, a professor at the Białystok University, President of the Supreme Court; Barbara Wagner, a professor at the Jagiellonian University, judge of the Supreme Court; Jan Wojtyła, a pro- fessor at the K. 53 Żołyński, Ustawa, 93. 54 In the previous legal regime applicable in 1980ies, the Public Prosecutor General argued that purposive considerations (the need to amend awards) warrant the application of rem- edies provided for in civil procedure regulations but the Supreme Court did not share this position in its decision of 10 December 1986, III PZP 72/86, see Polish Supreme Court, Resolution of 23 May 1986, Ref. No. III PZP 72/86, unreported. The labour law doctrine also indicated that, even though the nature of arbitration proceedings is different than the nature of litigation, this circumstance does not entail the right to conclude that it is not possible to appeal against an arbitration award on the basis of autonomous findings of the parties, see Andrzej Marian Świątkowski, “Spory zbiorowe (I),” Praca i Zabezpieczenie Społeczne, no. 8 (1987): 13–17. 55 See Cudowski, Spory, 112; Żołyński, Ustawa, 93; Żołyński, Ustawa, 438. 56 Http://www.mpips.gov.pl/gfx/mpips/userfiles/File/Departament%20Prawa%20Pracy/kod- eksy%20pracy/ZKP_04.08..pdf., accessed May 9, 2016, hereinafter: CLC. The CLC draft was submitted to the President of the Council of Ministers on 5.12.2006 even though its contents refer to “April 2007”, while the draft description found at the website provides the information that the draft originated in April 2008. The Labour Law Codification Commission had been preparing the draft for a few years on the basis of the Ordinance of the Council of Ministers of 20th August 2002 on the establishment of a labour law codifica- tion commission, Journal of Laws 2002, No. 139 item 1167, as amended. The Commission initially worked under the leadership of Tadeusz Zieliński. As of 5.12.2003, the Commis- sion consisted of: Michał Seweryński, a professor at the Łódz University (the chairman); Ludwik Florek, a professor at the Warsaw University (deputy chairman); Grzegorz Goździewicz, a professor at the M. Kopernik University in Toruń; Zbigniew Hajn, a profes- sor at the Łódz University, a judge at the Supreme Court; Andrzej Kijowski, a professor at the A. Mickiewicz University in Poznań, a judge of the Supreme Court; Walerian Sanetra, a professor at the Białystok University, President of the Supreme Court; Barbara Wagner, a professor at the Jagiellonian University, judge of the Supreme Court; Jan Wojtyła, a pro- fessor at the K. Adamiecki University of Economics in Katowice; Jerzy Wratny, a professor at the Rzeszów University; dr Eugenia Gienieczko, Director of the Labour Law Department in the then Ministry of Labour and Social Policy. In 2005, Teresa Liszcz, a professor of the Maria Curie-Skłodowska University in Lublin, later a judge in the Constitutional Tribu- nal, joined the Commission. t 57 Art. 158 § 2 of the draft Collective Labour Code from mid 1990ies suggested that each of the parties and the labour inspector could be granted the right to appeal against the arbitra- tor’s award violating the laws, see Cudowski, Spory, 115. 58 The text of the draft collective labour agreement, see https://www.gov.pl/web/rodzina/ bip-teksty-projektu-kodeksu-pracy-i-projektu-kodeksu-zbiorowego-prawa-pracy-opra- cowane-przez-komisje-kodyfikacyjna-prawa-pracy, accessed January 23, 2021. The last La- bour Law Codification Commission was established pursuant to the Ordinance of the Coun- cil of Ministers of 9th August 2016 on the establishment of a labour law codification commission, Journal of Laws 2016, item 1366. The Commission consisted of: Prof. UG dr hab. Marcin Zieleniecki – the Chairman, Prof. dr hab. Arkadiusz Sobczyk, Prof. UJ dr hab. Leszek Mitrus, Prof. UW dr hab. Łukasz Pisarczyk, Prof. UAM dr hab. Michał Skąpski, Prof. UG dr hab. Jakub Stelina, Prof. UW dr hab. Jacek Męcina, Prof. UKSW dr hab. Mon- ika Gładoch, Prof. UAM dr hab. Anna Musiała, Prof. WSH dr hab. Marek Pliszkiewicz, 5. Arbitration awards in selected European states vs. Polish legal realities Adamiecki University of Economics in Katowice; Jerzy Wratny, a professor Review of European and Comparative Law \| 2022 Vol. 48, No. 1 20 Issues of awards given as a part of social arbitration in a collective dispute arbitration as a resolution method for dispute of the voluntary nature of awards issued by an arbitration commission does not fulfil the role expect- ed by the legislature. In light of this fact, authors of the project observed that it was necessary to reinstate the importance of arbitration by consid- ering that the arbitrator’s decision would be binding for the parties and end the collective dispute. Additionally, authors of the draft believe that above-mentioned problems justify the introduction of the rule according to which an arbitration award would be subjected to judicial control when it comes to its legal compliance and interests of the parties, which would strengthen the rule of law and social peace in collective labour agreements. This position of the Labour Law Codification Commission was expressed in the suggestion contained in art. 156 § 2 CLC that assumed that each of the parties to a collective dispute would be able to appeal to a court against an arbitration award within 7 days of its receipt if that award blatantly vi- olates the party’s interest or the law57. The appeal would be made to a dis- trict court in the case of a one employer dispute and to a regional court in the case of a multi-employer dispute – both these courts having jurisdiction in the dispute initiation location. Even though an appeal from an arbitra- tion award is not provided for in the following draft of the collective labour code of 14 March 2018 by the new Labour Law Codification Commission58, there is no doubt that the concept worked out in 2006 is worth considering. arbitration as a resolution method for dispute of the voluntary nature of awards issued by an arbitration commission does not fulfil the role expect- ed by the legislature. In light of this fact, authors of the project observed that it was necessary to reinstate the importance of arbitration by consid- ering that the arbitrator’s decision would be binding for the parties and end the collective dispute. 57 Art. 158 § 2 of the draft Collective Labour Code from mid 1990ies suggested that each of the parties and the labour inspector could be granted the right to appeal against the arbitra- tor’s award violating the laws, see Cudowski, Spory, 115.ht Prof. UŁ dr hab. Mirosław Włodarczyk, Dr Jakub Szmit, legal counsel Marta Matyjek, attorney at law dr Liwiusz Laska. 59 The granting of this right to the labour inspector was postulated by Michał Seweryński, see: Michał Seweryński, “Wybrane zagadnienia rozwiązywania sporów zbiorowych w Polsce,” in Arbitraż i mediacja w prawie pracy, ed. Grzegorz Goździewicz (Lublin: Wydawnictwo KUL, 2005), 52. 5. Arbitration awards in selected European states vs. Polish legal realities Additionally, authors of the draft believe that above-mentioned problems justify the introduction of the rule according to which an arbitration award would be subjected to judicial control when it comes to its legal compliance and interests of the parties, which would strengthen the rule of law and social peace in collective labour agreements. hi 2 Review of European and Comparative Law \| 2022 Vol. 48, No. 1 21 Maciej Jarota One has to welcome the rule corresponding, among other things, with the German or French legislation, that is expressed in art. 156 § 2 CLC and guarantees the parties to a dispute the right to appeal against the arbitration award while satisfying conditions provided for in the laws. Considering that the appellate review of arbitration awards is necessary in complicated collective labour relationships, the suggestion presented in CLC is very de- sirable. However, it would be worthwhile to think about a prolongation of the 7-day deadline for the appeal to 14 days. Such a solution would make a professional preparation of the appeal possible, especially in cases with complicated factual and legal circumstances. p g One has to note that satisfying the appeal condition, i.e. blatant viola- tion of an interest of the party, can turn out to be ambiguous. It can be diffi- cult to define the blatant nature of the arbitrator’s undesirable resolution if the arbitrator has the freedom of decision with no statutory model imposed in advance. The grounds for an appeal will be evaluated by an independent court. It seems that, even though the court analysing the legal compliance of an award will be limited by the proper application of the principles of interpretation of the law, it will have the margin of decision when it comes to the analysis of the party’s interest. Therefore, the court hearing the appeal would have the particular responsibility for the correct determination of facts and an appropriate dispute resolution. While analysing the issue of an appeal from the arbitrator’s award, the participation of a labour inspector at this stage of the dispute is worth considering. 22 Review of European and Comparative Law \| 2022 Vol. 48, No. 1 5. Arbitration awards in selected European states vs. Polish legal realities In light of the 2006 proposal, the labour inspector would not have the right to appeal59 even though, in certain situations such as the an- nouncement of a strike, suspension of operations of a plant or its part by the employer for more than 3 months, creation of a major threat to public interest, the labour inspector would be able to initiate a collective dispute pursuant to art. 154 § 2 CLC. According to art. 156 § 1 CLC, the arbitra- tor would resolve a collective dispute by issuing an award to be delivered 22 Review of European and Comparative Law \| 2022 Vol. 48, No. 1 22 Issues of awards given as a part of social arbitration in a collective dispute to the parties, to an appropriate labour inspector and to the National La- bour Dialogue Consultant. Therefore, the labour inspector would receive the arbitrator’s award and, in certain cases, would in fact be able to initiate arbitration proceedings according to CLC but would not be able to appeal against the decision issued by way of social arbitration. Such a situation can cause doubts whether the suggested participation of the labour in- spector in social arbitration would be sufficient. It seems that the lack of interference from the labour inspector at the stage of the appeal against the arbitrator’s decision is justified in the context of the desirable autonomy of the will of parties to the dispute regarding the application of arbitration proceedings. The freedom of trade unions and employers should also cover the making of decisions regarding the undermining of arbitration awards. Review of European and Comparative Law \| 2022 Vol. 48, No. 1 6. Conclusionsh The above considerations allow us to formulate three principal conclusions. Firstly, the Polish legislature should offer an in-depth analysis of institu- tions lacking in PSA. A reference should be introduced to labour law regu- lations to state that in cases not regulated in the appropriate regulation on the amendment and supplementation of an arbitration award and the rejec- tion of an arbitration application, CPC provisions shall be applied accord- ingly. As a consequence, the amendment and supplementation of an arbi- tration award would be permitted in line with cases specified in art. 350 § 1 CPC and art. 351 § 1 CPC. Such an action would also remove doubts regarding the interpretation of the possibility that the social arbitration col- lege may reject the employee application for procedural reasons. Secondly, the failure of parties to the dispute to respect binding arbi- tration awards is also a problematic issue. Legal consequences of the par- ties’ failure to comply with the award issued by an arbitrator within specific time limits has to be set out precisely in the Polish act. The example of Russia is interesting here, where a fine is imposed for non-compliance with the award. Indeed, it would be worth adopting such a sanction in the Pol- ish legal reality – a pecuniary penalty could be imposed by the National Labour Inspectorate (PIP). Additionally, the arbitration award should be included in the catalogue of enforcement titles referred to in art. 777 § 1 of the Code of the Civil Procedure so that it could be efficiently enforced in practice. Review of European and Comparative Law \| 2022 Vol. 48, No. 1 23 Maciej Jarota It also seems appropriate to adopt the principle that an arbitration award is binding on the parties to a collective dispute and is made immedi- ately enforceable, as is the case, for example, in Spain. Such a legal construct could increase confidence in this method of collective dispute resolution. Legal certainty that the award will be enforced is essential from the per- spective of the effectiveness of social arbitration itself. h f Thirdly, it is worth considering whether it might be necessary to in- troduce the possibility of an appeal against an arbitration award in such socially important cases relating to the resolution of collective disputes. It seems that this solution would contribute to an increased importance of social arbitration. 24 Review of European and Comparative Law \| 2022 Vol. 48, No. 1 6. Conclusionsh Judicial control over the award issued in arbitration pro- ceedings would promote trust among parties to the dispute, which could mean that this method of resolution of collective disputes would be used more frequently. Award correctness verification would also make it possi- ble to eliminate errors that can appear in practice during the case assess- ment. It would not be unusual if such a possibility is introduced into Polish legislation. In France, Slovakia or Germany, an option to appeal against an arbitration award is guaranteed by law. It seems that, just like in Germa- ny, the Labour Court should be competent to hear appeals. Obviously, it re- mains an open point whether the case should be examined by the Court of Appeal or the Supreme Court. Given that social arbitration committees competent for in-company disputes operate at District Courts, it would not necessarily be desirable to make the District Court an appellate body. However, if awards are reviewed by the Court of professional judges, there is a reason to believe without any doubt that the process will be carried out with due diligence. In view of the foregoing, how should we assess the above-described regulations on the settlement of a collective dispute through social arbi- tration? First of all, do they provide the balance between social partners? It ought to be emphasized that seemingly these regulations affect the legal situation of both employers and trade unions to an equal extent. Neverthe- less, the provisions are unclear and the enforcement of an arbitration award uncertain, which makes the trade unions reluctant when it comes to this form of dispute resolution. However, this is a disadvantage for the employer itself as well. Failing successful mediation, the employer must be aware that a strike is forthcoming. 24 Issues of awards given as a part of social arbitration in a collective dispute If we interpreted the principle of equal treatment of parties to a col- lective dispute in its broad sense, we could not unambiguously claim that the rule is complied with, given that only a trade union party is vested with the right to institute arbitration proceedings. Still, the rule is not absolute, and we should share the view that it is permissible that in certain situations the legislator may intentionally differentiate the rights of the subjects of collective labour relations in order to achieve a legal balance in practice60. 60 Paweł Nowik, Pojęcie równowagi prawnej w zbiorowym prawie pracy (Lublin: Wydawnictwo KUL, 2016), 90–91. 6. Conclusionsh It seems legitimate whenever the legislator differentiates between the legal situation of the trade unions and the employers in this respect. It cannot be presumed that the employer could also submit a motion to initiate social arbitration in a binding manner. This would be an excessive interference in the collective dispute resolution procedure, giving the employer a real opportunity to block the trade union’s right to organise a strike for some indefinite period of time. Given the current problems in the application of arbitration proceedings and an illusory, non-binding nature of arbitration awards, this would be a highly dysfunctional step. Pre-arbitration meas- ures, i.e. negotiations and mediations give the parties to a collective dis- pute the opportunity to reach an agreement before a strike is initiated in the wake of a failed mediation. Postponing the possibility of resorting to a non-amicable action due to pending arbitration initiated by the employer could adversely affect the success of previous dispute resolution methods. The employer would then be basically deprived of any pressure in the event of a dispute, which could mean its lower involvement in the amicable set- tlement of the dispute during negotiations or mediations. It is particularly worth noting that the common objective of bargain- ing, mediation and arbitration is to prevent non-amicable actions, espe- cially strikes. However, arbitration is the last amicable stage of collective disputes in the light of RCD regulations. It does not necessarily mean that it is the most important method of dispute resolution even though, in the Polish legal reality, arbitration is the final tool making reconciliation of the parties possible. In turn, a strike can negatively impact various aspects of daily life, in particular, it can worsen the employer’s economic situation. Therefore, so as not to permit an automatic cessation of work by employees Review of European and Comparative Law \| 2022 Vol. 48, No. 1 25 Maciej Jarota after unsuccessful mediation, one has to guarantee complete and clear mechanisms of arbitration proceedings. From this perspective, changes in the legal nature of an arbitration award, the possibilities to appeal against it, enforce, supplement or amend it are unavoidable. A comprehensive anal- ysis of the arbitration award institution by the legislature is necessary to strengthen social arbitration as an amicable dispute resolution method. Review of European and Comparative Law \| 2022 Vol. 48, No. 1 References Baran, Krzysztof Wojciech. “Porozumienia zawierane w sporach zbiorowych jako źródła prawa pracy.” Monitor Prawa Pracy, no. 9 (2008): 452–455. Baran, Krzysztof Wojciech. Zbiorowe prawo pracy. Komentarz. Warszawa: Wol- ters Kluwer Polska sp. z o.o., 2010. Barancová, Helena, and Andrea Olšowská. Labour Law in Slovak Republic. 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https://openalex.org/W2147267195	https://academicjournals.org/journal/AJB/article-full-text-pdf/17649F123629.pdf	English	null	Effect of environment and cultivar on the expression of banana streak disease symptoms in Kenya	African journal of biotechnology	2,013	cc-by	5,266	Accepted 2 April, 2010 Accepted 2 April, 2010 Banana (Musa spp.) is grown for subsistence and income generation by 80% of small scale farmers all year round in Kenya hence it is an important food security crop. However viral diseases such as banana streak disease (BSD), caused by Banana streak virus, hamper the production of the crop. BSV has been reported to be present in all the commercial banana cultivars in Kenya. Tissue culture materials of 15 cultivars were evaluated for BSV expression so as to determine the tolerant cultivars under field and greenhouse conditions and stages of disease expression. A number of factors including plant age, cultivar, season and growth site were investigated in the greenhouse and field to assess their effect on BSD symptom expression. Statistical analysis system (SAS) for ANOVA was used for analysis of data for the experiments. Results indicated that BSD symptom expression is significantly influenced by all the factors under investigation. Significant correlation was also observed between symptom severity and plant height, girth, and number of leaves. Findings of this study will be invaluable to stakeholders and researchers in banana industry by laying a foundation for development and adoption of viable BSD management strategies. ana streak virus, Banana streak disease, symptoms expression, tissue culture, ELISA. Key words: Banana streak virus, Banana streak disease, symptoms expression, tissue c Corresponding author. E-mail: laurashali@yahoo.com. INTRODUCTION The banana industry in Kenya is entering a new era based on the demands of the farmers and consumers. There has been interest in the use of in vitro produced material to re-invigorate the banana industry. Reports point to the superior performance of tissue culture plants compared to suckers (Robinson, 1993; Qaim, 1999). Food quality, environmental degradation, increased pest and disease pressure and market competition are key issues favouring use of in vitro propagated banana material over the suckers (Qaim, 1999.; KARI, 1998). However, the occurrence of banana streak badnavirus (BSV) in recent years has negatively affected in vitro production and is considered a major constraint to banana improvement worldwide (Cote et al., 2010). integration and expression, it is difficult to develop an effective strategy in developing Musa hybrids with resistance to BSV. Therefore short term alternatives are required for the management of BSV in the farmers’ fields. Nine Cavendish and six East Africa Highland cultivars were evaluated for BSV symptom expression at Njoro, Kokoto and Rongai in Nakuru district in Kenya and in a greenhouse in 2005/2006. The objective was to establish the effect of the environment on the expression of BSV symptoms in tissue culture banana cultivars. African Journal of Biotechnology Vol. 12(16), pp. 1999-2005, 17 April, 2013 Available online at http://www.academicjournals.org/AJB DOI: 10.5897/AJB09.1433 ISSN 1684–5315 ©2013 Academic Journals African Journal of Biotechnology Vol. 12(16), pp. 1999-2005, 17 April, 2013 Available online at http://www.academicjournals.org/AJB DOI: 10.5897/AJB09.1433 ISSN 1684–5315 ©2013 Academic Journals Full Length Research Paper Effect of environment and cultivar on the expression of banana streak disease symptoms in Kenya Laura Karanja1, Anne Wangai2, Ram Sumer Pathak3 and Glyn Harper4 1Kenya Agricultural Research Institute, Njoro, P.O. Njoro, Kenya. 2Kenya Agricultural Research Institute, Biotechnology Program, P.O. Box 14733, NARL, Nairobi, Kenya. 3Department of Crops, Horticulture and Soil, Egerton University P.O. Njoro, Kenya. 4Department of Disease and Stress Biology, John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, UK. Full Length Research Paper Effect of environment and cultivar on the expression of banana streak disease symptoms in Kenya Laura Karanja1, Anne Wangai2, Ram Sumer Pathak3 and Glyn Harper4 1Kenya Agricultural Research Institute, Njoro, P.O. Njoro, Kenya. 2Kenya Agricultural Research Institute, Biotechnology Program, P.O. Box 14733, NARL, Nairobi, Kenya. 3Department of Crops, Horticulture and Soil, Egerton University P.O. Njoro, Kenya. 4Department of Disease and Stress Biology, John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, UK. Full Length Research Paper Laura Karanja1, Anne Wangai2, Ram Sumer Pathak3 and Glyn Harper4 Accepted 2 April, 2010 In vitro micropropagation of banana Due to integration of the BSV sequences into the plant genome and lack of understanding of the mechanism of Two suckers were selected from each mother plant of 15 cultivars: Chinese Dwarf, Dwarf Cavendish, Giant Cavendish, Gold Dessert, Gold finger, Grand Naine, Jamanga, Lacatan, Muraru, Namukhila, Ng’ombe, Ntobe, Kibuzi, Uganda Green, and Valary. Shoot tips (30 explants) were micropropagated as described by Vuylsteke (1989) Afr. J. Biotechnol. 2000 0. 1. 2. 3 4. 5. Figure 1. BSV disease rating scale, based on symptomatic expression where 0 = no symptoms, 1 = localised flecks, 2 = scattered discontinuous streaks, 3 = continuous streaks covering moderate portion of lamina, 4 = continuous and conspicuous chlorotic streaks, and 5 = necrotic streaks. 0. 1. 1. 2. 3 . 4. 5 5. 4. Figure 1. BSV disease rating scale, based on symptomatic expression where 0 = no symptoms, 1 = localised flecks, 2 = scattered discontinuous streaks, 3 = continuous streaks covering moderate portion of lamina, 4 = continuous and conspicuous chlorotic streaks, and 5 = necrotic streaks. of leaves with symptoms were recorded for each plant. and adapted by Acuña (1996). The photoperiod was fixed at 16 h of light (15500 Lux) and 8 h of darkness. Temperature was maintained at 26°C.The mother plants of each cultivar were sampled and indexed for BSV using enzyme-linked immune sorbent assay (ELISA) protocol as described by Clark and Adams (1977). Only BSV negative materials were selected for micropropagation. All the micropropagated plants were established in polythene bags containing 2 kg of soil. The effect of cultivar and environment (location, season) on BSV symptom expression The objectives of the experiment were to identify the effect of cultivar and environment on BSV symptom expression. Njoro, Kokoto and Rongai fall in the following agro-ecological zones: KARI j p y cultivar and environment on BSV symptom expression. Njoro, Kokoto and Rongai fall in the following agro-ecological zones: KARI - Njoro lies at an altitude of 2166 m above sea level (ASL) and is located within the agro-ecological zone low highlands (LH3) (Jaetzold and Schmidt, 1983), longitude 0° 20’ South and 35 56’ East with a mean annual rainfall of 931mm per annum. Temperature ranges from 7.9 - 21.9°C with a mean of 14.9°C (Jaetzold and Schmidt, 1983). Kokoto is in upper midland (UM4) zone at an altitude of 1830 ASL with annual rainfall of 700 mm and mean temperatures of 20°C. Rongai is in upper midland (UM6) at an altitude of 1620 ASL and receives annual rainfall of 600 mm with mean temperatures of 19°C. The soils are fertile vitric mollic Andosols that are well drained, deep to very deep, dark reddish brown in colour consisting of heavy textured friable silty clay to clay humid top soils (Jaetzold and Schmidt, 1983). Evaluation of fifteen in vitro propagated cultivars was carried out in the three sites from January, 2005 - December, 2006. The experiment was planted in a randomized complete block design (RCBD) replicated three times for each cultivar per site. The spacing and depth used was 3 m by 3 m and depth of 4 ft. Twenty tons per ha of manure and 250 kg/ha of diammonium phosphate fertiliser was used at planting time which are the current recommended quantities. Fortnight spraying was done with Bullock® containing active ingredients Beta-cyfluthrin 12.5g/l and Chlorpyrifos 250g/l for aphid control. The concentration used for spray was 20 ml/45 L of water. Mitigan® with an active ingredient Cypermetrin was used for the control of mites. The rates used for spray were 20mls/30 L of water. Rindomil® with active ingredients Mancozeb 640 g and Metalaacyl-M 40 g was used for Effect of seedling age on BSV symptom expression The objective of the experiment was to determine the stage in months of growth at which BSV expression is highest and the cultivars which are most affected. Out of the total 15 micro- propagated cultivars, five dessert cultivars that are most common were selected and tested in the greenhouse for BSV expression. Three months old micropropagated plants were used. A completely randomized design (CRD) was used. The layout was composed of two treatments, cultivars and time. The cultivar treatment included Giant Cavendish, Gold Dessert, Gold Finger, Lacatan and Grand Naine while the time treatment had six months, month 1, 2, 3, 4, 5, and 6. All the seedlings were planted in 30 by 24 cm polythene bags. Cultivar Grand Naine was used as a healthy control in this experiment. No virus inoculation was done. The soil used was mainly forest soils. The plants were treated with Furadan at 5 g per seedling in each bag to control nematodes. Watering was done daily and the plants were sprayed with fungicide and miticide fortnightly. The study was conducted during the six months growing period before on-set of flowers. Plants were observed for six months and data was collected monthly. The variables measured were symptom severity where individual leaves were scored on a disease rating scale of 0 - 5 (Figure 1) and the total number of leaves and number Karanja et al. 2001 2001 Karanja et al. Table 1. Serological analysis of 15 banana cultivars before and after in vitro micropropagation. Table 1. Serological analysis of 15 banana cultivars before and after in vitro micropropagation. Cultivar No of initial suckers tested No. Effect of seedling age on BSV symptom expression of in vitro seedlings tested after potting No of positive plants Negative mean A405nm values of ELISA for the initial suckers Positive mean A405nm Values of ELISA for in vitro seedlings Positive or negative Ng’ombe 2 5 2 0.094 0.169 + D/ Cavendish 2 5 2 0.093 0.132 + G/Cavendish 2 5 2 0.098 0.111 + Grand Naine 2 5 1 0.095 0.124 + Namukhila 2 5 1 0.070 0.128 + Valary 2 5 1 0.093 0.110 + C/Cavendish 2 5 1 0.098 0.131 + Gold finger 2 5 2 0.093 0.120 + Lacatan 2 5 1 0.100 0.112 + Gold Dessert 2 5 0 0.100 0.100 - Jamanga 2 5 0 0.083 0.084 - Ntobe 2 5 0 0.080 0.083 - Uganda Green 2 5 2 0.089 0.111 + Kibuzi 2 5 0 0.074 0.076 - Muraru 2 5 1 0.098 0.365 + Negative control (PBS buffer) 0.082 0.091 - ELISA protocol was used to screen for BSV and leaf samples with and without symptoms were used. All samples with absorbance levels of above 0.100 were considered positive while those with 0.100 or less were considered negative. ELISA protocol was used to screen for BSV and leaf samples with and without symptoms were used. All samples with absorbance levels of above 0.100 were considered positive while those with 0.100 or less were considered negative. ELISA protocol was used to screen for BSV and leaf samples with and without symptoms were used. All sampl 0.100 were considered positive while those with 0.100 or less were considered negative. of some of the cultivars might have been too low for the ELISA technique to detect especially the dessert bananas. Also, there is a possibility of presence of many different isolates with varying A405nm. However for the balbisiana (B genome)-containing cultivars such as Gold finger, there is a possibility of integrated viral sequences being activated after tissue culture (Ndowora et al., 1999). For all the cultivars three seedlings with absorbance levels of less than 0.1 were selected and planted in the field. fungal infection at the rate of 20 g / 40 L of water. Weeds were removed by uprooting regularly to maintain a clean stand. Infection and symptom expression was monitored as described in Figure 1. Data analysis All data on symptom severity, plant height, width, number of leaves and absorbance levels were subjected to ANOVA using the General Linear Model (GLM) of SAS computer package version 6.12 (SAS, 1999) and statistical model as described by Gomez and Gomez (1984). Means were separated using the Least Significant difference (LSD). Data were recorded on a monthly basis and observations done over a period of six months. Correlations between variables were done using Pearson’s correlation coefficient in SAS statistical package. All data were transformed before analysis with ANOVA procedure. BSV expression as influenced by age The results showed there was significant (P < 0.05) variation in symptom expression among the five cultivars during the third and fourth month of growth (Table 2). Cultivar, Gold finger, had highest symptom expression whereas Gold dessert had the lowest severity in com- parison to the rest of the cultivars. There was variation in symptom expression for all the five genotypes with significantly (P < 0.05) high BSV expression at sixth month compared to first month when the expression was lowest. Symptom expression fluctuated from month to month intermittently with appearance and disappearance of symptoms. Fluctuating temperatures in the green house may possibly have played a role (Dahal et al., 1998). With maturity of plants, there is a possibility of accelerated virus replication and increased virus concen- tration therefore higher symptom expression could easily be detected. Serological diagnostics of micropropagated seedlings When the potted seedlings were screened after 3 months and before transplanting into the field, results indicated that from the five seedlings of each cultivar screened, 0 - 40% were BSV positive even though the seedlings were initially symptomless. Over 73% overall of the micro- propagated cultivars showed BSV positive symptoms. However the absorbance value (A405nm) of BSV antigen varied with cultivar, Muraru had the highest followed by Ng’ombe and Kibusi had the lowest A405nm (Table 1). It is possible that during the initial screening the virus A405nm 2002 Afr. J. Biotechnol. Table 2. Effect of time and cultivar on expression of banana streak virus. Time (months) Cultivar Giant cavendish Gold dessert Gold finger Grand naine Lacatan Time means 1 1.00c 1.00c 1.00c 1.14bc 1.00c 1.03c 2 1.28abc 1.00c 1.28abc 1.24abc 1.28abc 1.21abc 3 1.24abc 1.00c 1.38ab 1.00c 1.14bc 1.15bc 4 1.24bc 1.00c 1.63a 1.14bc 1.24abc 1.25ab 5 1.00c 1.24bc 1.14bc 1.24abc 1.00c 1.13bc 6 1.38ab 1.28abc 1.52a 1.28abc 1.58a 1.41a Cultivar means 1.19xy 1.09y 1.32x 1.17xy 1.21xy Seedlings were grown from micropropagated plants. Disease severity rating was done as described in Figure 1.Numbers within the columns followed by the same letter (a, b, c) are not significantly different at 5% and numbers within the rows followed by the same letter (x, y) are not significantly different P < 0.05. LSD for cultivar is significant (P < 0.05); LSD for time is significant (P < 0.01). Cultivar Seedlings were grown from micropropagated plants. Disease severity rating was done as described in Figure 1.Numbers within the columns followed by the same letter (a, b, c) are not significantly different at 5% and numbers within the rows followed by the same letter (x, y) are not significantly different P < 0.05. LSD for cultivar is significant (P < 0.05); LSD for time is significant (P < 0.01). Table 3. Correlation between the growth characteristics and infection of banana streak virus under greenhouse conditions. Table 3. Correlation between the growth characteristics and infection of banana streak virus under greenhouse conditions. Parameter Symptom Height Girth Leaves Time Symptom Height Girth Leaves Time 1.000 -0.1408 1.0000 0.1178 0.05015 1.0000 -0.2329* 0.4285** 0.2110* 1.0000 0.280 -0.525 -0.30311 -0.68695 1.0000 Micropropagated plants were 6 months at determination of these parameters. Significant at 5% level of probability. Micropropagated plants were 6 months at determination of these parameters. Significant at 5% level of probability. Serological diagnostics of micropropagated seedlings Gold finger cultivar (AAAB_FHIA 01) had significantly (P < 0.05) higher expression of BSV during its growth period and Gold Dessert (AAA) had the lowest. The high incidence of BSV expression in Gold finger is probably due to the presence of a ‘B’ genome which has integrated viral sequences and could have been activated by micropropagation process. These results affirm those reported by Dallot et al. (2001) and Geering et al. (2005) who observed that expression of episomal BSV during in vitro procedure is correlated to the presence of an integrated form. However Gold Dessert (AAA genotype) might have had episomal virus with very low concentration or the meristem culture could have eliminated any BSV infection as has been reported before (Helliot et al., 2002). The higher expression shows that virus concen- tration increases with plant growth. Therefore cultivar and age of banana plants have a bearing in BSV expression. infection of BSV leaf numbers were reduced due to interruption of nutrient synthesis for the development of leaves. Highly significantly and (r = 0.43) positive correlation were also recorded between number of leaves and plant height; number of leaves and girth (r = 0.21). This indicated that taller plants and those with wider girth had more leaves. Leaf number can therefore be used to estimate the photosynthetic capacity and to predict the performance of a banana crop (Valladares et al., 2003). The significant (r = -0.140) negative correlation observed between symptom expression and height was an indication that BSV infection affected the growth rate in that there was slower growth rate with higher infection. These results gave an indication that with more infection and expression of symptoms there is reduced height of the plant which directly affects synthesis of nutrients for development of banana bunches. In the analysis of the five growth characteristics, signi- ficant and positive correlation (r > 0.28) was observed between BSV symptom expression and time taken. There was less infection and expression during the early months of growth but this progressively increased with age of the plant. This was an indication that the age of the plant contributed directly to expression of BSV. Significant negative correlations (r = 0.525; -0.303; - 0.687) were observed between plant height and time, Correlation between the growth characteristics and infection of banana streak virus under greenhouse conditions With each passing month there was reduction in the rate growth in height width and number of leaves. BSV virus infection could have slowed down growth. Wet conditions could have contributed to the activation of BSV in dessert cultivars as had also been reported by Dahal et al. (1998). However, more studies are required to establish the relationship of cultivar and moisture levels in BSV activation and symptom expres-sion. Ng’ombe, a cooking variety (EAH-AAA genotype) had relatively high BSV infection and expression in comparison to the rest of the varieties while Valary had the lowest infection and expression across the locations and years (Table 4). Most interesting however was that Ng’ombe cultivar had vigorous vegetative growth across the locations. Further investigations arerequired to confirm tolerance to BSV aspect in Ng’ombe cultivar. Correlation between the growth characteristics and infection of banana streak virus under greenhouse conditions A significant (P < 0.05) negative correlation was observed between BSV symptoms and number of leaves produced (r = -0.23) (Table 3). This indicated that with higher Karanja et al. 2003 2003 Karanja et al. Table 4. Banana streak virus symptom incidence in Njoro, Kokoto, and Rongai over two years’ period. Table 4. Banana streak virus symptom incidence in Njoro, Kokoto, and Rongai over two years’ period. Cultivar Njorob Kokotoa Rongaiab 2005 2006 2005 2006 2005 2006 Chinese Cavendish 1.00e 1.28cd 1.00e 1.41bc 1.41b 1.41bc Dwarf Cavendish 1.14de 1.14d 1.28cd 1.41bc 1.28cd 1.28cd Giant Cavendish 1.00e 1.41bc 1.14de 1.52b 1.28cd 1.41bc Gold Dessert 1.14de 1.47b 1.14de 1.61a 1.14de 1.14d Gold Finger 1.00e 1.14d 1.28cd 1.41bc 1.28cd 1.41bc Grande Naine 1.00e 1.14d 1.14de 1.61a 1.14de 1.28cd Jamanga 1.41b 1.28cd 1.41b 1.41bc 1.41b 1.41bc Lacatan 1.00e 1.28cd 1.14de 1.41bc 1.14de 1.28cd Muraru 1.00e 1.52b 1.00e 1.41bc 1.00e 1.00e Namukhila 1.28cd 1.41bc 1.75a 1.41bc 1.00e 1.00e Ng’ombe 2.24a 1.41bc 2.24a 1.28cd 2.24a 2.24a Ntobe 1.14de 1.52b 1.14de 1.00e 1.00e 1.00e Kibuzi 1.00e 1.41bc 1.00e 1.14d 1.00e 1.00e Uganda Green 1.14de 1.28cd 1.00e 1.41bc 1.14de 1.28cd Valary 1.14de 1.14d 1.00e 1.28cd 1.14de 1.14d Means for Locations 1.17xyz 1.32w 1.25wx 1.38w 1.24wx 1.28wx Seedlings were grown from micropropagated plants. Disease severity rating was done as described in Figure 1. Numbers within the columns followed by the same letter (a, b, c) are not significantly different at 5% using LSD and numbers within the rows followed by the same letter (w, x, y, z) are not significantly different at 5% using LSD. Seedlings were grown from micropropagated plants. Disease severity rating was done as described in Figure 1. Numbers within the columns followed by the same letter (a, b, c) are not significantly different at 5% using LSD and numbers within the rows followed by the same letter (w, x, y, z) are not significantly different at 5% using LSD. There is a possibility that dry conditions played a role in activation of BSV in the cooking cultivars (EAH-AAA) as had been also observed earlier by Daniells et al. (2001). In the second year when rainfall level recorded was approximately 1178.1 mm, highest expression was observed on the dessert bananas especially in Kokoto location. plant girth and time, and number of leaves and time (Table 3). Growth rate of all the three characteristics reduced with the time factor. DISCUSSION Detection in the field of de novo BSV infections in micropropagated Musa spp has led to the hypothesis of a possible effect of the in vitro multiplication procedure on BSV expression (Dahal et al., 1999). Factors contributing to the expression of BSV in the greenhouse and the field were identified. These factors included cultivars, plant age, season of the year and location (site). It was observed that under the greenhouse conditions, infected Gold finger cultivar expressed high symptoms during the third and fourth month, while Gold dessert, Giant Cavendish, Grand Naine and Lacatan had lower expression during the same period of time (Table 2). Infection of some isolates of BSV may arise de novo from viral sequences that are integrated into the ‘B’ genome of Musa (Ndowora et al., 1999; Harper et al., 1999). Tissue culture has been considered as one of the trigger factors for episomal expression of these integrants (Geering et al., 2001; Dallot et al., 2001; Ndowora and Lockhart, 1997). Gold finger, which is a cultivar of the AAAB genotype which contains the ‘B’ genome, may have been activated by the micropropagation process leading to high infection and expression of BSV. However, the integrants are absent from Cavendish banana (Geering et al., 2001) and this could have contributed to low infection or symptom expression observed in the rest of the five cultivars in the greenhouse. Optimum infection and symptom expression for the Gold finger in the greenhouse was observed 4 months after planting of seedlings. Plants that are four months old could be used for screening greenhouse material for BSV before field planting. However, more studies are required to establish the right time for final screening before field establishment of micropropagated seedlings. y These data suggest that specific genetic factors in cultivar (such as nature of integrated BSV sequences) and environmental conditions (moisture and type of isolates) may be key factors in the expression of BSV. The findings could be used as a guide in the management of banana streak disease in Kenya. Determining the effect of cultivar, location and season in banana streak virus (BSV) symptom expression From this study there is an indication that BSV expression might be affected by cultivar, location and year (wet or dry). It might therefore be possible to select cultivars suitable for the three locations. There were fluctuations in the BSV incidences from location to location and cultivar to cultivar. For example while Chinese Dwarf was less infected in Njoro there was high BSV infection in Kokoto and Rongai during the second season. From this study there is an indication that BSV expression might be affected by cultivar, location and year (wet or dry). It might therefore be possible to select cultivars suitable for the three locations. The interaction between cultivar and location was significant (P < 0.01). It was observed that the response of the banana cultivars to BSV expression varied with the location where it was planted. This probably was because the three locations fall under different ecological zones with different altitudes, rainfall and temperatures as was described previously. The information will be valuable in future for cultivar recommendation of specific areas. Additionally, more studies are required to validate this study. Determining the effect of cultivar, location and season in banana streak virus (BSV) symptom expression Results showed significant (P < 0.05) differences for infection and symptom expression based on 15 cultivars in Njoro, Kokoto and Rongai locations (Table 4). The significant effect of cultivar by location indicates that screening should be done at each location. g The cultivar by location effect also indicates that the potential for BSV infection and expression might be influenced by location due to differences in virus isolates within locations. BSV symptom expression was significantly (P < 0.05) higher in Kokoto compared to Njoro or Rongai locations (Table 4). In the first year, symptom expression was significantly (P < 0.05) lower compared to the second year in all the 15 cultivars However, high expression levels were observed predominantly in cooking varieties (Ng’ombe, Jamanga, and Namukhila) of genotype (EAH- AAA) across the locations. During the same period, there was low moisture recorded of approximately 778.7 mm. Based on the eight dessert cultivars and seven cooking cultivars evaluated in the three locations during the second year, the response of the cooking and dessert cultivars in Njoro indicated 88% dessert bananas (Cavendish and Gold finger) had lower infection in Njoro but high infection in Kokoto and Rongai locations while the cooking varieties (Muraru, Ntobe and Kibuzi) had significantly high infection in Njoro and relatively lower infection in Kokoto and Rongai location during the second Afr. J. Biotechnol. Afr. J. Biotechnol. 2004 activated the high symptom expression. These results contradict those obtained from a study carried out in Nigeria where greater symptom expression were observed when plants were grown in wet season compared to those grown in dry season (Dahal et al., 1998). This information could be used in the management of the BSV by ensuring no water stress (Daniells et al., 2001). year, 2006 (Table 4). The rest of the cooking cultivars however had either lower or similar rate of infection as was observed with Ng’ombe, Uganda Green, Namukhila and Jamanga cultivars across the locations and seasons. year, 2006 (Table 4). The rest of the cooking cultivars however had either lower or similar rate of infection as was observed with Ng’ombe, Uganda Green, Namukhila and Jamanga cultivars across the locations and seasons. There were fluctuations in the BSV incidences from location to location and cultivar to cultivar. For example while Chinese Dwarf was less infected in Njoro there was high BSV infection in Kokoto and Rongai during the second season. REFERENCES Acuña PI (1996). Micropropagación del cv. ‘Maqueño’ (Musa AAB) y de las especies M. acuminata (AA) y M. balbisiana (BB) a concentraciones bajas de 6-bencilaminopurina. Corbana 21:85-92. Jaetzold R, Schmidt H (1983a). Farm Management Handbook of Kenya. Ministry of Agriculture Kenya. Central Kenya (Rift Valley and Central Province. p. 11. Clark MF, Adams AN (1977). Characteristics of the microplate method of enzyme-linked immunosorbent assay for the detection of plant viruses. J. Gen. Virol. 34:475-483. Ndowora T, Dahal G, LaFleur D, Harper G, Hull R, Olszewski NE, Lockhart BEL (1999). Evidence that badnavirus infection in Musa can originate from integrated pararetroviral sequences. Virology 255:214- 220. Dahal G, Gauhl F, Pasberg-Gauhl C, Hughes Jd’A, Thottappilly G, Lockhart BEL (1999). Evaluation of micropropagated plantain and banana (Musa spp) for banana streak badnavirus incidence under field and screenhouse conditions in Nigeria. Annals Appl. Biol. 134:181-191. Ndowora TCR, Lockhart BEL (1997). Improved serological method for detecting banana streak virus. Musae Africa 11:15. Ortiz R (1996). The potential of AMMI analysis for field assessment of Musa genotypes to virus infection. Hort. Sci. 31:829-832. Dahal G, Hughes Jd’A, Thottappilly G, Lockhart BEL (1998a). Effect of temperature on symptom expression and reliability of banana streak badnavirus detection from naturally-infected plantain and banana (Musa spp). Plant Dis. 82:16-21. Qaim M (1999). Assessing the Impact of Banana Biotechnology in Kenya. Biotechnol. Dev. Monitor pp. 18-22. y pp Robinson JC (1993) Handbook of banana growing in South Africa. Institute of Tropical and Subtropical Crops. Nelspruit, S. Afr. p. 128. Dallot S, Acuna P, Rivera C, Ramirez P, Cote F, Lockhart BEL, Caruana ML (2001). Evidence that the proliferation stage of micropropagation procedure is determinant in the expression of banana streak virus integrated into the genome of the FHIA 21 hybrid (Musa AAAB). Arch. Virol. 146:2179-2190. SAS (1999). Statistical Analysis Systems, SAS Institute, Cary, NC. Stover RH, Simmonds N (1987). Bananas in the Longman Tropical Agriculture streak disease. J. Phytopathol. 76:995-999. Vuylsteke DR (1989). Shoot-tip culture for the propagation, conservation and exchange of Musa germplasm. In: Inter. Board Plant Gen. Res. Rome pp. 1-56. Daniells JW, Geering AD, Bryde NJ, Thomas JE (2001). The effect of banana streak virus on the growth and yield of dessert bananas in tropical Australia. Ann. Appl. Biol. 139:51-60. Geering ADW, Parry JN, Zhang L, Olszewski N, Lockhart BEL, Thomas JE (2001a). Is Banana streak virus strain OL the only activatable virus integrant in the Musa genome? growth would eventually translate into fruit production. Harper G, Osuji JO, Heslop-Harrison JS, Hull R (1999b). Integration of banana streak badnavirus into the Musa genome: molecular and cytogenetic evidence. Virology 255:207-213. Helliot B, Panis B, Poumay Y, Swennen R, Lepoivre P, Frison E (2002) Cryopreservation for the elimination of Cucumber mosaic and Banana streak viruses from banana (Musa spp). Plant Cell Rep. 20:1117-1122. Karanja et al. 2005 Conclusion During the screening of the 15 micropropagated banana cultivars, it was found that the BSV concentration varied with cultivars. When 5 banana cultivars were exposed to the greenhouse conditions they expressed varying degrees of BSV symptom expression. Symptoms were however not changing drastically within the six months because the initial screened samples could have had very low virus load which was activated by environmental factors with time. BSV infection affected the height, girth, and number of leaves negatively. This could have been due to toxicity and death of cells caused by the virus. As a result there were fewer resources available to the plant for growth. In the current study, BSV varied greatly between cultivars and locations and was significantly higher in landraces of cooking varieties which included Ng’ombe, Jamanga and Namukhila. These expressed severe symptoms under low moisture conditions while the dessert cultivars mostly Cavendish expressed severe symptoms under high moisture levels. This is of great concern since most landraces are of the AAA genome. From these findings, it may be possible that reducing moisture for Cavendish may result into low BSV symptom expression, while increasing moisture levels for landraces may also reduce BSV symptom expression. Moisture control can only be achieved either through irrigation or planting the appropriate cultivars in the right environment. Although there were minimal differences in BSV severity amongst cultivars, Ng’ombe had a higher expression of BSV compared to Valary (AAA genotype) across the three locations (Table 4). There is need to establish why Ng’ombe which is an EAH - AAA genotype had higher expression than the Cavendish Valary. It was observed that moisture level was a critical factor in BSV expression (Table 4). Dry conditions could have Although Ng’ombe expressed high BSV levels, vigorous vegetative growth was observed showing the level of tolerance. As was described previously, good vegetative Karanja et al. REFERENCES In: Proceedings of the Second International Conference on Molecular and Cellular Biology of Banana. InfoMusa 10:5. Geering ADW, Pooggin MM, Olszewski NE, Lockhart BEL, Thomas JE (2005b). Characterisation of Banana streak Mysore virus and evidence that it’s DNA is integrated in the B genome of cultivated Musa. Arch. Virol. 150:787-796. Gomez AK, Gomez AA (1984). Statistical Procedures for Agricultural Research. In: Wiley-Interscience Publication 2 nd Edition. Chapter 3:97-101.
https://openalex.org/W2057910823	https://europepmc.org/articles/pmc4094678?pdf=render	English	null	Circulating anti-filamin C autoantibody as a potential serum biomarker for low-grade gliomas	BMC cancer	2,014	cc-by	6,952	© 2014 Adachi-Hayama 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 credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. * Correspondence: iwadatey@faculty.chiba-u.jp †Equal contributors 1Department of Neurological Surgery, Chiba University, Graduate School of Medicine, 1-8-1, Inohana, Chuo-ku, Chiba 260-8670, Japan Full list of author information is available at the end of the article Abstract Background: Glioma is the most common primary malignant central nervous system tumor in adult, and is usually not curable due to its invasive nature. Establishment of serum biomarkers for glioma would be beneficial both for early diagnosis and adequate therapeutic intervention. Filamins are an actin cross-linker and filamin C (FLNC), normally restricted in muscle tissues, offers many signaling molecules an essential communication fields. Recently, filamins have been considered important for tumorigenesis in cancers. Methods: We searched for novel glioma-associated antigens by serological identification of antigens utilizing recombinant cDNA expression cloning (SEREX), and found FLNC as a candidate protein. Tissue expressions of FLNC (both in normal and tumor tissues) were examined by immunohistochemistry and quantitative RT-PCR analyses. Serum anti-FLNC autoantibody level was measured by ELISA in normal volunteers and in the patients with various grade gliomas. Results: FLNC was expressed in glioma tissues and its level got higher as tumor grade advanced. Anti-FLNC autoantibody was also detected in the serum of glioma patients, but its levels were inversely correlated with the tissue expression. Serum anti-FLNC autoantibody level was significantly higher in low-grade glioma patients than in high-grade glioma patients or in normal volunteers, which was confirmed in an independent validation set of patients’ sera. The autoantibody levels in the patients with meningioma or cerebral infarction were at the same level of normal volunteers, and they were significantly lower than that of low-grade gliomas. Total IgG and anti-glutatione S-transferase (GST) antibody level were not altered among the patient groups, which suggest that the autoantibody response was specific for FLNC. Conclusions: The present results suggest that serum anti-FLNC autoantibody can be a potential serum biomarker for early diagnosis of low-grade gliomas while it needs a large-scale clinical study. Keywords: Glioma, Filamin C, FLNC, Biomarker, Early diagnosis Keywords: Glioma, Filamin C, FLNC, Biomarker, Early diagnosis Adachi-Hayama et al. BMC Cancer 2014, 14:452 http://www.biomedcentral.com/1471-2407/14/452 Open Access Circulating anti-filamin C autoantibody as a potential serum biomarker for low-grade gliomas Masayo Adachi-Hayama1,3†, Akihiko Adachi1†, Natsuki Shinozaki1,2,3, Tomoo Matsutani1, Takaki Hiwasa2, Masaki Takiguchi2, Naokatsu Saeki1 and Yasuo Iwadate1* * Correspondence: iwadatey@faculty.chiba-u.jp Background advances in glioma diagnosis and therapy, two-year sur- vival for the grade IV glioblastoma is less than 30%. Even among patients with low-grade gliomas that usually confer a relatively good prognosis, treatment is almost never curative under the current diagnostic system [1]. Glioma is the most common type of primary brain tumor in adults. Currently, tumor grading by histological analysis of surgically-resected specimens is the most reliable pre- dictor of glioma prognosis. They are classified into four grades; low-grades including WHO Grade I (localized gli- omas) and WHO Grade II (diffuse gliomas), and high- grades including WHO Grades III (anaplastic gliomas) and WHO Grade IV (glioblastoma). Despite the recent Identification of specific glioma antigens is long awaited for the clinical management such as early diagnosis, more objective diagnosis, monitoring treatment response, and for novel therapeutic targets for glioma [2-5]. In the previ- ous study utilizing proteomics, we found several proteins that are overexpressed in high-grade gliomas and some were potentially applicable to serum biomarkers by ability of secretion [6]. Serum levels of these candidate proteins * Correspondence: iwadatey@faculty.chiba-u.jp †Equal contributors 1Department of Neurological Surgery, Chiba University, Graduate School of Medicine, 1-8-1, Inohana, Chuo-ku, Chiba 260-8670, Japan Full list of author information is available at the end of the article Adachi-Hayama et al. BMC Cancer 2014, 14:452 http://www.biomedcentral.com/1471-2407/14/452 Adachi-Hayama et al. BMC Cancer 2014, 14:452 http://www.biomedcentral.com/1471-2407/14/452 Page 2 of 10 Page 2 of 10 Table 1 Characteristics of the training set and validation set Training set Validation set Demographic Glioma (n=65) Control (n=38) Glioma (n=66) Control (n=39) Age Mean 46.6 47.6 49.2 51.2 Range 12-74 34-68 24-78 22-77 Sex Male 40 21 37 21 Female 25 17 29 18 WHO grade I 7 8 II 28 29 III 9 9 IV 21 20 Table 1 Characteristics of the training set and validation set were shown to correlate significantly with tumor grade, in- vasive nature of the tumor and patient survival periods [7,8]. However, detection of low-grade tumors is difficult by utiliz- ing the protein amount-based diagnostic system because their serum protein levels may not be sufficiently altered to be detectable by current proteomic technologies. One ap- proach for overcoming this difficulty and thereby enable early detection of slight changes in protein amount, protein structure or protein localization would be utilization of antigen-antibody interactions [9,10]. Background Detection of the host immune reactions which can respond to slight changes in the precursor cells that have started to transform into neo- plastic cells would be a breakthrough to enable early diagno- sis of cancers including glioma. To verify this concept, we utilized immunoscreening of cDNA libraries prepared from glioblastoma cells with IgG in the sera from glioma patients. We found filamin C (FLNC), which is normally restricted in muscle tissues but abundantly exists in fetal central ner- vous system [11,12], as a candidate protein for glioma anti- gen. Filamins are an actin cross-linker, and serve as scaffolds for many binding partners including channels, re- ceptors, intracellular signaling molecules, and transcription factors [13,14]. Because of these extensive fields of associat- ing proteins, mutations in filamin genes result in a wide range of cell and tissue anomalies. Especially they have a decisive role in cellular motility and migration [11-14]. Fur- thermore, Filamin genes mutations are common in human breast and colon cancers [15]. Many recent studies have suggested filamin A as an important factor for tumor ma- lignancy and invasiveness in various human cancers in- cluding primary brain tumors [16-20]. In addition, filamin A interacts with BRCA1/2 or other DNA repair-related proteins to affect the DNA repair process resulting in re- sistance to radiation and chemotherapy [21,22]. In this paper, we examined tissue expressions of FLNC (both in normal and tumor tissues), and investigated the serum levels of anti-FLNC autoantibody in glioma patients. 38 samples from healthy volunteers were used to develop a diagnostic model (training set) that was validated in an inde- pendent, blinded validation set using the serum samples from 66 glioma patients and 39 healthy volunteers. The protocol of this study was approved by the Institutional Review Board of Chiba University, and written informed consent was ob- tained from the patients or their guardians. Total RNAs of the lung, liver, spleen, testis and muscle were commercially obtained (Zyagen Laboratories, San Diego, CA). Total RNAs of lung, liver, spleen, testis and muscle were commercially ob- tained (Zyagen Laboratories, San Diego, CA). Serological analysis of recombinant cDNA expression libraries (SEREX) Total RNA was prepared from the U87MG glioblastoma cell line by the acid guanidium thiocyanate-phenol-chloroform method, and purified to poly(A) + RNA using the Oligotex- dT30 (Super) mRNA Purification Kit (Takara Biochemicals, Kyoto, Japan). cDNA was ligated into the EcoRI-XhoI site of the λZAP II phage. The original library size was 1 × 106. Escherichia coli XL1-Blue MRF’ was infected with the λZAP II phages which contained the U87MG cDNA library, and the expression of cDNA was induced by blotting on nitro- cellulose membranes which had been pretreated for 30 min with 10 mM IPTG (Wako Pure Chemicals, Osaka, Japan). After washing and blocking, the membranes were exposed in 1:2000-diluted sera from 18 glioma patients. Then, the membranes were treated with 1:5000-diluted al- kaline phosphatase-conjugated F(ab)’ fragment-specific goat antihuman IgG. Positive reactions were detected by incubation in a color development solution containing 0.3 mg/mL of nitroblue tetrazolium chloride and 0.15 mg/ mL of 5-bromo-4-chloro-3-indolyl-phosphate. Positive clones were re-cloned twice to obtain monoclonality and retested for the serum reactivity. Real-time RT-PCR The real-time quantitative RT-PCR with SYBR-green was performed using the Light Cycler (Roche Diagnostics, Meylan, France). The amplification was performed using 5’-GGACATGAGTGGCCGGTACAC-3’ as the forward primer and 5’-ACTGTGACGAGGCACTTGCTG-3’ as the reverse primer. A series of cDNA dilutions, 1/1, 1/10, 1/100, and 1/1000, were used in each run separately. Standard curves were obtained by doing serial dilutions of the same sample in each run. Then, 1 mM of each primer and 3 mM of MgCl2 in the total volume of 20 μl were used. The real-time RT-PCR cycle started with the initial denaturation at 95°C for 10 min, followed by 45 cycles of denaturation at 95°C for 10s, annealing at 61°C for 10s and then elongation at 72°C for 10s. As an internal quanti- tative control of the gene expression, the glyceraldehydes- 3-phosphate dehydrogenase (GAPDH) gene expression was used. The ratios of filamin C and GAPDH gene Sandwich ELISA for Serum FLNC measurement Sandwich ELISA for Serum FLNC measurement ELISA 96-well plates were coated with 20 μg/ml antihu- man FLNC antibody, and were filled overnight with 50 μl of patients’ sera diluted 1:100. The plates were developed with o-phenylene-diamine (Sigma-Aldrich, St Louis, MO) and were read at an absorbance of 490 nm. Figure 1 Tissue expression of filamin C mRNA was measured by quantitative real-time RT-PCR analysis in normal brain tissues, low-grade gliomas, and high-grade gliomas, and also in lung, liver spleen, testis and muscle which were commercially obtained. The filamin C mRNA expression was significantly up-regulated in high-grade gliomas compared with normal brain tissues. It was moderately upregulated in low-grade gliomas and normal muscle tissues. The mean values of duplicate experiments for each sample are presented. Figure 1 Tissue expression of filamin C mRNA was measured by quantitative real-time RT-PCR analysis in normal brain tissues, low-grade gliomas, and high-grade gliomas, and also in lung, liver spleen, testis and muscle which were commercially obtained. The filamin C mRNA expression was significantly up-regulated in high-grade gliomas compared with normal brain tissues. It was moderately upregulated in low-grade gliomas and normal muscle tissues. The mean values of duplicate experiments for each sample are presented. Purification of recombinant FLNC protein The cDNA insert of FLNC incorporated in pBlueScript was cleaved by EcoRI and XhoI, and then recombined in pGEX-4 T-3. E. coli JM109 cells containing either pGEX- 4 T-3- FLNC or control pGEX-4 T-3 were cultured in 200 mL of Luria broth and treated with 1 mM IPTG for 2.5 hrs. The cell lysate was centrifuged and GST-FLNC in the supernatant was directly purified with glutathione- Sepharose (Amersham Biosciences, Piscataway, NJ). The purified proteins were concentrated using Apollo centrifu- gal concentrators (Orbital Biosciences, Topsfield, MA). Sera and tissue specimens We analyzed 131 glioma patients’ sera (low-grade, 72; high- grade, 59) along with 77 sera from healthy volunteers, 19 sera from patients with meningioma, and 24 patients with cerebral infarction at chronic stage. These were newly- diagnosed patients, and had no other cancer or diseases at the time of sample collection. They had serum drawn at the time of initial diagnosis. The patient demographics and clinical profiles are presented in Table 1. Forty-eight glioma tissues surgically-resected from newly diagnosed glioma pa- tients (low-grade, 22; high-grade, 26) and 10 healthy brain tissues were analyzed for the tissue expression of FLNC. The normal brain tissues were obtained from the patients undergoing resection of extra-axial brain tumors or epilepsy surgery. Sixty-five serum samples from glioma patients and Adachi-Hayama et al. BMC Cancer 2014, 14:452 http://www.biomedcentral.com/1471-2407/14/452 Page 3 of 10 Page 3 of 10 Adachi-Hayama et al. BMC Cancer 2014, 14:452 http://www.biomedcentral.com/1471-2407/14/452 Extraction of mRNA and preparation of cDNA p p The mRNAs were extracted from the tumors and nor- mal brain tissues using the QIAzol Lysis Reagent and RNeasy® Lipid Tissue Mini Kit (QIAGEN, Tokyo, Japan), followed by DNase treatment. One μg of each mRNA was reversely transcribed using the oligo dT primer (Takara Biochemicals, Inc., Tokyo, Japan) and Super Script II (Invitrogen, CA). Monoclonalized phage cDNA clones were converted to pBluescript phagemids by in vivo excisions with ExAssist helper phage (Stratagene, La Jolla, CA). Plasmid DNA was obtained from E. coli SOLR strain transformed by the pha- gemid. The cDNA inserts were sequenced by the dideoxy chain termination method using the DNA sequencing kit BigDye Terminator (Applied Biosystems, Foster City, CA). Sequences were analyzed for homology with public data- bases of known genes and proteins using BLAST on the National Center for Biotechnology Information’s website (http://www.ncbi.nlm.nih.gov/gene or protein). ELISA for anti-FLNC autoantibody Fifty μl of antigen (GST or GST-tagged recombinant FLNC) was added to each well, and incubated at 4°C overnight. The plate was washed and blocked with 10% fetal calf serum in PBS (PBS-FCS). Fifty μl of sera diluted at 1:100 in 10% PBS-FCS was added to the wells and then they were incu- bated. The bound IgG antibodies were detected by incubat- ing with horseradish peroxidase-conjugated antihuman IgG antibody (Jackson Immuno Research Laboratories, West Grove, PA), followed by the addition of 100 μl of a peroxid- ase substrate (o-phenylenediamine, 0.4 mg/ml) in a citrate-phosphate buffer. Absorbance at 490 nm was de- termined using a microplate reader (Emax; Molecular Devices, Sunnyvale, CA). Figure 1 Tissue expression of filamin C mRNA was measured by quantitative real-time RT-PCR analysis in normal brain tissues, low-grade gliomas, and high-grade gliomas, and also in lung, liver spleen, testis and muscle which were commercially obtained. The filamin C mRNA expression was significantly up-regulated in high-grade gliomas compared with normal brain tissues. It was moderately upregulated in low-grade gliomas and normal muscle tissues. The mean values of duplicate experiments for each sample are presented. Immunohistochemistry IHC staining was performed on 4 μm paraffin-embedded sections. Antigenicity was recovered by the microwave method. Endogenic peroxidase was inactivated with 0.3% H2O2 methanol. After antigen blocking, the sections were incubated overnight with mouse monoclonal primary anti- body against FLNC (Lab Vision, Fremont, CA). The sec- tions were then incubated with mouse biotinylated secondary antibody followed by the ABC complex reaction. Total IgG measurement Serum total IgG was measured in the same samples as the anti-FLNC autoantibody measurements according to the manufacturer’s instructions using a human IgG ELISA quantitative kit (Bethyl, Montgomery, TX). Page 4 of 10 Adachi-Hayama et al. BMC Cancer 2014, 14:452 http://www.biomedcentral.com/1471-2407/14/452 expressions represented the normalized relative levels of filamin C expressions. Finally, the reaction was visualized using DAB and counter- stained with hematoxylin. To quantitate FLNC protein ex- pression, the mean percentage of positive tumor cells was determined in at least 5 random fields at x400 magnifica- tion in each section. Statistical analysis Results of ELISA were statistically analyzed by unpaired t- test. Receiver–operating characteristics (ROC) curve ana- lysis was used to determine the optimal cutoff values for differential diagnosis of low-grade gliomas and healthy vol- unteers. The survival rates were estimated using Kaplan- 100 90 80 70 60 50 40 30 20 10 0 A B Figure 2 Immunohistochemistry (IHC) for FLNC among normal brain, low-grade glioma and high-grade glioma. (A) FLNC protein expression level was higher in high-grade glioma (e, f) than in low-grade glioma (c, d) which expressed significantly higher level of FLNC than normal brain tissues (a, b). FLNC expression is only observed around the nucleus of glia cells in normal brains, whereas it spreads into the whole cytoplasm and the fibrous cellular processes of the glioma cells (magnification × 400). (B) Quantitative analysis of FLNC protein expressions in IHC shows that the mean percentage of positive tumor cells gets higher as the tumor grade advances. (normal brain vs low-grade glioma; p = 0.0132, low-grade glioma vs high-grade glioma; p = 0.0005). A A 100 90 80 70 60 50 40 30 20 10 0 B 100 90 80 70 60 50 40 30 20 10 0 B B Figure 2 Immunohistochemistry (IHC) for FLNC among normal brain, low-grade glioma and high-grade glioma. (A) FLNC protein expression level was higher in high-grade glioma (e, f) than in low-grade glioma (c, d) which expressed significantly higher level of FLNC than normal brain tissues (a, b). FLNC expression is only observed around the nucleus of glia cells in normal brains, whereas it spreads into the whole cytoplasm and the fibrous cellular processes of the glioma cells (magnification × 400). (B) Quantitative analysis of FLNC protein expressions in IHC shows that the mean percentage of positive tumor cells gets higher as the tumor grade advances. (normal brain vs low-grade glioma; p = 0.0132, low-grade glioma vs high-grade glioma; p = 0.0005). Adachi-Hayama et al. BMC Cancer 2014, 14:452 http://www.biomedcentral.com/1471-2407/14/452 Page 5 of 10 A B Figure 3 ELISA of anti-FLNC autoantibody levels in the sera from glioma patients and normal volunteers. (A) For a training set, anti-FLNC autoantibody concentration in the sera from low-grade gliomas was significantly higher than those from high-grade gliomas (p = 0.0101) or normal volunteers (p < 0.0001). (B) The ELISA result obtained in the training set was confirmed in an independent validation set (low-grade vs. Statistical analysis high-grade: p = 0.0036, low-grade vs. healthy: p = 0.0010). B A B Figure 3 ELISA of anti-FLNC autoantibody levels in the sera from glioma patients and normal volunteers. (A) For a training set, anti-FLNC autoantibody concentration in the sera from low-grade gliomas was significantly higher than those from high-grade gliomas (p = 0.0101) or normal volunteers (p < 0.0001). (B) The ELISA result obtained in the training set was confirmed in an independent validation set (low-grade vs. high-grade: p = 0.0036, low-grade vs. healthy: p = 0.0010). Meier method, and they were compared with the log-rank test. The correlation between the filamin C mRNA expres- sion levels and serum anti-FLNC autoantibody concentra- tions was analyzed using the non-parametric Spearman’s rank test. The statistical analyses were performed using Stat-View software and SAS software (SAS Institute Inc., Cary, NC). Meier method, and they were compared with the log-rank test. The correlation between the filamin C mRNA expres- sion levels and serum anti-FLNC autoantibody concentra- tions was analyzed using the non-parametric Spearman’s rank test. The statistical analyses were performed using Stat-View software and SAS software (SAS Institute Inc., Cary, NC). normal brain tissue, but it spread into the whole cyto- plasm and the fibrous cellular processes in the low-grade glioma cells (Figure 2-c). We measured anti-FLNC auto-antibody concentrations by ELISA in a training set consisting of 65 glioma patients (low-grade, 35; high-grade, 30) and 38 healthy volunteers focusing on FLNC (Table 1). The results showed the serum anti-FLNC autoantibody level was significantly higher in low-grade gliomas than in high-grade gliomas or healthy volunteers (low-grade vs. high-grade: p = 0.0101, low-grade vs. healthy: p < 0.0001) (Figure 3-a). We then an- alyzed the antibody level in a prospectively-collected valid- ation set consisting of 66 glioma patients (low-grade, 37; high-grade, 29) and 39 healthy volunteers (Table 1). The significantly increased autoantibody level was confirmed in the patients with low-grade gliomas as compared to those in high-grade tumors or healthy volunteers (low-grade vs. high-grade: p = 0.0036, low-grade vs. healthy: p = 0.0010) (Figure 3-b). Although the categorization of grade I and grade II gliomas into low-grade gliomas is generally used in the clinical setting, these two are different diseases bio- logically and clinically. So, the anti-FLNC autoantibody Results Screening of the patients’ sera by serological analysis of re- combinant cDNA expression libraries (SEREX) resulted in identification of filamin C (FLNC) as one of the candidate glioma antigens (Additional file 1: Table S1). The list in- cluded many signal-transduction molecules and transcrip- tion factors, which was also confirmed in a previous proteomic study. We first examined whether the expression of filamin C mRNA is elevated in the glioma tissues (Figure 1). Quantitative reverse transcription–PCR (qRT-PCR) analysis of various glioma tissues and normal brain tis- sues confirmed that filamin C mRNA expression was significantly up-regulated in low-grade gliomas com- pared with normal brain tissues. High-grade gliomas expressed higher level of filamin C mRNA than low-grade gliomas. Other normal tissues including lung, liver, spleen, and testis contained the same levels of filamin C mRNA as normal brain tissues. In contrast, muscle tissues had a higher level than the other normal tissues and the same level as low-grade glomas (Figure 1). Table 2 Sensitivity and specificity of anti-FLNC antibody Table 2 Sensitivity and specificity of anti-FLNC antibody Table 2 Sensitivity and specificity of anti-FLNC antibody n Mean value Standard deviation p-value for healthy volunteers Healthy volunteers 77 0.242 0.115 __ Low-grade gliomas Grade I 15 0.359 0.112 0.0021 Grade II 57 0.373 0.152 <0.0001 High-grade gliomas Grade III 18 0.254 0.159 0.9748 Grade IV 41 0.268 0.101 0.7503 Meningioma 19 0.273 0.081 0.2281 Cerebral infraction 24 0.273 0.082 0.5531 We then analyzed protein expression levels and distri- butions in paraffin-embedded clinical specimens utiliz- ing semiquantitative immunohistochemical analysis (Figure 2). FLNC protein expression level was higher in high-grade glioma than in low-grade gliomas which expressed significantly higher level of FLNC than normal brain tissues (Figure 2-a, 2-b). FLNC protein expression was only observable around the nucleus of glial cells in Adachi-Hayama et al. BMC Cancer 2014, 14:452 http://www.biomedcentral.com/1471-2407/14/452 Adachi-Hayama et al. BMC Cancer 2014, 14:452 http://www.biomedcentral.com/1471-2407/14/452 Page 6 of 10 Table 3 Sensitivity and specificity of anti-FLNC antibody n Positive Negative (absorbance ≥0.31) (absorbance<0.31) Low-grade gliomas 72 52 20 Healthy volunteers 77 15 62 and p = 0.5581, respectively) (Table 2). The ROC curve analysis showed that a cutoff value of 0.31 provided the best sensitivity and specificity for the differential diagnosis between patients with low-grade gliomas and healthy vol- unteers. By using this cutoff value, the anti-FLNC auto- antibody biomarker system correctly classified 52 (sensitivity; 72.2%) of 72 low-grade glioma patients and 62 (specificity; 80.5%) of 77 healthy volunteers (Table 3). Al- though the diagnostic ability was not sufficient for clinical application, the autoantibody-based tumor markers de- tected in the patients’ sera may contribute to the construc- tion of early diagnosis system for low-grade gliomas. levels were examined for each grade independently, which showed that those of grade I and grade II, and also of grade III and grade IV, were at the same levels (Table 2). To know whether the increase in anti-FLNC auto- antibody levels is specific to low-grade gliomas, the pa- tients with other brain lesions whose MRI manifests a hypointesity on T1-weighted image and hyperintensity on T2-weighted image were examined. The anti-FLNC auto- antibody levels for meningioma and cerebral infarction were at the same level of healthy volunteers (p = 0.2281, The filamin C mRNA expression level inversely corre- lated with the serum anti-FLNC autoantibody concentra- tions (p = 0.0094) (Figure 4-a). Table 2 Sensitivity and specificity of anti-FLNC antibody The serum anti-FLNC autoantibody levels showed no difference between large tumors (size ≥3 cm) and small tumors (size <3 cm) A B Figure 4 Serum anti-FLNC autoantibody levels and glioma progression. (A) Filamin C mRNA expression level inversely correlated with the serum anti-FLNC autoantibody concentrations (p = 0.0094, R = −0.433). (B) Serum anti-FLNC autoantibody levels were not difference between large tumors (size ≥3 cm) and small tumors (size <3 cm). A A B B Figure 4 Serum anti-FLNC autoantibody levels and glioma progression. (A) Filamin C mRNA expression level inversely correlated with the serum anti-FLNC autoantibody concentrations (p = 0.0094, R = −0.433). (B) Serum anti-FLNC autoantibody levels were not difference between large tumors (size ≥3 cm) and small tumors (size <3 cm). Adachi-Hayama et al. BMC Cancer 2014, 14:452 http://www.biomedcentral.com/1471-2407/14/452 Page 7 of 10 (Figure 4-b). These data collectively suggest that circulat- ing anti-FLNC autoantibody is induced at the early stage of glioma progression, and the serum level decreases with tumor progression. A B C Figure 6 Non-specific factors affecting serum autoantibody levels. (A) Serum FLNC protein concentration levels were measured utilizing the sandwich ELISA to be proved equivalent among low-grade gliomas, high-grade gliomas and normal volunteers. (B) Serum total IgG and (C) ant-GST autoantibody levels were not different among low- grade gliom patients, high-grade glioma patients and normal volunteers. A There was a discrepancy between the decreased level of anti-FLNC autoantibody and the increased tissue expres- sion of FLNC in the patients with high-grade gliomas. To investigate the mechanisms for the decreased level of anti- FLNC autoantibodies in high-grade glioma patients in spite of the high tissue expression, we first measured per- ipheral blood lymphocyte amount, which showed that the mean lymphocyte ratio in the peripheral blood of grade II glioma patients was equivalent level as healthy volunteers, but those in grade III and grade IV glioma patients were significantly decreased compared with healthy volunteers (both p < 0.0001) (Figure 5). The lymphocytopenia would contribute to the decreased anti-FLNC autoantibody level in high-grade glioma patients. Then, serum FLNC protein concentration levels were measured utilizing the sandwich ELISA to be proved equivalent among low-grade gliomas, high-grade gliomas and normal volunteers (Figure 6-a). Table 2 Sensitivity and specificity of anti-FLNC antibody Serum levels of total IgG and anti-GST autoantibody were also not different among the patient groups and normal volunteers (Figure 6-b and 6-c), which indicated that gen- eral B cell function was maintained and that the decreased level of anti-FLNC autoantibody production was not a non-specific reaction but was specific for FLNC. B B C C All the glioma patients included in this study were also subjected to survival analysis. Figure 7-a shows Kaplan- Meier overall survival curves for the two patients groups divided by the anti-FLNC antibody level of 0.31 in the ELISA. The group presenting higher anti-FLNC auto- antibody levels achieved significantly longer survival pe- riods (p < 0.01). When the survival analysis was focused Figure 6 Non-specific factors affecting serum autoantibody Figure 6 Non-specific factors affecting serum autoantibody levels. (A) Serum FLNC protein concentration levels were measured utilizing the sandwich ELISA to be proved equivalent among low-grade gliomas, high-grade gliomas and normal volunteers. (B) Serum total IgG and (C) ant-GST autoantibody levels were not different among low- grade gliom patients, high-grade glioma patients and normal volunteers. Figure 5 The mean lymphocyte ratio in the peripheral blood of grade II glioma patients was equivalent as healthy volunteers, but those in grade III and grade IV glioma patients were significantly decreased compared with healthy volunteers (both p < 0.0001). on low-grade gliomas, the patients with higher anti- FLNC autontibody level (≥0.31) lived significantly longer than those with lower levels (Figure 7-b). Likewise in high-grade gliomas, the patients with higher antibody level had significantly longer survival periods (Figure 7-c). Serum anti-FLNC autoantibody is a useful predictor for longer survival periods in patients with both low-grade gli- omas and high-grade gliomas. Figure 5 The mean lymphocyte ratio in the peripheral blood of grade II glioma patients was equivalent as healthy volunteers, but those in grade III and grade IV glioma patients were significantly decreased compared with healthy volunteers (both p < 0.0001). Figure 5 The mean lymphocyte ratio in the peripheral blood of grade II glioma patients was equivalent as healthy volunteers, but those in grade III and grade IV glioma patients were significantly decreased compared with healthy volunteers (both p < 0.0001). Discussion The present study showed that FLNC protein expression increased according to tumor histological grade in glioma and the circulating anti-FLNC autoantibody was induced Page 8 of 10 Adachi-Hayama et al. BMC Cancer 2014, 14:452 http://www.biomedcentral.com/1471-2407/14/452 A B C Figure 7 Correlations of serum anti-FLNC autoantibody concentration measured by ELISA and survival of glioma patients. Kaplan-Meier survival curves of glioma patients show that a group having higher values of serum anti-FLNC autoantibody (≥0.31) survived significantly longer than the other group with lower values (A) in all glioma patients (p = 0.0040), (B) in low-grade glioma patients (p = 0.0070), (C) and in high-grade glioma patients (p = 0.0310). efficiently worked as immune-surveillance system in the early stage of gliomagenesis. These results collectively pro- vide an evidence that serum anti-FLNC autoantibody can be a potential serum biomarker for early diagnosis of low- grade gliomas. However, a large-scale prospective study is required to confirm the usefulness of autoantibody- based biomarkers for gliomas. A A Filamins are large cytoplasmic proteins whose dimers provide cells with mechanical resilience by cross-linking the actin filaments into dynamic three-dimensional struc- tures [11-14]. Filamin A and B are ubiquitous, whereas FLNC is a muscle-restricted isoform. However, FLNC abundantly exists in both fetal brains and spinal cords [11,12]. Since many signaling molecules and receptors bind to the C-terminal region of filamins, it is now becom- ing clear that filamin is not merely a cytoskeletal support protein, but an important bridge between architecture and intracellular molecular signaling [23]. The C-terminal half of filamin A is reported to be a docking site for many intracellular signaling molecules, such as RalA, Rac, Rho, and Cdc42, and membrane receptors [24,25]. FLNC also binds some trans-membrane proteins such as sarcoglycan, presenilin, caveolin-1, and β1 integrin [26-29]. Recruit- ment of these signaling molecules to the C-terminal re- gion of filamin facilitates their functional communication [23]. All these molecular hub function of filamins contrib- utes to cancer progression, including metastasis and DNA damage response [15-22,30]. B B C C Actually, serum autoantibodies against filamins were re- ported to be detected in several cancers including colon and breast cancers [22]. In contrast, the central nervous system (CNS) is known to be an immunological privileged site and it was believed that antibody responses are only rarely elicited by the broad spectrum of antigens expressed by glioma [31]. Discussion We have shown that immune responses ef- fectively operate even for intracerebral antigens when the tumor was in an early stage. The antibody response was gradually suppressed as tumor malignancy progressed. Not the inherent immune privilege of the CNS but the acquired immunosuppressive effect in glioma progression was the main cause of the suppressed FLNC antibody response in high-grade gliomas. However, the precise mechanism of the discrepancy between the higher tissue expression of FLNC in high-grade gliomas and the lower serum antibody level in these patients remained to be known. Local im- munosuppression induced by T-cell specific lymphocytepe- nia is well documented in high-grade gliomas [31]. The impaired immunity is first occurred by immunosuppressive factors secreted by the tumors, such as vascular endothelial growth factor (VEGF), interleuikin-10 (IL-10), transform- ing growth factor-β (TGF-β), prostaglandin E2 (PGE2). Af- fected monocytes make a shift in cytokine secretion to favor a decrease in Th1-type cytokines and increase in Th2-type cytokines to induce T-cell signaling defect, IL-2 Figure 7 Correlations of serum anti-FLNC autoantibody concentration measured by ELISA and survival of glioma patients. Kaplan-Meier survival curves of glioma patients show that a group having higher values of serum anti-FLNC autoantibody (≥0.31) survived significantly longer than the other group with lower values (A) in all glioma patients (p = 0.0040), (B) in low-grade glioma patients (p = 0.0070), (C) and in high-grade glioma patients (p = 0.0310). y g patients. Kaplan-Meier survival curves of glioma patients show that a group having higher values of serum anti-FLNC autoantibody (≥0.31) survived significantly longer than the other group with lower values (A) in all glioma patients (p = 0.0040), (B) in low-grade glioma patients (p = 0.0070), (C) and in high-grade glioma patients (p = 0.0310). in low-grade gliomas or in an early stage of glioma progres- sion. In spite of the immunological privileged nature of the brain, the aberrant FLNC production could induce immune reaction. The induced serum autoantibody level decreased in high-grade gliomas or in a late stage of tumor progres- sion. The patients who had induced strong immune reac- tion with high level of circulating anti-FLNC autoantibody survived longer than those with weak immune reaction. Antitumor immune responses against glioma antigens Adachi-Hayama et al. BMC Cancer 2014, 14:452 http://www.biomedcentral.com/1471-2407/14/452 Page 9 of 10 Page 9 of 10 defect in T-cells, apoptosis of T-cell, and natural killer (NK) cell activity depression [31-33]. Conclusion 10. Schmits R, Cochlovius B, Treitz G, Regitz E, Ketter R, Preuss KD, Romeike BF, Pfreundschuh M: Analysis of the antibody repertorie of astrocytoma patients against antigens expressed by gliomas. Int J Cancer 2002, 98:73–77. In conclusion, we identified a novel glioma antigen, FLNC, utilizing SEREX and found that its autoantibody level is el- evated in low-grade glioma patients compared with nor- mal volunteers and high-grade glioma patients. Serum anti-FLNC autoantibody is thereby indicated to be a novel serum marker for the early diagnosis of low-grade gliomas. The conclusions of this study are limited by the small sample size and should be confirmed in a larger prospect- ive study with an independent cohort of patients. 11. 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Sjoblom T, Jones S, Wood LD, Parsons DW, Lin J, Barber TD, Mandelker D, Leary RJ, Ptak J, Silliman N, Szabo S, Buckhaults P, Farrell C, Meeh P, Markowitz SD, Willis J, Dawson D, Willson JK, Gazdar AF, Hartigan J, Wu L, Liu C, Parmigiani G, Park BH, Bachman KE, Papadopoulos N, Vogelstein B, Kinzler KW, Velculescu VE: The consensus coding sequences of human breast and colorectal cancers. Science 2006, 314:268–274. Discussion The long-term anti- gen exposure from a large tumor could induce immune tolerance through development of immune resistant tumor variants and the tumor microenvironment inducing im- mune cell anergy or death [34-36]. The induction and sup- pression of circulating anti-FLNC autoantibody would be individually regulated by the complex interaction between the host immune system and the tumor biology. 6. Iwadate Y, Sakaida T, Hiwasa T, Nagai Y, Ishikura H, Takiguchi M, Yamaura A: Molecular classification and survival prediction in human gliomas based on proteome analysis. Cancer Res 2004, 64:2496–2501. 7. Fukuda ME, Iwadate Y, Machida T, Hiwasa T, Seki N, Nimura Y, Nagai Y, Takiguchi M, Tanzawa H, Yamaura A, Seki N: Cathepsin D is a potential serum marker for poor prognosis in glioma patients. Cancer Res 2005, 65:5190–5194. 8. Iwadate Y, Hayama M, Adachi A, Matsutani T, Nagai Y, Hiwasa T, Saeki N: High serum level of plasminogen activator inhibitor-1 predicts histological grade of intracerebral gliomas. Anticancer Res 2008, 28:415–418. 9. Sahin U, Türeci O, Schmitt H, Cochlovius B, Johannes T, Schmits R, Stenner F, Luo G, Schobert I, Pfreundschuh M: Human neoplasms elicit multiple specific immune responses in the autologous host. Proc Natl Acad Sci U S A 2008, 92:11810–11813. Competing interests Competing interests Received: 1 October 2013 Accepted: 5 June 2014 20. Keshamouni VG, Michailidis G, Grasso CS, Anthwal S, Strahler JR, Walker A, Arenberg DA, Reddy RC, Akulapalli S, Thannickal VJ, Standiford TJ, Andrews PC, Omenn GS: Differential protein expression profiling by iTRAQ-2DLC-MS/MS of lung cancer cells undergoing epithelial-mesenchymal transition reveals a migratory/invasive phenotype. J Proteome Res 2006, 5:1143–1154. Authors’ contribution D d h Designed the experiments: TH, MT, NS, YI. Performed the experiments: MH-A, AA, NS, TM. Analyzed the data: TM, TH, YI. All authors read and approved the final manuscript. 17. Bedolla RG, Wang Y, Asuncion A, Chamie K, Siddiqui S, Mudryj MM, Prihoda TJ Siddiqui J, Chinnaiyan AM, Mehra R, de Vere White RW, Ghosh PM: Nuclear versus cytoplasmic localization of filamin A in prostate cancer: immunohistochemical correlation with metastases. Clin Cancer Res 2009, 15:788–796. Additional file Additional file 1: Table S1. Candidate glioma antigens identified with SEREX. 16. 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https://openalex.org/W2780412015	https://research-information.bris.ac.uk/ws/files/121713163/Gastro_paper.pdf	English	null	Safety and efficacy of parenteral iron in children with inflammatory bowel disease	BJCP. British journal of clinical pharmacology/British journal of clinical pharmacology	2,018	cc-by	5,552	Papadopoulos, M., Patel, D., Korologou-Linden, R., Eunice, G., Soondrum, K., Fell, J., & Epstein, J. (2017). Safety and Efficacy of Parenteral Iron in Children with Inflammatory Bowel Disease. Journal of Gastroenterology, Hepatology, and Endoscopy, 2(2), Article 1011. http://remedypublications.com/journal-gastroenterology-hepatology- endoscopy/articles/articles.php Papadopoulos, M., Patel, D., Korologou-Linden, R., Eunice, G., Soondrum, K., Fell, J., & Epstein, J. (2017). Safety and Efficacy of Parenteral Iron in Children with Inflammatory Bowel Disease. Journal of Gastroenterology, Hepatology, and Endoscopy, 2(2), Article 1011. http://remedypublications.com/journal-gastroenterology-hepatology- endoscopy/articles/articles.php Publisher's PDF, also known as Version of record License (if available): CC BY Publisher's PDF, also known as Version of record License (if available): CC BY Link to publication record on the Bristol Research Portal PDF-document Link to publication record on the Bristol Research Portal PDF-document This is the final published version of the article (version of record). It first appeared online via Remedy Publishing at http://remedypublications.com/journal-gastroenterology-hepatology-endoscopy/. Please refer to any applicable terms of use of the publisher. Papadopoulos, M., Patel, D., Korologou-Linden, R., Eunice, G., Soondrum, K., Fell, J., & Epstein, J. (2017). Safety and Efficacy of Parenteral Iron in Children with Inflammatory Bowel Disease. Journal of Gastroenterology, Hepatology, and Endoscopy, 2(2), Article 1011. http://remedypublications.com/journal-gastroenterology-hepatology- endoscopy/articles/articles.php Papadopoulos, M., Patel, D., Korologou-Linden, R., Eunice, G., Soondrum, K., Fell, J., & Epstein, J. (2017). Safety and Efficacy of Parenteral Iron in Children with Inflammatory Bowel Disease. Journal of Gastroenterology, Hepatology, and Endoscopy, 2(2), Article 1011. http://remedypublications.com/journal-gastroenterology-hepatology- endoscopy/articles/articles.php Citation: Papadopoulos M, Patel D, Korologou- Linden R, Goto E, Krishna Soondrum SEM, John ME Fell, et al. Safety and Efficacy of Parenteral Iron in Children with Inflammatory Bowel Disease. J Gastroenterol Hepatol Endosc. 2017; 2(2): 1011. Papadopoulos M, Patel D, Korologou- Linden R, Goto E, Krishna Soondrum SEM, John ME Fell, et al. Safety and Efficacy of Parenteral Iron in Children with Inflammatory Bowel Disease. J Gastroenterol Hepatol Endosc. 2017; 2(2): 1011. Keywords: Iron deficiency anemia; Ferric carboxymaltose; Iron sucrose; Intravenous iron; Pediatric Keywords: Iron deficiency anemia; Ferric carboxymaltose; Iron sucrose; Intravenous iron; Pediatric Abstract Objectives: Iron deficiency anemia (IDA) frequently complicates inflammatory bowel disease (IBD) in children and adults. Oral iron may exacerbate gastrointestinal symptoms and absorption may be insufficient in intestinal inflammation. Even where oral iron is successful, repletion of iron stores can be unacceptably slow. Intravenous (IV) iron compounds were in the past associated with serious adverse reactions and historically were considered a last resort in children. New generation preparations have a safer profile in adults although reluctance to use them in children may persist, where safety data is lacking. We investigate the safety and efficacy of ferric carboxymaltose (FCM) and iron sucrose (IS) in children. Methods: We retrospectively identified all children with IBD who received parenteral iron over a 38 month period in a single regional referral centre. Safety, tolerability and adverse events were established by case note review. Efficacy was assessed by change in hematinic indices pre- and post- treatment. Results: 47 children (18 male; median age 14yrs, range 3-17) received a total of 104 iron infusions. 44% (21) had Crohn’s disease (CD); 56% (27) ulcerative colitis (UC). 40 received FC, eight IS and one both. Three children developed mild rash post infusion which resolved quickly with chlorphenamine. Mean increase in hemoglobin (Hb) was 2.5g/dl (0.3-5.8). Iron levels increased by a mean of 8.4 g/dl (1-25), transfer in saturation by 16.2% (2-47). Transfer in decreased by 0.84 g/dl (0.3-3.4). Safety and Efficacy of Parenteral Iron in Children with Inflammatory Bowel Disease Michael Papadopoulos1, Deepa Patel2, Roxanna Korologou-Linden3, Eunice Goto1, Krishna Soondrum SEM1, John ME Fell1 and Jenny Epstein1* 1Department of Paediatric Gastroenterology, Chelsea and Westminster Hospital, UK 2Department of Paediatric Pharmacy, Chelsea and Westminster Hospital, UK 3Department of Medicine, St Mary’s Campus, Imperial College, UK Michael Papadopoulos1, Deepa Patel2, Roxanna Korologou-Linden3, Eunice Goto1, Krishna Soondrum SEM1, John ME Fell1 and Jenny Epstein1* 1Department of Paediatric Gastroenterology, Chelsea and Westminster Hospital, UK 2Department of Paediatric Pharmacy, Chelsea and Westminster Hospital, UK 3Department of Medicine, St Mary’s Campus, Imperial College, UK Journal of Gastroenterology, Hepatology and Endoscopy Journal of Gastroenterology, Hepatology and Endoscopy Research Article Published: 10 Jul, 2017 OPEN ACCESS Correspondence: Jenny Epstein, Department of Paediatric Gastroenterology, Chelsea and Westminster Hospital, London, SW10 9NH, UK, Tel: 0203 315 8628; Fax: 0203 315 8770; E-mail: j.epstein@imperial.ac.uk Received Date: 14 Apr 2017 Accepted Date: 03 Jul 2017 Published Date: 10 Jul 2017 Citation: Papadopoulos M, Patel D, Korologou- Linden R, Goto E, Krishna Soondrum SEM, John ME Fell, et al. Safety and Efficacy of Parenteral Iron in Children with Inflammatory Bowel Disease. J Gastroenterol Hepatol Endosc. 2017; 2(2): 1011. Correspondence: Jenny Epstein, Department of Paediatric Gastroenterology, Chelsea and Westminster Hospital, London, SW10 9NH, UK, Tel: 0203 315 8628; Fax: 0203 315 8770; E-mail: j.epstein@imperial.ac.uk Received Date: 14 Apr 2017 Accepted Date: 03 Jul 2017 Published Date: 10 Jul 2017 *Correspondence: Jenny Epstein, Department of Paediatric Gastroenterology, Chelsea and Westminster Hospital, London, SW10 9NH, UK, Tel: 0203 315 8628; Fax: 0203 315 8770; E-mail: j.epstein@imperial.ac.uk Received Date: 14 Apr 2017 Accepted Date: 03 Jul 2017 Published Date: 10 Jul 2017 Conclusion: New generation parenteral iron preparations are safe, well tolerated and efficacious in children with IDA and IBD. What is known: Iron deficiency anemia (IDA) is a common complication of inflammatory bowel disease (IBD) and a significant contributor to morbidity. Parenteral iron is effective and relatively safe in adults. What is new: The intravenous iron preparations ferric carboxymaltose (FCM) and iron sucrose (IS) are effective treatments for IDA in children with IBD. The safety profiles of FCM and IS are favourable, and similar in children to that in adults. University of Bristol – Bristol Research Portal General rights This document is made available in accordance with publisher policies. Please cite only the published version using the reference above. Full terms of use are available: http://www.bristol.ac.uk/red/research-policy/pure/user-guides/brp-terms/ Introduction Gastroenterol Hepatol Endosc. 2017; 2(2): 1011. Anemia is one of the commonest extra - intestinal complications of IBD. It is more common in children than adults [1]; affecting an estimated 70% of children with IBD [2]. It occurs more frequently in CD than UC [3]. Anemia in IBD is multifactorial and closely associated with IBD activity. The leading cause is IDA followed by anemia of chronic disease. ID results from gastrointestinal blood loss, poor dietary intake and reduced iron bioavailability. Iron malabsorption is a contributing factor especially in CD affecting the small bowel [4]. Nearly 90% of children with IBD have ID with or without associated anemia [1]. It is increasingly recognized that IDA is a significant contributor to morbidity in IBD. Chronic IDA in children causes fatigue, impairs cognitive development and is Copyright © 2017 Jenny Epstein. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 2017 \| Volume 2 \| Issue 2 \| Article 1011 Remedy Publications LLC. 1 Journal of Gastroenterology, Hepatology and Endoscopy Jenny Epstein, et al., Remedy Publications LLC. Remedy Publications LLC. 2017 \| Volume 2 \| Issue 2 \| Article 1011 Introduction 2017 \| Volume 2 \| Issue 2 \| Article 1011 2 Patient Age/Sex Disease Hb (g/dL) Iron (µmol/L) Dose (mg) No of infusions/preparation 1 16F CD 8.8 3 1500 2 FCM 2 14M UC 8.2 2 1000 1 FCM 3 15F CD 8.4 4 900 1 FCM 4 17M CD 9.4 3 2000 2 FCM 5 16F CD 9.2 8 1500 2 FCM 6 13F UC 8.8 2 500 1 FCM 7 15M UC 8.3 4 1500 2 FCM 8 17M CD 11.0 8 1000 1 FCM 9 13M CD 9.2 3 1500 2 FCM 10 14F UC 10.3 7 1000 1 FCM 11 13M UC 9.0 3 800 1 FCM 12 13F CD 7.7 2 800 1 FCM 13 15F UC 9.1 5 1500 2 FCM 14 13F CD 11.1 4 1000 1 FCM 15 12M UC 9.3 3 1500 2 FCM 16 13F CD 9.6 2 1500 2 FCM 17 16F CD 8.6 9 1500 2 FCM 18 13F UC 8.8 6 1500 2 FCM 19 15F CD 8.9 3 500 1 FCM 20 12F UC 9.3 3 1500 2 FCM 21 16M CD 12.1 4 1000 1 FCM 22 12M UC 9.7 3 1500 2 FCM 23 16F UC 9.5 3 1000 1 FCM 24 16M CD 8.9 4 1500 2 FCM 25 15F UC 8.2 3 1500 2 FCM 26 16F CD 10.3 3 1000 1 FCM 27 14F CD 7.5 3 800 1 FCM 28 16F UC 10.3 4 1000 1 FCM 29 12F UC 9.8 2 500 1 FCM 30 15M CD 9.7 2 1500 2 FCM 31 16F UC 11.6 2 1000 1 FCM 32 14F CD 10 2 1000 1 FCM 33 16M CD 9.6 3 3000 2 FCM 34 13M UC 10 3 850 1 FCM 35 15F UC 8.6 3 1500 2 FCM 36 16F CD 10.3 3 930 1 FCM 37 14M CD 10.4 6 500 1 FCM 38 14F UC 8.3 unavailable 1500 2 FCM 39 16M UC 8.8 2 1000 1 FCM 40 13F UC 8.5 2 1000 1 FCM 41 12M UC 8.5 2 320 4 IS 42 8M UC 8.4 3 420 6 IS 16 12F CD 7.3 2 600 6 IS 43 10F UC 9.1 3 630 7 IS 44 10F UC 8.6 2 640 8 IS 45 3M UC 8.8 1 390 8 IS 46 5F UC 8.3 2 45 1 IS 47 9M UC 10.2 5 528 6 IS Table 1: Patient demographics, initial haematinics and iron therapy. Introduction 2017 \| Volume 2 \| Issue 2 \| Article 1011 2 Journal of Gastroenterology, Hepatology and Endoscopy Jenny Epstein, et al., as HB below reference range for age and gender according to the World Health Organization (WHO) [16]. ID was diagnosed by the combination of low serum iron, low mean red cellular volume (MCV), high transfer in and low iron saturation. Ferritin falls in ID, but rises in the acute phase [17], complicating its interpretation as a marker for IDA in IBD. Ferritin was therefore defined as low in the context of CRP (ferritin <30 if CRP <10 and ferritin <100 if CRP >10). Children with severe atopy or allergies or history of prior anaphylaxis were excluded from the study, and were not offered IV iron therapy. Choice of iron preparation was based primarily on patient age, with those under 12 years of age receiving IS and those over 12 years FCM. Demographic data, disease status, iron preparation type, dose and number of infusions were recorded. Dosage was calculated as a function of body weight and HB, as per manufacturers’ recommended dosing schedules. Iron preparations were infused according to British National Formulary guidelines. FCM infusions were administered over 15 to 30 minutes; IS over 50 to 70 minutes. All patients completed a two hour inpatient observation period post infusion during which vital signs were recorded at regular intervals. Table 2: Haematinic indices pre- and post-treatment. Data were imported into STATA version 14. Descriptive statistics: mean, standard deviation (SD), minimum and maximum were calculated for pre- and post-treatment values of Hb, Tfer saturation, CRP, ESR, transferrin, ferritin and iron. Paired sample Student’s T Tests for paired differences were calculated for pre- and post-treatment values for each variable. Hb, transferrin, transferrin saturation, Fe and ESR: p=<0.0001. Results Only a small number of studies on the safety and efficacy of modern IV iron products have been conducted in children. Most of these studied IS not FCM and concerned small numbers of children with chronic kidney disease [11-14]. More recently investigators have shown encouraging safety and efficacy of IS in 24 children with IBD and IDA [15]. 47 children with IBD received 104 IV iron infusions over the study period (Table 1). 29 children were female and 18 male. Mean age was 13.5 years, ranging from 3 to 17 years. 20 had CD (44%) and 27 UC (56%). 40 patients received 58 FCM infusions, average 1.5 infusions per patient. Eight patients received 46 IS infusions; average 5.8 infusions per patient. One patient initially completed a course of IS infusions and 11 months later received FCM as she moved age group (thus total number of infusion episodes studied = 48). The doses ranged from (500 to 1000) mg per infusion for FCM and 70 to 100 mg per infusion for IS. All children over 12 years of age received FCM. Patients under 12 years received IS, which, although also off licence, is in line with greater past pediatric experience and wide spread pediatric practice in the United Kingdom. Choice of iron preparation for children between their 12th and 13th birthdays was made on a case by case basis. Three 12 year olds received FCM and two received IS. Safety. 2/40 patients who received FCM developed rash after completing their first infusion, giving a reaction rate of 5%. Both reactions were mild. In patient 3 rash appeared 30 minutes post infusion, with a small drop in systolic blood pressure which resolved rapidly. The rash resolved after a single dose of IV chlorphenamine with no further complications. Subsequent doses of FCM were not given and the patient continued on oral iron therapy. Patient 10 developed rash one hour post infusion with no systemic compromise. She received a single dose of IV chlorphenamine after which the rash resolved. She did not require a second dose of FCM as adequate increment in HB was achieved with single dose. One patient (case 46) of the eight who received IS developed facial itching and mild eye puffiness 15 minutes into her first infusion, giving a reaction rate of 12.5%. There was no systemic compromise and symptoms resolved with single dose IV chlorphenamine. Results Here we describe our 38-month experience of FCM and IS in children with IBD with a focus on safety and efficacy. Introduction Ferritin: p=<0 0002 CRP: p=0 94 n= Mean SD Min Max Index 48 13.52 2.88 3 17 Hb Pre 48 9.26 1.01 7.3 12.1 Post 44 11.8 1.28 9.3 15 Tfer saturation Pre 44 4.66 2.91 1 15 Post 40 20.95 11.02 3 47 CRP Pre 20 8.87 15.47 .2 65.0 Post 19 8.66 12.10 .2 46.0 ESR Pre 16 37.50 23.67 5.0 112.0 Post 18 22.06 17.80 2.0 73.0 Transferrin Pre 42 3.06 0.50 2.3 4.0 Post 35 2.24 0.42 1.7 4.0 Ferritin Pre 30 11.3 11.88 2 56 Post 23 146.43 133.08 10 446 Iron Pre 47 3.38 1.78 1 9 Post 40 11.78 5.21 3 25 p p Adverse reactions were identified by detailed review of written case notes, nursing observations and nursing, pharmacy and medical electronic records. Efficacy was assessed by pre- treatment and post- treatment iron, transfer in saturation, ferritin, transfer in and HB levels. Change in CRP and ESR were also recorded. Remedy Publications LLC. Results associated with lower intelligence quotients (IQ), attention deficit and other behavioral disorders [5]. Immune regulation and growth are also negatively impacted [6]. These effects are apparent in ID even in the absence of anemia. Adult IBD patients who are anemic report low quality of life (QOL) indices and there is a marked improvement in QOL when HB is corrected in adults with IBD [7]. For these reasons early recognition and prompt and effective treatment of ID (A) are of paramount importance in IBD care. Oral iron supplementation may be considered first line therapy, although issues with tolerance and adherence to prolonged treatment courses often impair therapeutic success. Blood transfusion as a therapy for IDA carries costs and risks which are well recognized. Newer intravenous (IV) iron preparations with a more favorable side effect profile have become available in the last decade including iron sucrose (IS) and ferric carboxymaltose (FCM) which have been shown to be safe and effective in adults [8-10]. Only a small number of studies on the safety and efficacy of modern IV iron products have been conducted in children. Most of these studied IS not FCM and concerned small numbers of children with chronic kidney disease [11-14]. More recently investigators have shown encouraging safety and efficacy of IS in 24 children with IBD and IDA [15]. associated with lower intelligence quotients (IQ), attention deficit and other behavioral disorders [5]. Immune regulation and growth are also negatively impacted [6]. These effects are apparent in ID even in the absence of anemia. Adult IBD patients who are anemic report low quality of life (QOL) indices and there is a marked improvement in QOL when HB is corrected in adults with IBD [7]. For these reasons early recognition and prompt and effective treatment of ID (A) are of paramount importance in IBD care. Oral iron supplementation may be considered first line therapy, although issues with tolerance and adherence to prolonged treatment courses often impair therapeutic success. Blood transfusion as a therapy for IDA carries costs and risks which are well recognized. Newer intravenous (IV) iron preparations with a more favorable side effect profile have become available in the last decade including iron sucrose (IS) and ferric carboxymaltose (FCM) which have been shown to be safe and effective in adults [8-10]. Methods Even in the adherent patient, several months of oral supplementation may be required to achieve target HB. The first IV iron introduced in the 1930s, an iron oxyhydroxide complex [22], had major side effects and was only indicated in rare circumstances [23]. In the 1950s a high molecular weight iron dextran (HMW-ID) was developed in which the iron oxyhydroxide complex was surrounded by a shell of dextran polymers [24]. This improved its bioavailability and side effect Figure 1: A) Hbpre- and post-treatment (g/dL) (normal ranges(NR) differ with age and sex). p=<0.0001. B) Iron pre- and post-treatment (NR 7-27 µg/dL). p=<0.0001. C) Ferritin pre- and post-treatment (NR 7-140 µg/dL). p=<0.0002. D) Transferrin saturation pre- and post-treatment (NR 16-55%). p=<0.0001. E) Transferrin pre- and post-treatment (NR 1.7-3.4 g/L). p=<0.0001. Figure 2: A) ESR (mm/hr) p=<0.0001. B) CRP (mg/ml) p=0.94 pre- and post-treatment. profile, however allergic reactions were frequent [25]. Gradually, low molecular weight iron dextran compounds became available which caused fewer adverse reactions [26]. Later ferric gluconate (FG) Figure 1: A) Hbpre- and post-treatment (g/dL) (normal ranges(NR) differ with age and sex). p=<0.0001. B) Iron pre- and post-treatment (NR 7-27 µg/dL). p=<0.0001. C) Ferritin pre- and post-treatment (NR 7-140 µg/dL). p=<0.0002. D) Transferrin saturation pre- and post-treatment (NR 16-55%). p=<0.0001. E) Transferrin pre- and post-treatment (NR 1.7-3.4 g/L). p=<0.0001. age and sex). p=<0.0001. B) Iron pre- and post-treatment (NR 7-27 µg/dL). D) Transferrin saturation pre- and post-treatment (NR 16-55%). p=<0.0001. E) Figure 2: A) ESR (mm/hr) p=<0.0001. B) CRP (mg/ml) p=0.94 pre- and post-treatment. profile, however allergic reactions were frequent [25]. Gradually, low molecular weight iron dextran compounds became available which caused fewer adverse reactions [26]. Later ferric gluconate (FG) Figure 1: A) Hbpre- and post-treatment (g/dL) (normal ranges(NR) differ with age and sex). p=<0.0001. B) Iron pre- and post-treatment (NR 7-27 µg/dL). p=<0.0001. C) Ferritin pre- and post-treatment (NR 7-140 µg/dL). p=<0.0002. D) Transferrin saturation pre- and post-treatment (NR 16-55%). p=<0.0001. E) Transferrin pre- and post-treatment (NR 1.7-3.4 g/L). p=<0.0001. Figure 2: A) ESR (mm/hr) p=<0.0001. B) CRP (mg/ml) p=0.94 pre- and post-treatment. profile, however allergic reactions were frequent [25]. Gradually, low molecular weight iron dextran compounds became available which caused fewer adverse reactions [26]. Later ferric gluconate (FG) case the second dose was cancelled because of adverse reaction; one was less than 35 kg –as per administration guidelines and 17 achieved adequate increment in HB with single dose. Methods HB was recorded pre- infusion in all 48 infusion episodes and post-infusion in 44/48 cases. Mean HB pre-infusion was 9.3 (range 7.3 to 12.1) g/dl. Mean HB post treatment was 11.8, an increase of 2.5 per patient (p=<0.0001). There was significant improvement in all hematinic indices following infusion (Table 2 ) (Figure 1,2). Remedy Publications LLC. Methods All children with IBD who received IV iron in a single regional referral centre between April 2013 and May 2016 were retrospectively identified via pharmacy records. Inclusion criteria were therefore IBD and IDA. In most cases oral iron was used as first line treatment for IDA. IV iron was used in the following circumstances: (a) patient reported side effects with oral iron; (b) non-resolution of IDA with 3 months of oral iron; (c) physician assessment of severity of either IBD or IDA contraindicates trial of oral iron. Anemia was defined Efficacy of the 40 patients who received FCM, 18 received a second dose 1-4 weeks later. Of the 22 children who received single dose, three did not attend for their scheduled second infusion, in one 2017 \| Volume 2 \| Issue 2 \| Article 1011 3 Jenny Epstein, et al., Journal of Gastroenterology, Hepatology and Endoscopy case the second dose was cancelled because of adverse reaction; one was less than 35 kg –as per administration guidelines and 17 achieved adequate increment in HB with single dose. HB was recorded pre- infusion in all 48 infusion episodes and post-infusion in 44/48 cases. Mean HB pre-infusion was 9.3 (range 7.3 to 12.1) g/dl. Mean HB post treatment was 11.8, an increase of 2.5 per patient (p=<0.0001). There was significant improvement in all hematinic indices following infusion (Table 2 ) (Figure 1,2). Discussion Published evidence strongly supports both proactive treatment of IDA in IBD, and the use of newer IV iron preparations which are efficacious with fewer doses and less allergenic than their predecessors. As in many other areas of pediatric practice, treatment of children has relied upon the extrapolation of adult research. Here we contribute safety and efficacy data for IV iron therapy in pediatric IBD. Oral ferrous supplements are oxidized in the gut, produce activated hydroxyl radicals that affect the mucosa [18] and may provoke gastrointestinal symptoms including bloating, nausea and pain which can be exacerbated in patients with IBD [19]. In inflammatory diseases, iron absorption is reduced and increased hepcidin, an acute phase protein, plays a key role. In health, hepcidin levels rise with total body iron stores and protect against iron overload. Hepcidin is up-regulated in systemic inflammatory states by interleukin (IL) 6 and IL-1, and impairs absorption of oral iron via multiple mechanisms [20]. Adherence to oral iron therapy, particularly in adolescent IBD patients, is frequently suboptimal [21]. References 1. Goodhand JR, Kamperidis N, Rao A, Laskaratos F, McDermott A, Wahed M et al. Prevalence and management of anaemia in children, adolescents and adults with IBD. Inflamm Bowel Dis. 2012;18(3):513-9. 1. Goodhand JR, Kamperidis N, Rao A, Laskaratos F, McDermott A, Wahed M et al. Prevalence and management of anaemia in children, adolescents and adults with IBD. Inflamm Bowel Dis. 2012;18(3):513-9. 2. Gerasimidis K, Barclay A, Papangelou A, Missiou D, Buchanan E, Tracey C et al. The epidemiology of anemia in pediatric inflammatory bowel disease: prevalence and associated factors at diagnosis and follow- up and the impact of exclusive enteral nutrition. Inflamm Bowel Dis. 2013;19(11):2411-22. 3. Bager P, Befrits R, Wikman O, Lindgren S, Moum B, Hjortswang H, et al. The prevalence of anemiaand iron deficiency in IBD outpatients in Scandinavia.Scand J Gastroenterol. 2011;46(3):304-9. 4. Bartels U, Pedersen NS, Jarnum S. Iron absorption and serum ferritin in chronic inflammatory bowel disease. Scand J Gastroenterol. 1978;13(6):649–56. 5. Agaoglu L,Torun O, Unuvar E, Sefil Y, Demir D. Effects of iron deficiency anemia on cognitive function in children. Arzneimittelforschung. 2007;57(6A):426-30. 6. Reinisch W, Staun M, Bhandari S, Munoz M. State of the iron: how to diagnose and efficiently treat iron deficiency anemia in inflammatory bowel disease. J Crohns colitis. 2013;7(6):429-40. 7. Wells CW, Lewis S, Barton JR, Corbett S. Effects of changes in hemoglobin level on quality of life and cognitive function in inflammatory bowel disease patients. Inflamm Bow Dis. 2006;12(2):123-30. 8. Anker SD, Comin Colet J, Filippatos G, Ronnie Willenheimer, Kenneth Dickstein, Helmut Drexler, et al. FAIR-HF Trial Investigators. Ferric carboxymaltose in patients with heart failure and iron deficiency. N Engl J Med. 2009;361:2436-48. 9. Van Wyck DB, Mangione A, Morrison J, Hadley PE, Jehle JA, Goodnough LT. Large-dose intravenous ferric carboxymaltose injection for iron deficiency anemia in heavy uterine bleeding: a randomized controlled trial. Transfusion. 2009;49(12):2719-28. We acknowledge the limitations of this study associated with its retrospective design and heterogeneous clinical nature. Whilst prospective randomized trial design is needed in the field, the current study provides supporting evidence for the safety and efficacy of new generation IV iron preparations in the treatment of IBD-associated IDA in children. 10. Covic A, Mircescu G. The safety and efficacy of intravenous iron carboxymaltose in anaemic patients underoing haemodialysis: a multi-centre, open-label, clinical study. Nephrol Dial Transplant. 2010;25(8):2722-30. JE designed the study, interpreted the data and co-wrote the paper. Whilst the safety profile of parenteral iron in children is probably comparable to that in adults, oral iron remains a safer and less costly option, and should be trialed in the first instance in most cases, where IDA is mild to moderate, IBD activity is quiescent and there is no history of intolerance. All authors approved the final version of the manuscript and are accountable for the accuracy and integrity of the work. el sponsorship with Falk. Ethical Review and Approval 11. Moorani KN, Asim S. Parenteral iron sucrose in iron deficiency anaemia of paediatric chronic kidney disease. J Ayub Med Coll Abbottabad. 2011;23(3):47-50. The study was approved as a registered clinical audit by the Chelsea and Westminster and West Middlesex University Hospitals Clinical Governance Department reference LA187. 12. Pinsk V, Levy J, Moser A, Yerushalmi B, Kapelushnik J. Efficacy and safety of intravenous iron sucrose therapy in a group of children with iron deficiency anemia. Isr Med Assoc J. 2008;10(5):335-8. Conflicts of interest and sources of funding: JF is on the advisory board with Janssen and has received travel sponsorship with Falk. 13. Goldstein SL, Morris D, Warady BA. Comparison of the safety and efficacy of 3 iron sucrose iron maintenance regimens in children, adolescents, and young adults with CKD: a randomized controlled trial. Am J Kidney Dis. 2013;61(4):588-97. JE designed the study, interpreted the data and co-wrote the paper. JE designed the study, interpreted the data and co-wrote the paper. JE designed the study, interpreted the data and co-wrote the paper. was introduced and was safer than iron dextran [27]. In 2000 iron sucrose (IS) came into clinical use, whose incidence of anaphylaxis was lower at 0.002%; compared to 0.04% with FG and 0.6-2.3% with HMW-ID [28]. The biggest drawback of IS its low maximum single administrable dose, resulting in multiple separate infusions to reach therapeutic dose. Finally in 2007 ferric carboxymaltose (FCM) became available with a license in Europe for patients above 14 yrs. A single dose of 1000 mg of iron can be given, and administered relatively fast, over 15-60 minutes [29], with no requirement for test dose, thus reducing hospital attendance times and costs. Of the 47 children in our series who received IV iron none experienced major or lasting adverse event. In our series FCM was associated with a 5% mild reaction rate (rash) and IS with a 12.5% mild reaction rate (itch and eye swelling). These numbers do not allow comparative conclusions to be drawn between the two iron preparations in terms of safety, however we provide evidence that parenteral iron and in particular FCM is safe and well tolerated in children. We show that IV iron is a highly efficacious treatment for IDA in children with IBD, inducing clinically and statistically significant increments in HB, Fe and transfer in saturation and reductions in transfer in and ESR. The reduction in ESR was not paralleled by significant reduction in CRP and reflects resolution of IDA rather than change in IBD activity. As well as its lower reported incidence of anaphylaxis, FCM offers a less onerous dosing schedule, no requirement for test dose and shorter infusion time in contrast to IS. In our series we demonstrate safety of FCM in children at least as young as 12 years. This is an off-label use of this medication (<14 years of age). Other investigators have more recently demonstrated safety of FCM in even younger age groups, not specific to IBD [30]. We have selected lower risk cases by excluding those with a personal history of severe allergy, atopy or anaphylaxis to other agents. Remedy Publications LLC. Discussion Published evidence strongly supports both proactive treatment of IDA in IBD, and the use of newer IV iron preparations which are efficacious with fewer doses and less allergenic than their predecessors. As in many other areas of pediatric practice, treatment of children has relied upon the extrapolation of adult research. Here we contribute safety and efficacy data for IV iron therapy in pediatric IBD. Oral ferrous supplements are oxidized in the gut, produce activated hydroxyl radicals that affect the mucosa [18] and may provoke gastrointestinal symptoms including bloating, nausea and pain which can be exacerbated in patients with IBD [19]. In inflammatory diseases, iron absorption is reduced and increased hepcidin, an acute phase protein, plays a key role. In health, hepcidin levels rise with total body iron stores and protect against iron overload. Hepcidin is up-regulated in systemic inflammatory states by interleukin (IL) 6 and IL-1, and impairs absorption of oral iron via multiple mechanisms [20]. Adherence to oral iron therapy, particularly in adolescent IBD patients, is frequently suboptimal [21]. Even in the adherent patient, several months of oral supplementation may be required to achieve target HB. The first IV iron introduced in the 1930s, an iron oxyhydroxide complex [22], had major side effects and was only indicated in rare circumstances [23]. In the 1950s a high molecular weight iron dextran (HMW-ID) was developed in which the iron oxyhydroxide complex was surrounded by a shell of dextran polymers [24]. This improved its bioavailability and side effect Figure 2: A) ESR (mm/hr) p=<0.0001. B) CRP (mg/ml) p=0.94 pre- and post-treatment. Figure 2: A) ESR (mm/hr) p=<0.0001. B) CRP (mg/ml) p=0.94 pre- and post-treatment. profile, however allergic reactions were frequent [25]. Gradually, low molecular weight iron dextran compounds became available which caused fewer adverse reactions [26]. Later ferric gluconate (FG) 2017 \| Volume 2 \| Issue 2 \| Article 1011 4 Jenny Epstein, et al., Journal of Gastroenterology, Hepatology and Endoscopy Authorship Statement MP acquired and interpreted the data and drafted the paper. MP acquired and interpreted the data and drafted the paper. DP acquired the data and revised the manuscript. DP acquired the data and revised the manuscript. 14. Laass MW, Straub S, Chainey S, Virgin G, Cushway T. Effectiveness and safety of ferric carboxymaltose treatment in children and adolescents with inflammatory bowel disease and other gastrointestinal diseases. BMC Gastroenterol. 2014;14:184. RKL performed the statistical analyses and revised the manuscript. RKL performed the statistical analyses and revised the manuscript. EG acquired the data. KS revised the manuscript. 15. Danko I, Weidkamp M. Correction of iron deficiency anemia with intravenous iron sucrose in children with inflammatory bowel disease. J Pediatr Gastroenterol Nutr. 2016 Nov;63(5):e107-e111. JF interpreted the data and revised the manuscript. JF interpreted the data and revised the manuscript. 2017 \| Volume 2 \| Issue 2 \| Article 1011 Remedy Publications LLC. 5 Journal of Gastroenterology, Hepatology and Endoscopy Jenny Epstein, et al., 16. WHO. Iron deficiency anaemia: assessment, prevention, and control. A guide for programme managers. 24. Auerbach M, Ballard H. Clinical use of intravenous iron. Administration efficacy and safety. Hematology Am Soc Hematol Educ Program. 2010;2010:338-47. 17. Gasche C, Berstad A, Befrits R, Beglinger C, Dignass A, Erichsen K et al. Guidelines on the diagnosis and management of iron deficiency and anemia in inflammatory bowel diseases. Inflamm Bowel Dis. 2007;13(12):1545–53. 25. Cançado RD, Muñoz M. Intravenous iron therapy: how far have we come?Rev Bras HematolHemoter. 2011;33(6):461-9. 18. Millar AD, Rampton DS, Blake DR. Effects of iron and iron chelation in vitro on mucosal oxidant activity in ulcerative colitis. Aliment Pharmacol Ther. 2000;14(9):1163–8. 26. Fishbane S, Ungureanu VD, Maesaka JK, Kaupke CJ, Lim V, Wish J. The safety of intravenous iron dextran in hemodialysis patients. Am J Kidney Dis. 1996;28(4):529-34. 19. Erichsen K, Hausken T, Ulvik RJ et al. Ferrous fumarate deteriorated plasma antioxidant status in patients with Crohn disease. Scand J Gastroenterol. 2003;39(8):543-8. 27. Faich G, Strobos J. Sodium ferric gluconate complex in sucrose: safer intravenous iron therapy than iron dextrans. Am J Kid Dis. 1999;33(3):464- 70. 20. Bergamaschi G, Di Sabatino A, Pasini A, Cristina Ubezio, Filippo Costanzo, Davide Grataroli, et al. Intestinal expression of genes implicated in iron absorption and their regulation by hepcidin. Clin Nutr. 2016. 28. Chertow GM, Mason PD, Vaage-Nilsen O, Ahlmén J. Update on adverse drug events associated with parenteral iron. Nephrol Dial Transplant. 2017 \| Volume 2 \| Issue 2 \| Article 1011 Authorship Statement 2006;21(2):378-82. 29. Lyseng-Williamson KA, Keating GM. Ferric carboxymaltose: a review of its use in iron-deficiency anaemia. Drugs. 2009;69(6):739-56. 21. Greenley RN, Stephens KA, Nguyen EU, Kunz JH, Janas L, Goday P, Schurman JV. Vitamin and mineral supplement adherence in pediatric inflammatory bowel disease. J Pediatr Psychol. 2013;38(8):883-92. 30. Powers JM, Shamoun M, McCavit TL, Leah Adix, George R Buchanan. Intravenous ferric carboxymaltose in children with iron deficiency anemia who respond poorly to oral iron. J Pediatr. 2016;180:212-6. 22. Ganz T, Nemeth E. Iron sequestration and anemia of inflammation. Semin Hematol. 2009;46(4):387-93. 23. Macdougall IC. Evolution of iv iron compounds over the last century. J Ren Care. 2009;35(2):8-13. 2017 \| Volume 2 \| Issue 2 \| Article 1011 Remedy Publications LLC. 6
https://openalex.org/W2973913147	https://ieeexplore.ieee.org/ielx7/18/8907537/08839856.pdf	English	null	Overflow Probability of Variable-Length Codes With Codeword Cost	IEEE transactions on information theory	2,019	cc-by	12,296	8194 8194 IEEE TRANSACTIONS ON INFORMATION THEORY, VOL. 65, NO. 12, DECEMBER 2019 Overﬂow Probability of Variable-Length Codes With Codeword Cost Ryo Nomura , Member, IEEE On the other hand, as is well known, if we impose unequal costs on code symbols, it makes no sense to use the codeword length as a measure. In this setting, we have to consider the codeword cost, instead of the codeword length. The average codeword cost, which is a generalization of the average codeword length, have been ﬁrst analyzed also by Shannon [2]. Moreover, several researchers have studied on the average codeword cost [7]–[9]. Karp [10] has given the variable-length code, which minimizes the average codeword cost. Golin and Rote [11], and Golin and Li [12] have proposed the efﬁcient algorithm for constructing the optimal variable-length code for i.i.d. sources while Uyematsu et al. [13] have proposed the asymptotic optimal universal variable-length code for stationary ergodic sources, with respect to the aver- age codeword cost. The inﬁmum of the average codeword cost has been determined by Krause [9] for i.i.d. sources and extended to general sources by Han and Uchida [14]. Recently, Yagi and Nomura [15] have determined the general formula of the inﬁmum of the average codeword cost in the variable-length code allowing non-vanishing error probability. Among others, Uchida and Han [5] have proposed the overﬂow probability of the codeword cost. They have considered the overﬂow probability as the probability of the codeword cost per symbol being above a threshold. Then, they have shown the inﬁmum of achievable thresholds, where the achievable threshold means that there exists a variable-length code whose overﬂow probability of the codeword cost decreases with given exponent r. Abstract—Lossless variable-length source coding with code- word cost is considered for general sources. The problem setting, where we impose on unequal costs on code symbols, is called the variable-length coding with codeword cost. In this problem, the inﬁmum of average codeword cost have already been deter- mined for general sources. On the other hand, the overﬂow probability, which is deﬁned as the probability of codeword cost being above a threshold, have not been considered yet. In this paper, we ﬁrst determine the inﬁmum of achievable threshold in the ﬁrst-order sense and the second-order sense for general sources with additive memoryless codeword cost. Then, we compute it for some special sources such as i.i.d. sources and mixed sources. A generalization of the codeword cost is also discussed. Index Terms—Codeword cost, general source, information- spectrum, overﬂow probability, variable-length coding. Manuscript received October 29, 2013; revised September 14, 2018; accepted August 20, 2019. Date of publication September 16, 2019; date of current version November 20, 2019. This work was supported in part by JSPS KAKENHI under Grant JP26420371 and Grant JP18K04150. This article was presented in part at the 2012 IEEE International Symposium on Information Theory. The author is with the Center for Data Science, Waseda University, Tokyo 162-0042, Japan (e-mail: nomu@waseda.jp). his work is licensed under a Creative Commons Attribution 4.0 License. For more information, see http://creativecommo er a Creative Commons Attribution 4.0 License. For more information, see http://creativecommons.org/licenses/by/4.0/ I. INTRODUCTION L OSSLESS variable-length coding problem is quite impor- tant not only from the theoretical viewpoint but also from the viewpoint of its practical applications. To evalu- ate the performance of variable-length codes, several criteria have been proposed. The most fundamental criterion is the average codeword length proposed by Shannon [2]. Many variable-length codes have been proposed and its performance has been evaluated by using the average codeword length [3]. The overﬂow probability of codeword length is one of other criteria, which denotes the probability of codeword length per symbol being above a threshold R > 0. Merhav [4] has ﬁrst determined the optimal exponent of the overﬂow probability given R for uniﬁlar sources. Uchida and Han [5] have shown the inﬁmum of achievable threshold R under constraints that the overﬂow probability vanishes with given exponent r. Their analyses are based on the information-spectrum methods and the results are valid for general sources. Nomura and Matsushima [6] have computed the inﬁmum of ε-achievable overﬂow threshold for general sources. Here, ε-achievable overﬂow threshold means that there exists a variable-length code, whose overﬂow probability is smaller than or equal to ε. All the results mentioned here are in the meaning of the codeword length. L p In this paper, we also deal with the overﬂow probability of the codeword cost. In particular, we consider the ε-achievable threshold, which means that there exists a variable-length code, whose overﬂow probability is smaller than or equal to ε. We ﬁrst determine the inﬁmum of ﬁrst-order and second-order achievable thresholds for general sources. The ﬁner evaluation of the achievable rate, called the second-order achievable rate, has been investigated in several contexts. In the variable-length source coding problem, Kontoyiannis [16] has established the second-order source coding theorem on the codeword length for i.i.d. sources and Markov sources. In the channel coding problem, Strassen [17] (see, Csiszär and Körner [8]), Hayashi [18], and Verdú et al. [19] have determined the second-order capacity rate. Hayashi [20] has also shown the second-order achievability theorems for the ﬁxed-length source coding problem for general sources and compute the optimal second-order achievable rates for i.i.d. sources by using the asymptotic normality. Nomura and Han [21] have also computed the optimal second-order achievable rates in Manuscript received October 29, 2013; revised September 14, 2018; accepted August 20, 2019. Date of publication September 16, 2019; date of current version November 20, 2019. Communicated by Y. Oohama, Associate Editor for Source Coding. Color versions of one or more of the ﬁgures in this article are available online at http://ieeexplore.ieee.org. Manuscript received October 29, 2013; revised September 14, 2018; accepted August 20, 2019. Date of publication September 16, 2019; date of current version November 20, 2019. This work was supported in part by JSPS KAKENHI under Grant JP26420371 and Grant JP18K04150. This article was presented in part at the 2012 IEEE International Symposium on Information Theory. The author is with the Center for Data Science, Waseda University, Tokyo 162-0042, Japan (e-mail: nomu@waseda.jp). Communicated by Y. Oohama, Associate Editor for Source Coding. Color versions of one or more of the ﬁgures in this article are available online at http://ieeexplore.ieee.org. Digital Object Identiﬁer 10.1109/TIT.2019.2941888 This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see http://creativecommons.org/licenses/by/4.0/ I. INTRODUCTION This work was supported in part by JSPS KAKENHI under Grant JP26420371 and Grant JP18K04150. This article was presented in part at the 2012 IEEE International Symposium on Information Theory. Digital Object Identiﬁer 10.1109/TIT.2019.2941888 NOMURA: OVERFLOW PROBABILITY OF VARIABLE-LENGTH CODES 8195 ﬁxed-length source coding for mixed sources by using the two-peak asymptotic normality. of n-dimensional random variables Xn, where each com- ponent random variable X(n) i takes values in a countable set X . It should be noted that each component of Xn may change depending on block length n. This implies that even consistency condition, which means that for any integers m, n (m < n), X(m) i = X(n) i holds, may not hold. Analogously to these settings, we deﬁne the second-order achievable threshold on the overﬂow probability and derive the inﬁmum of the second-order achievable thresholds. Notice here that Nomura and Matsushima [6] have already considered the ﬁrst-order and the second-order achievabilities with respect to the overﬂow probability of the codeword length. One of contributions of this paper is to extend the results in [6] to the case of the codeword cost. Our analysis is based on the information-spectrum methods and hence our results are valid for general sources. Furthermore, we apply our results to i.i.d. sources and mixed sources as special cases and compute the inﬁmum of the ﬁrst- and second-order achievable thresholds for these important sources. ( ) i i y Variable-length codes are characterized as follows. Let ϕn : X n →U∗, ψn : {ϕn(x)}x∈X n →X n, be a variable-length encoder and a decoder, respectively, where U = {1, 2, . . . , K} is called the code alphabet and U∗is the set of all ﬁnite-length strings over U excluding the null string. We consider the situation that there are unequal costs on code symbols. Let us deﬁne the cost function over U considered in this paper. Each code symbol u ∈U is assigned the corresponding cost c(u) such that 0 < c(u) < ∞, and the additive cost c(u) of u = (u1, u2, . . . , uk) ∈Uk is deﬁned by be a variable-length encoder and a decoder, respectively, where U = {1, 2, . . . , K} is called the code alphabet and U∗is the set of all ﬁnite-length strings over U excluding the null string. I. INTRODUCTION (2) (2) Throughout this paper, the logarithm is taken to the base K. Throughout this paper, the logarithm is taken to the base K. B. Overﬂow Probability of Codeword Cost The Overﬂow Probability of the Codeword Length is Deﬁned as Follows: This paper is organized as follows. In Section II, we state the problem settings and deﬁne two achievabilities treated in this paper. In Section III, we prove two lemmas which play the key role in the subsequent analysis. In Section IV, we determine the inﬁmum of the ﬁrst-order achievable thresh- olds. In Section V, we derive the inﬁmum of the second-order achievable threshold and compute it for some special sources. In Section VI, we extend our results to the conditional cost. Finally, in Section VII, we conclude our results. Deﬁnition 2.1: [4] Given a threshold R, the overﬂow probability of the variable-length encoder ϕn is deﬁned by εn(ϕn, R) = Pr 1 nl(ϕ(Xn)) > R , (3) (3) where l(·) denotes the length function. In this paper, we generalize the above overﬂow probability not only to the case for unequal costs on code symbols but also for the ﬁner evaluation of the overﬂow probability. To this end, we consider the overﬂow probability of codeword cost as follows: I. INTRODUCTION We consider the situation that there are unequal costs on code symbols. Let us deﬁne the cost function over U considered in this paper. Each code symbol u ∈U is assigned the corresponding cost c(u) such that 0 < c(u) < ∞, and the additive cost c(u) of u = (u1, u2, . . . , uk) ∈Uk is deﬁned by A generalization of the codeword cost is also considered. The codeword cost considered in the above is the memo- ryless cost, which means that the component cost function is ﬁxed and the cost capacity is uniquely determined. This property helps us to derive fundamental limits of the ﬁrst- and second-order achievable thresholds explicitly. On the other hand, we also consider the case that the cost function may vary according to the previous sequence. This is a setting inherence in the variable-length coding with cost, and hence it is meaningful to discuss. We try to extend our results to this general cost function and demonstrate the upper and lower bounds of the inﬁmum of the ﬁrst-order achievable thresholds. c(u) = k i=1 c(ui). In particular, we denote cmax = maxu∈U c(u) for short. This cost function is called the memoryless cost function in this paper. A generalization of this cost function is discussed in Section VI. We only consider variable-length codes satisfying preﬁx condition. It should be noted that every variable-length code with preﬁx condition over unequal costs, satisﬁes Related works include works by Kontoyiannis and Verdú [22], and Kosut and Sankar [23], [24]. They have also considered the similar quantity with the overﬂow probability. Kontoyiannis and Verdú [22] have derived the fundamental limit of this quantity without the preﬁx con- ditions. Kosut and Sankar [23], [24] have also derived the upper-bound of the overﬂow probability in universal setting. It should be emphasized that they have considered the overﬂow probability of codeword length for some special sources and derived bounds up to the third-order. On the other hand, in this paper we consider the overﬂow probability of codeword cost for general sources and address the fundamental limit of the achievable threshold up to the second-order. x∈X n K −αcc(ϕn(x)) ≤1, (1) (1) where αc is called cost capacity and deﬁned as the positive unique root α of the equation [8]: u∈U K −αc(u) = 1. III. FINITE BLOCKLENGTH BOUNDS 1) βn = nR, 2) βn = na + √nL. 1) βn = nR, 2) βn = na + √nL. Our objective in this paper is to demonstrate the inﬁmum of the ε-achievable overﬂow thresholds as well as the inﬁmum of the (ε, a)-achievable overﬂow thresholds. To do so, in this section we derive two lemmas. Remark 2.2: If we set βn = nR for all n = 1, 2, . . . , that is, in the ﬁrst case, the overﬂow probability can be written as εn(ϕn, nR) = Pr c(ϕn(Xn)) > nR = Pr 1 n c(ϕn(Xn)) > R . Lemma 3.1: For any general sources X and any sequence of positive number {βn}∞ n=1, there exists a variable-length encoder ϕn that satisﬁes Thus, in the case of βn = nR, the overﬂow probability deﬁned by (4) means the probability that the codeword cost per symbol exceeds some constant R. This is a natural extension of the overﬂow probability of the codeword length to the overﬂow probability of the codeword cost deﬁned by (3). εn(ϕn, βn) < Pr zn PXn(Xn) ≤K −αcβn +znK αccmax+1, (5) (5) for n = 1, 2, . . . , where {zn}∞ n=1 is a given sequence of an arbitrary number satisfying zi > 0 for i = 1, 2, . . . and αc denotes the cost capacity deﬁned in (2). p y y ( ) On the other hand, in the analysis of the ﬁxed-length coding problem, Hayashi [20] has shown the second-order asymptotics, which enables us a ﬁner evaluation of the achiev- able coding rate. A coding theorem from the view point of the second-order asymptotics have been also analyzed by Kontoyiannis [16]. Analogously to their results, we evaluate the overﬂow probability in the second-order sense. To do so, we consider the second case: βn = na + L√n for all n = 1, 2, . . . . Hereafter, if we consider the overﬂow proba- bility in the case of βn = na + L√n, we call it the second- order overﬂow probability given a, while in the ﬁrst case it is called the ﬁrst-order overﬂow probability. The second-order overﬂow probability given a of the variable-length encoder ϕn with threshold L is written as Proof: Here, we use the code proposed by Han and Uchida [14]. II. OVERFLOW PROBABILITY OF VARIABLE-LENGTH CODING WITH COST Deﬁnition 2.2 (Overﬂow Probability of Codeword Cost): Given some sequence {βn}∞ n=1, where 0 < βn < ∞for each n = 1, 2, . . . , the overﬂow probability of variable-length encoder ϕn is deﬁned by A. Variable-Length Codes With Codeword Cost for General Source The general source is deﬁned as an inﬁnite sequence X = Xn = X(n) 1 X(n) 2 . . . X(n) n ∞ n=1 εn(ϕn, βn) = Pr c(ϕ(Xn)) > βn . (4) (4) IEEE TRANSACTIONS ON INFORMATION THEORY, VOL. 65, NO. 12, DECEMBER 2019 8196 if there exists a sequence of variable-length code (ϕn, ψn) such that Remark 2.1: Nomura and Matsushima [6] have considered the overﬂow probability with respect to the codeword length, that is, Pr {l(ϕ(Xn)) > βn} and derived the achievability of the ﬁrst-order and the second-order sense. Kosut and Sankar [23] have also deﬁned the similar probability in the case of code- word length and derived the upper bound in universal setting. Since {βn}∞ n=1 is an arbitrary sequence, the above deﬁnition is general. In particular, we shall consider the following two types of overﬂow probability in this paper: Remark 2.1: Nomura and Matsushima [6] have considered the overﬂow probability with respect to the codeword length, that is, Pr {l(ϕ(Xn)) > βn} and derived the achievability of the ﬁrst-order and the second-order sense. Kosut and Sankar [23] have also deﬁned the similar probability in the case of code- word length and derived the upper bound in universal setting. lim sup n→∞ εn ϕn, na + L√n ≤ε. Deﬁnition 2.6 ((ε, a)-achievable overﬂow threshold): Deﬁnition 2.6 ((ε, a)-achievable overﬂow threshold): L(ε, a\|X) Since {βn}∞ n=1 is an arbitrary sequence, the above deﬁnition is general. In particular, we shall consider the following two types of overﬂow probability in this paper: := inf {L\|L is an (ε, a)-achievable overﬂow threshold} . IV. INFIMUM OF ε-ACHIEVABLE OVERFLOW THRESHOLD Lemma 3.2: For any variable-length code and any sequence {βn}∞ n=1, it holds that III. FINITE BLOCKLENGTH BOUNDS Substituting (9) into (8), we obtain Substituting (9) into (8), we obtain εn(ϕn, βn)<Pr Xn ∈An +znK αcβn K −αc(βn−cmax)+log2 ≤Pr Xn ∈An + znK αccmax+1, Therefore, we have proved the lemma. Therefore, we have proved the lemma. Therefore, we have proved the lemma. because log 2 ≤1 holds. Therefore, we have proved the lemma. III. FINITE BLOCKLENGTH BOUNDS Notice here that from the property of the code, it holds that 1 c(ϕ∗ n(x)) ≤−1 αc log PXn(x) + log 2 αc + cmax, (6) (6) for all n = 1, 2, . . . , where ϕ∗ n denotes the encoder of the code. Furthermore, we set the decoder as the inverse mapping of ϕ∗ n that is, ψn = ϕ∗−1 n . Please note that the code is a uniquely decodable variable-length code for general sources with countably inﬁnite source alphabet. Next, we shall evaluate the overﬂow probability of this code. Set An = x ∈X n zn PXn(x) ≤K −αcβn , Sn = x ∈X n c(ϕ∗ n(x)) > βn . εn ϕn, na + L√n = Pr c(ϕn(Xn)) > na + L√n = Pr c(ϕn(Xn)) −na √n > L . The overﬂow probability is given by The overﬂow probability is given by It should be noted that since we assume that βn satisﬁes 0 < βn < ∞, R is a positive number while L can be negative. εn(ϕn, βn) = Pr Xn ∈Sn = x∈Sn PXn(x) = x∈Sn∩An PXn(x) + x∈Sn∩Acn PXn(x) ≤Pr Xn ∈An + x∈Sn∩Acn PXn(x), In the ﬁrst-order case, we are interested in the inﬁmum of the threshold R that we can achieve. This is formalized as follows. Deﬁnition 2.3: Given 0 ≤ ε < 1, R is called an ε-achievable overﬂow threshold for the source if there exists a sequence of variable-length code (ϕn, ψn) such that (7) where Ac denotes the complement set of the set A. where Ac denotes the complement set of the Since (6) holds, for ∀x ∈Sn, we have lim sup n→∞ εn(ϕn, nR) ≤ε. Since (6) holds, for ∀x ∈Sn, we have −1 αc log PXn(x) + log 2 αc + cmax > βn. Deﬁnition 2.4 (ε-achievable overﬂow threshold): Deﬁnition 2.4 (ε-achievable overﬂow threshold): R(ε\|X) := inf {R\|R is an ε-achievable overﬂow threshold} . Thus, we have Also, in the analysis of the second-order overﬂow probabil- ity, we deﬁne the achievability: PXn(x) < K −αc(βn−cmax)+log 2, Deﬁnition 2.5: Given 0 ≤ε < 1 and 0 < a < ∞, L is called an (ε, a)-achievable overﬂow threshold for the source, 1In Section VI, we have introduced the modiﬁed version of this code. For the derivation of this inequality, see also the proof of Lemma 6.2. III. FINITE BLOCKLENGTH BOUNDS NOMURA: OVERFLOW PROBABILITY OF VARIABLE-LENGTH CODES 8197 for ∀x ∈Sn. Substituting the above inequality into (7), we have Thus, we have for ∀x ∈Sn. Substituting the above inequality into (7), we have Thus, we have for ∀x ∈Sn. Substituting the above inequality into (7), we have Thus, we have εn(ϕn, βn) < Pr Xn ∈An + x∈Sn∩Acn K −αc(βn−cmax)+log2 = Pr Xn ∈An + Sn ∩Ac n K −αc(βn−cmax)+log2. (8) Here, from the deﬁnition of An, for ∀x ∈Ac n, it holds that Pr PXn(Xn) ≤znK −αcβn ≤εn(ϕn, βn) + x∈Bn∩Scn PXn(x) ≤εn(ϕn, βn) + x∈Bn∩Scn znK −αcβn ε (ϕ β ) + B ∩Sc z K −αcβn (11) εn(ϕn, βn) < Pr Xn ∈An + x∈Sn∩Acn K −αc(βn−cmax)+log2 = Pr Xn ∈An + Sn ∩Ac n K −αc(βn−cmax)+log2. (8) Here, from the deﬁnition of An, for ∀x ∈Ac n, it holds that x∈Bn∩Scn = εn(ϕn, βn) + Bn ∩Sc n znK −αcβn. (11) Here, from the deﬁnition of An, for ∀x ∈Ac n, it holds that Here, from the deﬁnition of An, for ∀x ∈Ac n, it holds that = εn(ϕn, βn) + Bn ∩Sc n znK −αcβn. (11) (11) PXn(x) > K −αcβn zn . n(ϕn, βn) n n n Here, from (1), we have ( ( )) PXn(x) > K −αcβn zn . PXn(x) > K −αcβn zn . Here, from (1), we have ( ( PXn(x) > K −αcβn zn . Here, from (1), we have 1 K α c(ϕ (x)) Here, from (1), we have 1 ≥ x∈X n K −αcc(ϕn(x)) ≥ x∈Scn K −αcc(ϕn(x)) ≥ x∈Scn K −αcβn = Sc n K −αcβn. Thus, we have Thus, we have 1 ≥ x∈X n PXn(x) ≥ x∈Acn PXn(x) > x∈Acn K −αcβn zn = Ac n K −αcβn zn . n > x∈Acn K −αcβn zn = Ac n K −αcβn zn . x∈Sn This mean that Bn ∩Sc n ≤ Sc n ≤K αcβn. (12) (12) This means that This means that Hence, substituting (12) into (11), we have Hence, substituting (12) into (11), we have Sn ∩Ac n ≤ Ac n < znK αcβn. (9) (9) Pr PXn(Xn) ≤anK −βn ≤εn(ϕn, βn) + Bn ∩Sc n znK −αcβn ≤εn(ϕn, βn) + K αcβnznK −αcβn = εn(ϕn, βn) + zn. 2For any sequence {Zn}∞ n=1 of real-valued random variables, we deﬁne the limit superior in probability of {Zn}∞ n=1 by p- lim supn→∞ Zn = inf {β\| limn→∞Pr{Zn > β} = 0} (cf. [25]). A. General Formula εn(ϕn, βn) ≥Pr PXn(Xn) ≤znK −αcβn −zn, In this subsection, we determine R(ε\|X) for general sources. Before showing the theorem, we deﬁne the function F(R) as follows: for n = 1, 2, . . . , where {zn}∞ n=1 is a given sequence of an arbitrary number satisfying zi > 0 for i = 1, 2, . . . . F(R) := lim sup n→∞ Pr 1 nαc log 1 PXn(Xn) ≥R . Proof: Let ϕn be an encoder of the variable-length code. Set Bn = x ∈X n PXn(x) ≤znK −αcβn , Sn = x ∈X n \|c(ϕn(x)) > βn . The following theorem is one of our main results: Theorem 4.1: For 0 ≤∀ε < 1, it holds tha Theorem 4.1: For 0 ≤∀ε < 1, it holds that (10) (10) R(ε\|X) = inf {R \|F(R) ≤ε } . R(ε\|X) = inf {R \|F(R) ≤ε } . Then, we have Proof: The proof consists of two parts. (Direct Part) Let R0 be as Proof: The proof consists of two parts. (Direct Part) Let R0 be as Pr PXn(Xn) ≤znK −αcβn = x∈Bn PXn(x) = x∈Bn∩Sn PXn(x) + x∈Bn∩Scn PXn(x) ≤ x∈Sn PXn(x) + x∈Bn∩Scn PXn(x) ≤εn(ϕn, βn) + x∈Bn∩Scn PXn(x). R0 = inf {R \|F(R) ≤ε } , for short. Then, in this part we show that for short. Then, in this part we show that R(ε\|X) ≤R0 + γ, (13) (13) for any γ > 0 by showing that R0 is an ε-achievable overﬂow threshold for the source. Let βn be as βn = n(R0 + γ ), then from Lemma 3.1 there exists a variable-length code (ϕn, ψn) that satisﬁes εn(ϕn, n(R0 + γ )) < Pr zn PXn(Xn) ≤K −nαc(R0+γ ) +znK αccmax+1, On the other hand, for ∀x ∈Bn it holds that PXn(x) ≤znK −αcβn. IEEE TRANSACTIONS ON INFORMATION THEORY, VOL. 65, NO. 12, DECEMBER 2019 8198 Since we assume that (14) holds, it is obvious that there exists γ > 0 that satisﬁes the above inequality. Then, we have for n = 1, 2, . . . . A. General Formula p (Converse Part) p (Converse Part) B. Strong Converse Property Assuming that R1 satisfying Strong converse property is one of important properties for the source in the ﬁxed-length source coding problem [25]. When we consider the second-order achievability, we have to give an appropriate ﬁrst-order term. In many cases the ﬁrst-order term is determined by considering the strong con- verse property and hence the strong converse property has an important meaning in the analysis of the second-order achievability. R1 < inf {R \|F(R) ≤ε } , (14) A. General Formula Thus, we have εn(ϕn, n(R0 + γ )) < Pr zn PXn(Xn) ≤K −nαc(R0+γ ) + znK αccmax+1 = Pr 1 n log 1 zn PXn(Xn) ≥αc(R0 + γ ) + znK αccmax +1 = Pr 1 nαc log 1 PXn(Xn) ≥R0 + γ + 1 nαc log zn +znK αccmax+1, εn(ϕn, nR1) > Pr 1 nαc log 1 PXn(Xn) ≥R1 + γ αc −K −nγ . Hence, we have lim sup n→∞ εn(ϕn, nR1) ≥lim sup n→∞ Pr 1 nαc log 1 PXn(Xn) ≥R1 + γ αc > ε, for n = 1, 2, . . , , Notice here that zn > 0 is an arbitrary number. Setting zn = K −√nγ , we have where the last inequality is derived from (15) and the deﬁnition of F(R). εn(ϕn, n(R0 + γ )) < Pr 1 nαc log 1 PXn(Xn) ≥R0+γ − √nγ nαc +K −√nγ +αccmax +1 < Pr 1 nαc log 1 PXn(Xn) ≥R0+ γ 2 + K−√nγ +αccmax+1, On the other hand, since we assume that R1 is an ε-achievable overﬂow threshold, it holds that lim sup n→∞ εn (ϕn, nR1) ≤ε. This is a contradiction. Therefore, the proof of the converse part has been completed. for sufﬁciently large n, because γ 2 > γ √nαc as n →∞. Thus, since αc and cmax are positive constants, by taking lim supn→∞, we have for sufﬁciently large n, because γ 2 > γ √nαc as n →∞. Thus, since αc and cmax are positive constants, by taking lim supn→∞, we have From the above theorem, we can show a corollary. Before describing the corollary, we deﬁne the spectral sup-entropy rate [25]2: lim sup n→∞ εn(ϕn, n(R0 + γ )) ≤lim sup n→∞ Pr 1 nαc log 1 PXn(Xn) ≥R0 + γ 2 . lim sup n→∞ εn(ϕn, n(R0 + γ )) H(X) = p- lim sup n→∞ 1 n log 1 PXn(Xn). n→∞ ≤lim sup n→∞ Pr 1 nαc log 1 PXn(Xn) ≥R0 + γ 2 . Then, the following corollary holds. Since γ > 0 is an arbitrary constant, from the deﬁnition of R0 we have Corollary 4.1: R(0\|X) = 1 αc H(X). (16) lim sup n→∞ εn(ϕn, n(R0 + γ )) ≤ε. (16) Noting that γ > 0 is arbitrarily small, the direct part has been proved. holds. Proof: This theorem can be proved by using the similar argument with the proof of [25, Theorem 1.5.1]. For the completeness of the paper we give the proof in Appendix A. The theorem reveals that the strong converse property only depends on the source X and is independent on the cost function. Remark 4.1: For an i.i.d. source, the following relationship holds [25], H(X) = H(X) = H(X), Let zn be as zn = K −4√nγ , then we have εn ϕn, na + √n(L0 + γ ) < Pr 1 √nαc log 1 PXn(Xn) ≥√na + L0 + γ − 4√nγ √nαc +K αccmax+1K −4√nγ = Pr −log PXn(Xn) −nαca √nαc ≥L0 + γ − γ 4√nαc +K αccmax+1−4√nγ < Pr −log PXn(Xn) −nαca √nαc ≥L0 + γ 2 +K αccmax+1−4√nγ , where H(X) denotes the entropy of the source. Thus, any i.i.d. source satisﬁes the strong converse property. This means that the inﬁmum of ε-achievable overﬂow thresholds R(ε\|X) is constant and is independent of ε. V. INFIMUM OF (ε, a)-ACHIEVABLE OVERFLOW THRESHOLD A. General Formula (14) ) there exists a variable-length encoder ϕn that satisﬁes In order to state the strong converse theorem, we deﬁne the dual quantity of H(X) as In order to state the strong converse theorem, we deﬁne the dual quantity of H(X) as H(X) = p- lim inf n→∞ 1 n log 1 PXn(Xn), εn ϕn, na + √n(L0 + γ ) < Pr zn PXn(Xn) ≤K −αc(na+√nL0+√nγ ) +znK αccmax+1. which is called the spectral inf-entropy rate [25].3 Then, we have the following theorem on the strong converse property. Theorem 4.2: Source X satisﬁes the strong converse prop- erty if and only if Thus, we have H(X) = H(X) (17) (17) εn ϕn, na + √n(L0 + γ ) <Pr zn PXn(Xn) ≤K −√nαc(√na+L0+γ ) +znK αccmax+1 =Pr 1 √nαc log 1 zn PXn(Xn) ≥√na + L0 + γ +znK αccmax+1 =Pr 1 √nαc log 1 PXn(Xn) ≥√na+L0+γ + 1 √nαc log zn +znK αccmax+1. (14) (14) is an ε-achievable overﬂow threshold, then we shall show a contradiction. Let βn be as βn = nR1. Then, from Lemma 3.2 for any sequence {zn}∞ n=1 (zi > 0, i = 1, 2, . . . ) and any variable-length code it holds that εn(ϕn, nR1) > Pr PXn(Xn) ≤znK −nαc R1 −zn, Analogously to the ﬁxed-length coding problem, we can consider the strong converse property in the meaning of the overﬂow probability in variable-length codes. In this subsec- tion, we establish the strong converse theorem on the overﬂow probability of the variable-length coding with codeword cost. Let us begin with the deﬁnition of the strong converse property treated in this paper. for n = 1, 2, . . . . Thus, for any variable-length code it holds that for n = 1, 2, . . . . Thus, for any variable-length code it holds that εn(ϕn, nR1) > Pr PXn(Xn) ≤znK −nαc R1 −zn = Pr 1 nαc log 1 PXn(Xn) ≥R1 − 1 nαc log zn −zn. Deﬁnition 4.1: Source X is said to satisfy the strong con- verse property, if any variable-length code (ϕn, ψn) with the Set zn = K −nγ , where γ > 0 is a small constant that satisﬁes Set zn = K −nγ , where γ > 0 is a small constant that satisﬁes Set zn = K −nγ , where γ > 0 is a small constant that satisﬁes R1 + γ αc < inf {R \|F(R) ≤ε } . (15) R1 + γ αc < inf {R \|F(R) ≤ε } . (15) (15) NOMURA: OVERFLOW PROBABILITY OF VARIABLE-LENGTH CODES 8199 for short. Then, in this part we shall show that overﬂow probability εn(ϕn, nR), where R is an arbitrary rate satisfying R < R(0\|X), necessarily yields L(ε, a\|X) ≤L0 + γ, (18) (18) lim n→∞εn(ϕn, nR) = 1. for any γ > 0 by showing that L0 is an ε-achievable overﬂow threshold for the source. Let βn be as βn = na +√n(L0 +γ ), then from Lemma 3.1, for any sequence {zn}∞ n=1 (zi > 0, i = 1, 2, . . . IEEE TRANSACTIONS ON INFORMATION THEORY, VOL. 65, NO. 12, DECEMBER 2019 8200 is an (ε, a)-achievable second-order overﬂow threshold, we shall show a contradiction. For an i.i.d. source, from Remark 4.1, we are inter- ested in L ε, 1 αc H(X) X . To specify this quantity for an i.i.d. source, we need to introduce the variance of the self- information as follows: From Lemma 3.2 for any sequence {zn}∞ n=1 (zi > 0, i = 1, 2, . . . ) and any variable-length encoder, it holds that εn ϕn, na + √nL1 ≥Pr PXn(Xn) ≤znK −αc(na+√nL1) −zn, σ 2 := E (−log PX(X) −H(X))2 . εn ϕn, na + √nL1 ≥Pr PXn(Xn) ≤znK −αc(na+√nL1) −zn, Here, we assume that the above variance exists and σ 2 > 0. Then, from Theorem 5.1 we obtain the following theorem. for n = 1, 2, . . . . Thus, for any variable-length encoder, we have g Theorem 5.2: For any i.i.d. source, it holds that L ε, 1 αc H(X) X = 1 αc σ −1(1 −ε), εn ϕn, na + √nL1 ≥Pr PXn(Xn) ≤znK −αc(na+√nL1) −zn = Pr 1 √nαc log 1 PXn(Xn) ≥√na + L1 −log zn √nαc −zn = Pr −log PXn(Xn) −nαca √nαc ≥L1 −log zn √nαc −zn. where −1 denotes a inverse function of and (T) is the Gaussian cumulative distribution function with mean 0 and variance 1, that is, (T ) is given by where −1 denotes a inverse function of and (T) is the Gaussian cumulative distribution function with mean 0 and variance 1, that is, (T ) is given by = Pr 1 √nαc log 1 PXn(Xn) ≥√na + L1 −log zn √nαc −zn = Pr −log PXn(Xn) −nαca √nαc ≥L1 −log zn √nαc −zn. (T ) = T −∞ 1 √ 2π exp −y2 2 dy. (21) (21) Proof: From the deﬁnition of Fa(L), we have Proof: From the deﬁnition of Fa(L), we have Set zn = K −√nγ , where γ > 0 is a small constant that satisﬁes Set zn = K −√nγ , where γ > 0 is a small constant that satisﬁes FH(X)/αc(L) L1 + γ αc < inf {L \|Fa(L) ≤ε } . IEEE TRANSACTIONS ON INFORMATION THEORY, VOL. 65, NO. 12, DECEMBER 2019 (20) FH(X)/αc(L) = lim sup n→∞ Pr −log PXn(Xn) −nH(X) √nαc ≥L = lim sup n→∞ Pr −log PXn(Xn) −nH(X) √nσ ≥Lαc σ . (20) (20) Here, since we assume (19), it is obvious that there exists γ > 0 satisfying the above inequality. Then, we have Here, since we assume (19), it is obvious that there exists γ > 0 satisfying the above inequality. Then, we have εn ϕn, na + √nL1 ≥Pr −log PXn(Xn) −nαca √nαc ≥L1 + γ αc −K −√nγ . his implies that lim sup n→∞ εn ϕn, na + √nL1 ≥lim sup n→∞ Pr −log PXn(Xn) −nαca √nαc ≥L1 + γ αc > ε, εn ϕn, na + √nL1 ≥Pr −log PXn(Xn) −nαca √nαc ≥L1 + γ αc −K −√nγ . On the other hand, since we consider the i.i.d. source , from the asymptotic normality (due to the central limit theorem) it holds that This implies that This implies that lim n→∞Pr −log PXn(Xn)−nH(X) √nσ ≤U = U −∞ 1 √ 2π exp −y2 2 dy. lim sup n→∞ εn ϕn, na + √nL1 ≥lim sup n→∞ Pr −log PXn(Xn) −nαca √nαc ≥L1 + γ αc > ε, This means that FH(X)/αc(L) = +∞ Lαc σ 1 √ 2π exp −y2 2 dy. where the last inequality is derived from (20) and the deﬁnition of Fa(L). Thus, L ε, 1 αc H(X) X is given by L ε, 1 αc H(X) X = inf L +∞ Lαc σ 1 √ 2π exp −y2 2 dy ≤ε = inf L 1 − Lαc σ ≤ε . , L ε, 1 αc H(X) X is given by On the other hand, since we assume that L1 is (ε, a)-achievable, it holds that lim sup n→∞ εn ϕn, na + √nL1 ≤ε. This is a contradiction. Therefore, the proof of converse part has been completed. Remark 5.1: As we have shown in the above, the proof of Theorem 5.1 proceeds in parallel with that of Theorem 4.1 by using Lemma 3.1 and Lemma 3.2. We observe that we can use the same argument for other sequence {βn}∞ n=1 such that βn →∞. IEEE TRANSACTIONS ON INFORMATION THEORY, VOL. 65, NO. 12, DECEMBER 2019 Thus, these lemmas are of great use to analyze the overﬂow probability of the variable-length code with codeword cost. Since Lαc σ is a continuous function and monotonically increases as L increases, we have L ε, 1 αc H(X) X αc σ = −1(1 −ε). A. General Formula So far, we have considered the ﬁrst-order achievable thresh- old. In this section, we consider the second-order achievability. In the second-order case, the inﬁmum (ε, a)-achievable over- ﬂow threshold for general sources is also determined by using Lemma 3.1 and Lemma 3.2. for sufﬁciently large n, because γ 2 > γ 4√nαc holds for sufﬁ- ciently large n. for sufﬁciently large n, because γ 2 > γ 4√nαc holds for sufﬁ- ciently large n. We deﬁne the function Fa(R) given a as follows, which is analogous to the function F(R) in the ﬁrst-order case, y g By taking lim supn→∞, we have Fa(L) := lim sup n→∞ Pr −log PXn(Xn) −nαca √nαc ≥L . lim sup n→∞ εn ϕn, na + √n(L0 + γ ) ≤lim sup n→∞ Pr −log PXn(Xn) −nαca √nαc ≥L0 + γ 2 . Then, we have Then, we have m 5.1: For 0 ≤∀ε < 1, it holds that L(ε, a\|X) = inf {L \|Fa(L) ≤ε } . Theorem 5.1: For 0 ≤∀ε < 1, it holds that Hence, from the deﬁnition of L0 we have L(ε, a\|X) = inf {L \|Fa(L) ≤ε } . lim sup n→∞ εn ϕn, na + √n(L0 + γ ) ≤ε. Proof: The proof is similar to the proof of Theorem 4.1. (Direct Part) Let L0 be as This means that (18) holds. Therefore, the direct part has been proved. This means that (18) holds. Therefore, the direct part has been proved. proved. L0 = inf {L \|Fa(L) ≤ε } , (Converse Part) Assuming that L1 satisfying 3For any sequence {Zn}∞ n=1 of real-valued random variables, we deﬁne the limit inferior in probability of {Zn}∞ n=1 by p- lim infn→∞Zn = sup {α\| limn→∞Pr{Zn < α} = 0} (cf. [25]). L1 < inf {L \|Fa(L) ≤ε } , (19) (19) IEEE TRANSACTIONS ON INFORMATION THEORY, VOL. 65, NO. 12, DECEMBER 2019 NOMURA: OVERFLOW PROBABILITY OF VARIABLE-LENGTH CODES NOMURA: OVERFLOW PROBABILITY OF VARIABLE-LENGTH CODES 8201 C. Computation for Mixed Sources In Case I, we shall compute L ε, 1 αc H(X1) X (this is equal to L ε, 1 αc H(X2) X ). In Case II and Case III, we shall show L ε, 1 αc H(X1) X and where w(i) are constants satisfying w(1) + w(2) = 1 and w(i) > 0 (i = 1, 2). Since two i.i.d. sources Xi (i = 1, 2) are completely speciﬁed by giving just the ﬁrst component Xi (i = 1, 2), we may write simply as Xi = {Xi} (i = 1, 2) and deﬁne the variances: In Case II and Case III, we shall show L ε, 1 αc H(X1) X and c L ε, 1 αc H(X2) X , respectively. Then, from Theorem 5.1 we obtain the following theorem: σ 2 i := E log 1 PXi (Xi) −H(Xi) 2 (i = 1, 2), g Theorem 5.4: For any mixed source, it holds that Case I Case I L ε, 1 αc H(X1) X = T1, where we assume that these variances exist and σ 2 i > 0 (i = 1, 2). where T1 is speciﬁed by where T1 is speciﬁed by Before showing second-order analysis we shall consider the ﬁrst-order case. Without loss of generality, we assume that H(X1) ≥H(X2) holds. ε = 1 − 2 i=1 w(i) αcT1 σi . (25) (25) Theorem 5.3: For any mixed source deﬁned by (22), we have Case II R(ε\|X) = H(X1) αc if 0 ≤ε < w(1), H(X2) αc if w(1) ≤ε < 1. (23) ε, 1 αc H(X1) X = T2, ε, 1 αc H(X1) X = T2, (23) where T2 is speciﬁed by where T2 is speciﬁed by Proof: This theorem can be obtained as an immediate consequence of Theorem 4.1. ε = w(1) 1 − αcT2 σ1 . (26) (26) By using the above theorem we shall compute the second-order case. As we have mentioned in the above, the asymptotic normality of self information does not hold for mixed sources. However, since we consider the case where Xi = {Xi} (i = 1, 2) is an i.i.d. source, the following asymptotic normality holds for each component i.i.d. B. Computation for i.i.d. Sources Therefore, the proof has been completed. Therefore, the proof has been completed. Therefore, the proof has been completed. Therefore, the proof has been completed. Theorem 5.1 is a quite general result, because there is no restriction about the probability structure for the source. However, to compute the function L (ε, a\|X) is hard in general. Next, we consider a simple case such as an i.i.d. source with countably inﬁnite alphabet and we address the above quantity explicitly. Remark 5.2: As shown in the proof, the derivation of Theorem 5.2 is based on of the asymptotic normality of the self-information. This means that the similar argument is valid for any source for which the asymptotic nor- mality of self-information holds such as Markov sources (see, Hayashi [20]). C. Computation for Mixed Sources source X it holds that, for i = 1, 2 source X it holds that, for i = 1, 2, rce X it holds that, for i = 1, 2, Pr −log PXn(Xn i ) √n ≥zn ≥Pr −log PXn i (Xn i ) √n ≥zn + γn −e−√nγn, Pr −log PXn(Xn i ) √n ≥zn ≤Pr −log PXn i (Xn i ) √n ≥zn −γn , In this subsection we consider mixed sources. The class of mixed sources is very important, because all of stationary sources can be regarded as forming mixed sources obtained by mixing stationary ergodic sources with respect to appropriate probability measures. Notice here that, in general, the mixed source does not have the asymptotic normality of the self- information. So, we can not simply apply Theorem 5.2. The second-order achievable rates for mixed sources has been ﬁrst considered by Nomura and Han [21] in the ﬁxed-length source coding problem. The result in this sub- section is a variable-length coding counterpart of the result in [21]. where γn > 0 satisﬁes γ1 > γ2 > · · · > 0, γn →0, √nγn →∞. In the sequel, we also assume that H(X1) ≥H(X2) holds without loss of generality. Then, given ε (0 ≤ε < 1) we classify the problem into three cases: We consider the mixed source consisting of two stationary memoryless sources Xi = {Xn i }∞ n=1 with i = 1, 2. Then, the mixed source X = {Xn}∞ n=1 is deﬁned by I H(X1) = H(X2) holds. II H(X1) > H(X2) and w(1) > ε hold. III H(X1) > H(X2) and w(1) < ε hold. PXn(x) = w(1)PXn 1(x) + w(2)PXn 2 (x), (22) (22) (22) See Remark 5.3, in the case where H(X1) > H(X2) and w(1) = ε hold. In Case I, we shall compute L ε, 1 αc H(X1) X (this is equal to L ε, 1 αc H(X2) X ). In Case II and Case III, we shall show L ε, 1 αc H(X1) X and L ε, 1 αc H(X2) X , respectively. Then, from Theorem 5.1 we obtain the following theorem: See Remark 5.3, in the case where H(X1) > H(X2) and w(1) = ε hold. C. Computation for Mixed Sources source: Case III L ε, 1 αc H(X2) X = T3, where T3 is speciﬁed by ε = w(1) + w(2) 1 − αcT3 σ2 . (27) lim n→∞Pr −log PXn i (Xn i ) −nH(Xi) √nσi ≤U = (U). (24) (27) Proof: This theorem can be shown substantially same with [21, Theorem 5.1]. We only show the proof of Case I in Appendix B. The following lemma plays the key role in dealing with mixed sources in the proof of Theorem 5.4. Remark 5.3: In the case where H(X1) > H(X2) and w(1) = ε hold, it is not difﬁcult to check that T2 = −∞ Lemma 5.1 (Han [25], (see also [21, Lemma 4.1])): Let {zn}∞ n=1 be any real-valued sequence. Then for the mixed IEEE TRANSACTIONS ON INFORMATION THEORY, VOL. 65, NO. 12, DECEMBER 2019 8202 Fig. 1. Illustrative ﬁgure of Case I: Two Gaussian distributions with mean 0.097 and σ = 0.051, and mean 0.097 and σ = 0.041. T1 ≈0.07 is the unique point at which the probability of the weighted sum of shaded areas equals to ε = 0.06. Fig. 1. Illustrative ﬁgure of Case I: Two Gaussian distributions with mean 0.097 and σ = 0.051, and mean 0.097 and σ = 0.041. T1 ≈0.07 is the unique point at which the probability of the weighted sum of shaded areas equals to ε = 0.06. Fig. 2. Illustrative ﬁgure of Case II: Two Gaussian distributions with mean 1.169 and σ = 0.023, and mean 0.910 and σ = 0.046. T2 is the unique point at which the probability of the weighted sum of shaded areas equals to ε = 0.025. Fig. 2. Illustrative ﬁgure of Case II: Two Gaussian distributions with mean 1.169 and σ = 0.023, and mean 0.910 and σ = 0.046. T2 is the unique point at which the probability of the weighted sum of shaded areas equals to ε = 0.025. Fig. 2. Illustrative ﬁgure of Case II: Two Gaussian distributions with mean 1.169 and σ = 0.023, and mean 0.910 and σ = 0.046. T2 is the unique point at which the probability of the weighted sum of shaded areas equals to ε = 0.025. Fig. 1. Illustrative ﬁgure of Case I: Two Gaussian distributions with mean 0.097 and σ = 0.051, and mean 0.097 and σ = 0.041. C. Computation for Mixed Sources T1 ≈0.07 is the unique point at which the probability of the weighted sum of shaded areas equals to ε = 0.06. Fig. 3. Illustrative ﬁgure of Case III: Two Gaussian distributions with mean 1.169 and σ = 0.023, and mean 0.910 and σ = 0.046. T3 is the unique point at which the probability of the weighted sum of shaded areas equals to ε = 0.52. and T3 = +∞(cf. [21, Remark 5.2]). Hence, in this case we obtain L ε, 1 αc H(X1) X = −∞, L ε, 1 αc H(X2) X = +∞. Example 5.1: We here give numerical examples of Theorem 5.4. Let us consider the mixed sources PXn(x) = 0.5PXn 1(x) + 0.5PXn 2 (x) with alphabets X = {0, 1, 2} and U = X. Moreover, we con- sider the cost function satisfying c(0) = 1, c(1) = 1, and c(2) = 2. In this setting, the cost capacity αc is given by the unique solution of Fig. 3. Illustrative ﬁgure of Case III: Two Gaussian distributions with mean 1.169 and σ = 0.023, and mean 0.910 and σ = 0.046. T3 is the unique point at which the probability of the weighted sum of shaded areas equals to ε = 0.52. 3−α + 3−α + 3−2α = 1. Thus, we have αc = log3(1 + √ 2). In this case, we have H(X1) ≈0.937, H(X2) ≈0.730, σ 2 1 ≈0.11, and σ 2 2 ≈0.41. Hence, we have In this case, we have H(X1) ≈0.937, H(X2) ≈0.730, σ 2 1 ≈0.11, and σ 2 2 ≈0.41. Hence, we have g3( ) First, we consider Case I, that is, H(X1) = H(X2) = 0.8 with PX1(0) = 0.2, PX1(1) ≈0.145, PX1(2) ≈0.655, PX2(0) = 0.4, PX2(1) ≈0.065 and PX2(2) ≈0.535. Then, we have σ 2 1 ≈0.334 and σ 2 2 ≈0.214. Hence, we have H(X1) αc ≈1.169, H(X2) αc ≈0.910. H(X1) αc = H(X2) αc ≈0.997. Moreover, let us consider the case n = 300. Then, it holds that σ1 αc √n ≈0.023, σ2 αc √n ≈0.046. Moreover, let us consider the case n = 200. In this case it holds that σ1 αc √n ≈0.051, σ2 αc √n ≈0.041. Fig. 2 and Fig. 3 illustrate Case II and Case III in this numerical example. VI. GENERALIZATION OF THE COST FUNCTION So far, we have considered the additive cost consisting of the ﬁxed memoryless cost function. In this section, we will extend our results to more general cost function, that is, the conditional cost function. The conditional cost function is the function that the cost of the code symbol ui depends on the previous sequence ui−1 1 . In the application such as the video coding or the coding on magnetic data storage, constrained codes have been utilized [26]–[28]. In particular, the symmetric ﬁx-free code, the run-length constraint codes are representative. The symmetric constraint is the constraint that each codeword is required to be a palindrome. The run-length constraint code is the code in which the number of consecutive ones in a codeword is restricted. In each of these cases, it can be considered that the cost of ui depends on the previous sequence ui−1 1 . Furthermore, the conditional cost function con- sidered in this section includes the regular cost function (see, Remark 6.1 for the deﬁnition of the regular cost function). The relationship between the variable-length coding with regular cost function and the simulating Markov process has been clariﬁed [14]. Hence, the conditional cost function is important and meaningful to discuss from both of the practical and theoretical viewpoints. u∈D q(u) ≤1 holds (cf. [14, Lemma 1]). holds (cf. [14, Lemma 1]). holds (cf. [14, Lemma 1]). Secondly, we have the following lemmas which are gener- alizations of Lemmas 3.1 and 3.2. Lemma 6.2: For any general sources X and any sequence of positive number {βn}∞ n=1, there exists a variable-length encoder ϕn that satisﬁes εn(ϕn, βn) < Pr zn PXn(Xn) ≤K −αcβn + znK αccmax+1, (31) (31) for n = 1, 2, . . . , where {zn}∞ n=1 is a given sequence of an arbitrary number satisfying zi > 0 for i = 1, 2, . . . . Proof: First, we extend the code ϕ∗ n proposed by Han and Uchida [14] (see also, Yagi and Nomura [15]) to the cost function treated in this section. To do so, we assume that all the elements in X n are indexed as {x1, x2, . . . }. NOMURA: OVERFLOW PROBABILITY OF VARIABLE-LENGTH CODES NOMURA: OVERFLOW PROBABILITY OF VARIABLE-LENGTH CODES 8203 for any u ∈D. Then, the preﬁx condition and (29) assure that VI. GENERALIZATION OF THE COST FUNCTION We deﬁne Pk := k−1 j=1 PXn(x j), Qk := Pk + 1 2 PXn(xk), Pk := k−1 j=1 PXn(x j), Qk := Pk + 1 2 PXn(xk), The conditional cost function treated in this section is deﬁned as follows. The codeword cost c(ul) of a codeword ul ∈Ul is deﬁned by for k = 1, 2, . . . , where P1 = 0. Moreover set for k = 1, 2, . . . , where P1 = 0. Moreover set q(u) = K −l(u) i=1 αc(ui−1 1 )c(ui\|ui−1 1 ), c(ul) = l i=1 c(ui\|ui−1 1 ), (28) (28) for ∀u ∈U∗and also deﬁne for ∀u ∈U∗and also deﬁne β(u) := u′:u′≺u q(u′), γ (u) := β(u) + q(u), where c(ui\|ui−1 1 ) is called the conditional cost of ui given ui−1 1 such that 0 < cmin ≤c(ui\|ui−1 1 ) ≤cmax < ∞ (∀i, ∀ui ∈U, ∀ui−1 1 ∈Ui−1), where cmin and cmax are some constants. Here, we set u0 1 is the null string. We call this the conditional cost in this paper. 1 1 and I(u) := [β(u), γ (u)), where ≺denotes the lexicographic order on the set Ul(u). Now, to each xk we uniquely assign uk such as The conditional cost capacity αc(ui−1 1 ) given ui−1 1 is deﬁned by the positive unique root α of the equation ui∈U K −αc(ui\|ui−1 1 ) = 1, (29) uk = arg min u∈Kk l(u), (32) (32) (29) where Kk denotes the set of u ∈U∗such that the interval I(u) includes Qk but neither Pk nor Pk+1. Here, we can verify the existence uk for ∀xk ∈X n as follows. From (29) we obtain for ui−1 1 ∈Ui−1. It should be noted that αc(ui−1 1 ) may vary according to ui−1 1 . Moreover, we set u∈Um q(u) = u∈Um K −m i=1 αc(ui−1 1 )c(ui\|ui−1 1 ) = 1, αc = sup u∈U∗αc(u), αc = inf u∈U∗αc(u), and assume that 0 < αc ≤αc < ∞holds. for all m = 1, 2, . . . . Furthermore, from the assumption that αc > 0 and cmin > 0 hold, it holds that I(ui+1 1 ) ⊊I(ui 1) for any ui+1 1 ∈U∗. Hence, for ∀xk ∈X n there exists uk satisfying (32). C. Computation for Mixed Sources It should be noted that the area of shaded regions is equal to ε = 0.025 (Fig. 2) and ε = 0.52 (Fig. 3). Then, from Theorem 5.4, T2 ≈0.038 and T3 ≈0.071 are computed by using two Gaussian distributions with mean 1.169 and σ = 0.023, and mean 0.910 and σ = 0.046. Fig. 1 illustrates this case with ε = 0.06. From Theorem 5.4, T1 ≈0.07 is computed by using two Gaussian distributions with mean 0.097 and σ = 0.051, and mean 0.097 and σ = 0.041 such that the probability of the weighted sum of shaded regions equals to ε = 0.06. We also observe that the inﬁmum of the second-order achievable rate may be negative, when ε > 0.5. We also observe that the inﬁmum of the second-order achievable rate may be negative, when 0.25 < ε < 0.5 or 0.75 < ε < 1 holds. Remark 5.4: In [21], the countably inﬁnite mixture of i.i.d. sources and the general mixture of i.i.d. sources are treated. We can also obtain the inﬁmum of (ε, a)-achievable overﬂow threshold in these cases by using the similar argument. Secondly, we shall give examples of Case II and Case III. We assume that the probability of each source symbol is given by PX1(0) = 0.5, PX1(1) = 0.3, PX1(2) = 0.2, PX2(0) = 0.7, PX2(1) = 0.2, PX2(2) = 0.1, respectively. VI. GENERALIZATION OF THE COST FUNCTION c In order to establish the coding theorem in this setting, we ﬁrst have the following lemma instead of (1). In order to establish the coding theorem in this setting, we ﬁrst have the following lemma instead of (1). Lemma 6.1: The variable-length code D = {u1, u2, . . . } with the preﬁx condition over the conditional cost satisﬁes Then, clearly (Pk, Pk+1) ⊃ I(u) holds and each interval I(u1), I(u2), . . . is disjoint. This implies that {u1, u2, . . . , } satisﬁes the preﬁx condition. Thus, we can deﬁne the preﬁx encoder u∈D K −l(u) i=1 αc(ui−1 1 )c(ui\|ui−1 1 ) ≤1, (30) (30) where l(u) denotes the length of the codeword u. Proof: We deﬁne ϕ∗ n(xk) = uk. We next evaluate the cost of this code c(ϕ∗ n(xk)) as fol- lows. For each sequence uk = (u1, u2, . . . , ul), we deﬁne q(u) := K −l(u) i=1 αc(ui−1 1 )c(ui\|ui−1 1 ), IEEE TRANSACTIONS ON INFORMATION THEORY, VOL. 65, NO. 12, DECEMBER 2019 8204 Proof: By using Lemmas 6.2 and 6.3 it sufﬁces to proceed in parallel with the arguments as made in the proof of Theorem 4.1. Proof: By using Lemmas 6.2 and 6.3 it sufﬁces to proceed in parallel with the arguments as made in the proof of Theorem 4.1. uk = (u1, u2, . . . , ul−1). Since I(uk) ⊂I(uk) holds, we have Qk ∈I(uk). Moreover, from the deﬁnition we also have Pk ∈I(uk) or Pk+1 ∈I(uk). This means that the width \|I(uk)\| is larger than PXn(xk)/2. Thus, in view of (28), we have uk = (u1, u2, . . . , ul−1). Since I(uk) ⊂I(uk) holds, we have Qk ∈I(uk). Moreover, from the deﬁnition we also have Pk ∈I(uk) or Pk+1 ∈I(uk). This means that the width \|I(uk)\| is larger than PXn(xk)/2. Thus, in view of (28), we have uk = (u1, u2, . . . , ul−1). Since I(uk) ⊂I(uk) holds, we have Q ( ) Pk ∈I(uk) or Pk+1 ∈I(uk). This means that the width \|I(uk)\| is larger than PXn(xk)/2. Thus, in view of (28), we have We immediately have the following corollary, which subsumes Theorem 4.1. VI. GENERALIZATION OF THE COST FUNCTION Corollary 6.1: Assuming that αc = αc holds, then we have PXn(xk) 2 < \|I(uk)\| PXn(xk) 2 < \|I(uk)\| = K −l(uk) i=1 αc(ui−1 1 )c(ui\|ui−1 1 ) ≤K −αc l(uk) i=1 c(ui\|ui−1 1 ) = K −αcc(uk). R(ε\|X) = inf R lim sup n→∞ Pr 1 nαc log 1 PXn(Xn) ≥R ≤ε , R(ε\|X) = K −l(uk) i=1 αc(ui−1 1 )c(ui\|ui−1 1 ) ≤K −αc l(uk) i=1 c(ui\|ui−1 1 ) = K −αcc(uk). for 0 ≤ε < 1. Remark 6.1: As a special case of this conditional cost, we can consider the case that the conditional cost capacity αc(u) = α holds for all u ∈U∗. Such a class of cost functions, said to be regular, has been ﬁrst considered in [29]. Han and Uchida [14] also have treated the regular cost function. This is a typical example where αc = αc holds. Then, we obtain btain c(uk) < −1 αc log PXn(xk) + log 2 αc . c(uk) < −1 αc log PXn(xk) + log 2 αc . (33) (33) αc(u) = α holds for all u ∈U∗. Such a class of cost functions, said to be regular, has been ﬁrst considered in [29]. Han and Uchida [14] also have treated the regular cost function. This is a typical example where αc = αc holds. Hence, we obtain c(ϕ∗ n(xk)) ≤c(uk) + cmax < −1 αc log PXn(x) + log 2 αc + cmax. (34) Remark 6.2: In this section, we have only discussed the inﬁmum of the ﬁrst-order achievable threshold. It should be noted that we can also derive the upper and lower bounds of the inﬁmum of the second-order achievable threshold by using Lemmas 6.2 and 6.3. (34) Now, we can prove the lemma by using the similar argument as that in the proof of Lemma 3.1. Note that we use the inequality (34) instead of (6). VII. CONCLUDING REMARKS Theorem 6.1: For 0 ≤∀ε < 1, it holds that Theorem 6.1: For 0 ≤∀ε < 1, it holds that Theorem 6.1: For 0 ≤∀ε < 1, it holds that inf R F(R) ≤ε ≤R(ε\|X) ≤inf {R \|F(R) ≤ε } , where F(R) and F(R) are respectively deﬁned by VII. CONCLUDING REMARKS Lemma 6.3: For any variable-length code and any sequence {βn}∞ n=1, it holds that We have so far dealt with the overﬂow probability of the variable-length coding with codeword cost. As shown in the present paper, the information-spectrum approach is substantial in the analysis of the overﬂow probability of the variable-length coding. εn(ϕn, βn) ≥Pr PXn(Xn) ≤znK −αcβn −zn, for n = 1, 2, . . . , where {zn}∞ n=1 is a given sequence of an arbitrary number satisfying zi > 0 for i = 1, 2, . . . . for n = 1, 2, . . . , where {zn}∞ n=1 is a given sequence of an arbitrary number satisfying zi > 0 for i = 1, 2, . . . . In particular, Lemmas 3.1 and 3.2 are quite fundamental, because the ﬁrst-order coding theorem (Theorem 4.1) and the second-order coding theorem (Theorem 5.1) have been established by invoking these lemmas. It should be emphasized that these theorems can be proved by using the similar argument. In the channel coding problem, the third-order optimal achievable rate has been discussed [30], [31]. Also in the variable-length coding regime, the optimal achievable overﬂow threshold with respect to the codeword length has been discussed in detail in [22]. If we set βn appropriately in Lemmas 3.1 and 3.2, we can also derive the general formula of the optimal third- or higher-order overﬂow threshold for the variable-length coding with codeword cost. Proof: From Lemma 6.1, for any preﬁx code D we obtain 1 ≥ u∈D K −l(u) i=1 αc(ui−1 1 )c(ui\|ui−1 1 ) ≥ u∈D K −αc l(u) i=1 c(ui\|ui−1 1 ) = u∈D K −αcc(u). This means that 1 ≥ x∈X n K −αcc(ϕn(x)). (35) (35) Then, the proof of the lemma proceeds in parallel with the proof of Lemma 3.2. Note that we use the inequality (35) instead of (1). Finally, we have extended our results for the memoryless cost to the conditional cost. Another typical but important cost is the ﬁnite-state cost [7] which admits cmin = 0. It is an interesting problem to extend our result to this ﬁnite-state cost. Finally, we have the following theorem, which reveals the upper and lower bounds of the inﬁmum of the ﬁrst-order achievable overﬂow threshold. PROOF OF THEOREM 5.4 We only show Case I. The proofs of Case II and Case III are similar to that of Case I and [21, Theorem 5.1] We only show Case I. The proofs of Case II and Case III are similar to that of Case I and [21, Theorem 5.1] From the deﬁnition of Fa(L), we have εn(ϕn, nR) > Pr 1 nαc log 1 PXn(Xn) ≥R − 1 nαc log zn −zn, for any sequence {zn}∞ n=1 (zi > 0, i = 1, 2, . . . ). Let zn be as K −nγ , then we have εn(ϕn, nR) > Pr 1 nαc log 1 PXn(Xn) ≥R − 1 nαc log zn −zn, for any sequence {zn}∞ n=1 (zi > 0, i = 1, 2, . . . ). Let zn be as K −nγ , then we have εn(ϕn, nR) > Pr 1 nαc log 1 PXn(Xn) ≥R − 1 nαc log zn −zn, From the deﬁnition of Fa(L), we have FH(X1)/αc(L) = lim sup n→∞ Pr −log PXn(Xn) −nH(X1) √nαc ≥L = lim sup n→∞ 2 i=1 Pr −log PXn(Xn i ) −nH(X1) √nαc ≥L w(i) for any sequence {zn}∞ n=1 (zi > 0, i = 1, 2, . . . ). Let zn be as K −nγ , then we have for any sequence {zn}∞ n=1 (zi > 0, i = 1, 2, . . . ). Let zn be as K −nγ , then we have εn(ϕn, nR) > Pr 1 nαc log 1 PXn(Xn) ≥R + γ αc −K −nγ = Pr 1 nαc log 1 PXn(Xn) ≥1 αc H(X) −γ αc −K −nγ = Pr 1 nαc log 1 PXn(Xn) ≥1 αc H(X) −γ αc −K −nγ . The last equality is derived from the deﬁnition of the mixed source. Thus, from Lemma 5.1 we have 2 i=1 lim sup n→∞ Pr −log PXn i (Xn i ) −nH(X1) √nαc ≥L−γn w(i) ≥FH(X1)/αc(L) ≥ 2 i=1 lim inf n→∞Pr −log PXn i (Xn i )−nH(X1) √nαc ≥L+γn w(i), (B.1) Noting that γ > 0 is a constant, from the deﬁnition of H(X), we have lim n→∞εn(ϕn, nR) = 1. (B.1) Therefore, the sufﬁciency has been proved. PROOF OF THEOREM 5.4 , y p (Necessity) Set R = R(0\|X)−γ and zn = K −nγ , where γ > 0 is an arbitrary constant. From Lemma 3.1 with βn = nR, there exists a variable-length encoder ϕn that satisﬁes where γn is speciﬁed in Lemma 5.1. where γn is speciﬁed in Lemma 5.1. Then, noting that H(X1) = H(X2) holds, from the asymp- totic normality, we have εn(ϕn, nR) < Pr 1 nαc log 1 PXn(Xn) ≥R −γ αc +K αccmax+1K −nγ , lim n→∞Pr −log PXn i (Xn i ) −nH(X1) √nσi ≥Lαc σi = ∞ Lαc σi 1 √ 2π exp −z2 2 dz = 1 − Lαc σi , for n = 1, 2, . . . . We ﬁx this variable-length encoder ϕn. Then, assuming that the source satisﬁes the strong converse property, we have for i = 1, 2. Noting that γn →0 as n →∞and the continuity of the normal distribution function, we have 1 = lim inf n→∞εn(ϕn, nR) ≤lim inf n→∞Pr 1 nαc log 1 PXn(Xn) ≥R −γ αc + lim sup n→∞ K −nγ +αccmax+1 = lim inf n→∞Pr 1 nαc log 1 PXn(Xn) ≥R −γ αc . means that lim n→∞Pr 1 nαc log 1 PXn(Xn) < R −γ αc = 0. 1 = lim inf n→∞εn(ϕn, nR) ≤lim inf n→∞Pr 1 nαc log 1 PXn(Xn) ≥R −γ αc + lim sup n→∞ K −nγ +αccmax+1 = lim inf n→∞Pr 1 nαc log 1 PXn(Xn) ≥R −γ αc . 2 i=1 lim sup n→∞ Pr −log PXn i (Xn i )−nH(X1) √nαc ≥L −γn w(i) = 1 − 2 i=1 w(i) Lαc σi . Similarly, the last term in (B.1) is evaluated as This means that This means that 2 i=1 lim inf n→∞Pr −log PXn i (Xn i )−nH(X1) √nαc ≥L + γn w(i) = 1 − 2 i=1 w(i) Lαc σi . lim n→∞Pr 1 nαc log 1 PXn(Xn) < R −γ αc = 0. Thus, R −γ αc ≤ 1 αc H(X) holds from the deﬁnition of H(X). On the other hand, from Corollary 4.1, it holds that R = R(0\|X) −γ = 1 αc H(X) −γ. Hence, we have proved Case I of the theorem. APPENDIX B PROOF OF THEOREM 5.4 APPENDIX B PROOF OF THEOREM 5.4 PROOF OF THEOREM 5.4 APPENDIX A PROOF OF THEOREM 4.2 Theorem 6.1: For 0 ≤∀ε < 1, it holds that inf R F(R) ≤ε ≤R(ε\|X) ≤inf {R \|F(R) ≤ε } , inf R F(R) ≤ε ≤R(ε\|X) ≤inf {R \|F(R) ≤ε } , The proof consists of two parts. (Sufﬁciency) We assume that (17) holds. Set R = R(0\|X)−2γ αc , where F(R) and F(R) are respectively deﬁned by p p (Sufﬁciency) We assume that (17) holds. Set R = R(0\|X)−2γ αc , F(R) := lim sup n→∞ Pr 1 nαc log 1 PXn(Xn) ≥R , F(R) := lim sup n→∞ Pr 1 nαc log 1 PXn(Xn) ≥R . where γ > 0 is an arbitrary constant. Then, from Corollary 4.1 it holds that 1 2 R = 1 αc H(X) −2γ αc . NOMURA: OVERFLOW PROBABILITY OF VARIABLE-LENGTH CODES 8205 On the other hand, from Lemma 3.2 with βn = nR, it holds that REFERENCES [20] M. Hayashi, “Second-order asymptotics in ﬁxed-length source coding and intrinsic randomness,” IEEE Trans. Inf. Theory, vol. 54, no. 10, pp. 4619–4637, Oct. 2008. [1] R. Nomura and T. 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From 2000 to 2003 he was a Research Associate with School of Science and Engineering, Waseda University, Tokyo, Japan; from 2004 to 2010, Research Associate then Assistant Professor with College of Science and Engineering, Aoyama Gakuin University, Kanagawa, Japan; from 2012 to 2018, lecturer, Associate Professor and then Professor with School of Network and Information, Senshu University, Kanagawa, Japan. ACKNOWLEDGMENT Thus, we have 1 αc H(X)−γ −γ αc ≤1 αc H(X). Notice that γ > 0 is arbitrarily, we have (X)−γ −γ αc ≤1 αc H(X). Notice that γ > 0 Thus, we have 1 αc H(X) is arbitrarily, we have Thus, we have 1 αc H(X) is arbitrarily, we have Thus, we have 1 αc H(X)−γ −γ αc ≤1 αc H(X). Notice that γ > 0 is arbitrarily, we have The author is grateful to Prof. Toshiyasu Matsushima of Waseda University for his valuable discussions and com- ments. 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https://openalex.org/W2914952739	https://link.springer.com/content/pdf/10.1007/s00420-019-01404-9.pdf	English	null	Need for recovery and different types of early labour force exit: a prospective cohort study among older workers	International archives of occupational and environmental health	2,019	cc-by	12,842	Abstract When early retirement schemes were available, strong and significant associations between NFR and LFE were observed (HR 2.79, 95% CI 1.29–6.02), whereas no significant associations were found when such schemes were unavailable. Older workers with a higher NFR also had earlier retirement intentions and lower self-assessed abilities (both physical and mental) to prolong their working life until the mandatory retirement age. Conclusions Because this study shows that NFR is a precursor of LFE among older workers, monitoring NFR is important for timely interventions aimed at reducing NFR to facilitate extended labour participation. Keywords Older workers · Need for recovery · Early labour force exit · Retirement intentions Need for recovery and different types of early labour force exit: a prospective cohort study among older workers D. Stynen1 · N. W. H. Jansen1 · J. J. M. Slangen1 · A. de Grip2 · IJ. Kant1 Received: 29 June 2016 / Accepted: 14 January 2019 / Published online: 11 February 2019 © The Author(s) 2019 Abstract Purpose This study examines the relationship between need for recovery (NFR) and labour force exit (LFE) among older workers. Different types of LFE (early retirement, work disability and unemployment) are considered, and the role of poten- tial confounding and modifying factors, including the availability of early LFE schemes, is examined. Also, associations between NFR and the intention and ability to prolong one’s working life, which are known determinants of LFE, are assessed. Methods A subsample of older workers from the Maastricht Cohort Study was examined (n = 2312). The relationship between NFR and LFE was investigated by means of Cox regression analyses. Logistic regression analyses were performed to investigate cross-sectional associations between NFR and the intention and ability to prolong working life. Purpose This study examines the relationship between need for recovery (NFR) and labour force exit (LFE) among older workers. Different types of LFE (early retirement, work disability and unemployment) are considered, and the role of poten- tial confounding and modifying factors, including the availability of early LFE schemes, is examined. Also, associations between NFR and the intention and ability to prolong one’s working life, which are known determinants of LFE, are assessed. Methods A subsample of older workers from the Maastricht Cohort Study was examined (n = 2312). The relationship between NFR and LFE was investigated by means of Cox regression analyses. Logistic regression analyses were performed to investigate cross-sectional associations between NFR and the intention and ability to prolong working life. Results Elevated NFR was associated with a higher risk of overall LFE during a 4-year follow-up period (HR 1.39, 95% CI 1.09–1.78), and specifically with a higher risk of leaving the labour force through early retirement and work disability. When early retirement schemes were available, strong and significant associations between NFR and LFE were observed (HR 2.79, 95% CI 1.29–6.02), whereas no significant associations were found when such schemes were unavailable. Older workers with a higher NFR also had earlier retirement intentions and lower self-assessed abilities (both physical and mental) to prolong their working life until the mandatory retirement age. Results Elevated NFR was associated with a higher risk of overall LFE during a 4-year follow-up period (HR 1.39, 95% CI 1.09–1.78), and specifically with a higher risk of leaving the labour force through early retirement and work disability. * D. Stynen dave.stynen@maastrichtuniversity.nl 1 Department of Epidemiology, CAPHRI School for Public Health and Primary Care, Faculty of Health, Medicine and Life Sciences, Maastricht University, P.O. Box 616, 6200 MD Maastricht, The Netherlands 2 Research Centre for Education and the Labour Market (ROA), Maastricht University, Maastricht, The Netherlands International Archives of Occupational and Environmental Health (2019) 92:683–697 https://doi.org/10.1007/s00420-019-01404-9 International Archives of Occupational and Environmental Health (2019) 92:683–697 https://doi.org/10.1007/s00420-019-01404-9 International Archives of Occupational and Environmental Health (2019) 92:683–697 https://doi.org/10.1007/s00420-019-01404-9 ORIGINAL ARTICLE ORIGINAL ARTICLE Introduction The model also suggests that long-term effects occur via a pathway of short-term effects involving insuf- ficient recovery from work (van Veldhoven and Broersen 2003; Josten and Schalk 2010).f Second, beside demographic, work environment and per- sonal factors, the association between NFR and LFE might be influenced by contextual factors on a national (e.g. social security system) and organizational level (e.g. availability of early retirement schemes), since they might affect employ- ees’ LFE decisions. Whereas the determinants of LFE discussed earlier mainly have to do with workers’ ability and motivation to work, the factors that are discussed here, rather, involve the opportunity structure surrounding LFE. Different associations might be observed between those older workers employed in companies in which early retire- ment schemes are available and those employed in com- panies in which such schemes are unavailable. Therefore, this study will also examine the role of such schemes in an explorative way. The concept of NFR represents the short-term effects of a work day and has been defined as the need to recuperate from work-induced fatigue, generally experienced after a day of work (Jansen et al. 2002). The concept involves the intensity of work-induced fatigue, both mental and physi- cal, as well as the time required to return to a normal or pre-stressor level of functioning. Employees with elevated NFR are characterized by temporary feelings of overload, lack of energy for new effort and reduced performance (van Veldhoven 2008). Elevated NFR might hinder labour par- ticipation among older workers, as it was identified as a risk factor for work-related outcomes such as absence due to sickness (de Croon et al. 2003), occupational disability (Otten et al. 2012), reduced working hours (de Raeve et al. 2009) and early retirement intentions (Oude Hengel et al. 2012). Also, NFR was considered a possible link in the pathway between high physical job demands and the risk of losing employment in older workers (Gommans et al. 2016). However, to our knowledge, direct effects of NFR on LFE of older workers have not been investigated. The present study addresses this omission and primarily aims to Finally, in this study, we also investigate associations between NFR and intentions and perceived ability to prolong working life. Introduction of pension systems worldwide (van Soest and Vonkova 2014). Consequently, public policies are aimed at increas- ing the labour participation of older workers and extend- ing the eligible retirement age (de Grip et al. 2013). Such measures have resulted in a gradual increase in the average Dutch retirement age from 61.0 years in 2007 to 64.8 years in 2017 (Statistics Netherlands 2018a). However, compared to younger workers, the labour force participation of older workers—defined as workers aged 45 years or older (World Health Organization 1993)—still remains at a lower, sub- optimal level (Statistics Netherlands 2018b). Retirement can be regarded as a transition between work roles (like turnover or changing occupations), involving a process that workers undergo and in which an individual’s ability, motivation and opportunity to work play a role (Forrier et al. 2009; de Wind et al. 2015). Despite similarities across career transitions, the specific predictors of transitions like early retirement and turnover are, to an important extent, different (Adams and Beehr 1998). Earlier studies on the specific factors affecting Due to demographic shifts, the work force in many industri- alized countries is characterized by a growing proportion of older workers approaching eligible retirement age (Organi- sation for Economic Co-operation and Development 2014). Increased longevity further undermines the sustainability Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00420-019-01404-9) contains supplementary material, which is available to authorized users. 1 Department of Epidemiology, CAPHRI School for Public Health and Primary Care, Faculty of Health, Medicine and Life Sciences, Maastricht University, P.O. Box 616, 6200 MD Maastricht, The Netherlands 1 Department of Epidemiology, CAPHRI School for Public Health and Primary Care, Faculty of Health, Medicine and Life Sciences, Maastricht University, P.O. Box 616, 6200 MD Maastricht, The Netherlands (0121 3456789) 3 International Archives of Occupational and Environmental Health (2019) 92:683–697 684 identify NFR as a potential precursor of LFE among older workers, making NFR an important indicator in occupational health surveillance concerned with the labour market par- ticipation of older workers. In our study, different types of LFE, including early retirement, work disability or becom- ing unemployed will be distinguished because, aside from pure early retirement routes, some older workers settle into retirement through a (longer) period of work inability or unemployment (Schils 2008). Introduction Omitting these alternatives— which may act as complements or substitutes—from analysis may therefore result in an underestimation of the group of older workers at risk for permanent LFE. To enhance our understanding of this subject, three additional considerations will be taken into account. First, as indicated earlier, both NFR and LFE might be influenced independently by differ- ent demographic, work environment and personal factors, and may therefore also influence the developing relationship between NFR and LFE over time. As the latter association has not been investigated earlier, we take an explorative stance to investigate this issue by including relevant con- founding and modifying factors when assessing the asso- ciation between NFR and LFE, or by performing stratified analyses, respectively. decisions like early retirement in older workers have stressed its multi-factorial etiology, involving factors from different domains (de Wind et al. 2014). A first important factor stems from the personal domain, as Robroek et al. (2013) and de Wind et al. (2014) found that poor health was associated with a higher risk of earlier exit from paid employment. Second, several work-related factors have been associated with decreased labour participation, including high psycho- logical job demands (Canivet et al. 2013), low job control (Robroek et al. 2013) and low appreciation at work (de Wind et al. 2014). In addition, demographic factors, such as a low education level, have been associated with a higher risk of early exit from paid employment (Robroek et al. 2015). Although our understanding of the determinants of labour force exit (LFE) is growing, it will in practice be challenging to identify older workers with an elevated risk for LFE, as many older workers are simultaneously exposed to multiple hindering or facilitating factors, and insight into possible indicators of where an older worker is in the process of LFE is still sparse (de Wind et al. 2015). The work load–work capacity model of van Dijk et al. (1990) may be useful in identifying ‘precursors’ of emer- gent LFE. According to this model, long-term impacts on older workers’ health and labour participation result from an imbalance between the work load (e.g. exposure to high psychological or physical job demands) and workers’ carry- ing capacity (e.g. low job control, poor health, work motiva- tion, and lack of skills) to manage these demands (Heerkens et al. 2004). Introduction Employees scoring below the cutoff point were considered to have a low–medium NFR, employees with a score of 6 or higher were considered to have a high NFR (the latter also referred to as ‘NFR caseness’). Study population Data from the Maastricht Cohort Study were used. This is an ongoing prospective cohort established in May 1998, which included 12,140 respondents from 45 different companies at baseline measurement. Data were collected by means of self-administrated questionnaires. The design of the cohort is described elsewhere in detail (Kant et al. 2003; Mohren et al. 2007). The study was conducted in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments. In this study, follow-up wave October 2008 (n = 6082) was considered the starting point for the analyses, and two follow-up waves (October 2012 and October 2014) were included, resulting in a total follow-up duration of 6 years. At wave 2012, n = 5894 questionnaires were sent out, and n = 4783 questionnaires were returned, resulting in a response rate of 81%. At wave 2014, n = 3450 question- naires were sent out to those who were not fully retired at wave 2012. A total of n = 2945 questionnaires was returned (response rate of 85%). All respondents who indicated they were fully retired at wave 2012 received a specific question- naire assessing only questions on retirement at wave 2014 (n = 1284 sent out, response rate 90%) and were therefore not eligible for inclusion. Included in the study population were all respondents aged 45–59 years who indicated they were employed at wave 2008 (n = 3932). Employees aged 59 years and older were excluded (n = 349), as they could reach the mandatory retirement age of 65 years during the follow-up period. Workers under the age of 45 years were not consid- ered older workers and were therefore excluded (n = 981). Those who were employed but also indicated they were (par- tially) receiving disability insurance or unemployment ben- efits, were retired and/or were not involved in paid employ- ment, were excluded at wave 2008 (n = 32). Also excluded at wave 2008 were those with multiple jobs (n = 118), as well as those who were employed but (partly) disabled for work, on sabbatical leave, on pregnancy or parental leave (n = 120) or were self-employed (n = 20). This resulted in a total study population of 2312 employees at wave 2008. Labour force exit Labour force exit was assessed at waves 2012 and 2014. Respondents were asked to provide an overview of their work status(es) since the preceding questionnaire. This means that at wave 2012, employees described their work status(es) from October 2008 through October 2012; and at wave 2014, employees described their work status(es) from October 2012 through October 2014. First, the current work status of the employee was assessed. Respondents could indicate that they were currently employed, self-employed, receiving disability insurance, receiving unemployment ben- efits, retired and/or not involved in paid employment. At wave 2014, respondents could additionally indicate if they were actively seeking employment. For each work status, employees indicated whether this was fully or partly appli- cable. If changes in work status had taken place since the preceding questionnaire, employees were asked to describe their earlier work status(es) using the items listed above. For each specific work status, the start and end time were requested by month and year.i Employees were classified as having exited the labour force early if they indicated non-employment status (i.e. disability insurance, unemployment benefits, retirement, no paid employment and/or actively seeking employment) at any point during the follow-up period and were not involved in any type of (self) employment. Introduction On the one hand, the intentions and perceived ability of employees to prolong their working life might be less prone to contextual opportunities for LFE as compared to actual LFE (van Soest et al. 2007). On the other hand, as we have positioned NFR in the pathway to LFE, investigat- ing its associations with other key determinants involving individuals’ appraisals of their ability and motivation to work may add further insight into older workers’ retirement decision-making processes (de Wind et al. 2015). In sum, the aim of this study is to prospectively investi- gate the relationship of NFR with overall and specific types of LFE, as well as its associations with workers’ intentions and ability to prolong working life among a sample of older 1 3 1 3 International Archives of Occupational and Environmental Health (2019) 92:683–697 685 Dutch workers, while taking into account factors from the demographic, work environment and personal domains as well as the availability of opportunities for LFE within companies. on the Experience and Evaluation of Work (QEEW) (van Veldhoven and Broersen 2003). This scale is composed of 11 dichotomous items (yes/no), which represent the short- term effects of a working day, and scores range from 0 to 11. Example items are: ‘At the end of a working day I am really feeling worn out’, ‘I find it hard to relax at the end of a working day’ and ‘When I get home, people should leave me alone for some time’. The subscale score was transformed into a scale ranging from 0 to 100: a higher score indicates a higher NFR. This transformation is in line with the QEEW procedure to increase comparability across subscales ini- tially measured on different scale points. Thus, irrespective of the subscale, ‘0’ indicates the most desirable score and ‘100’ indicates the most undesirable score. The reliability of the scale is good (Cronbach’s alpha of 0.90). A cutoff point of 6 (out of 11 items) was used to define employees with an elevated NFR, as previous research showed that work- ers who score at this point have a higher risk of developing psychological complaints than those who score below that cutoff point (Broersen et al. 2004; van der Starre et al. 2013). Need for recovery The total scores for the psychological job demands (range 12–48) and decision latitude (range 24–96) subscales were grouped into tertiles of low, medium and high levels of psy- chological job demands and decision latitude for the strati- fied analyses. Supervisor social support was measured with a subscale of the JCQ, resulting in a range of 4–16 (Cron- bach’s alpha of 0.86). The total score of this subscale was dichotomized at the median, resulting in low and high super- visor social support for the stratified analyses. Self-perceived health was measured with one item from the SF-36 Health Survey (Aaronson et al. 1998): ‘How would you rate your health in general?’. In line with an earlier study by Dalstra et al. (2002), the response options ‘excellent’, ‘very good’ and ‘good’ were grouped into ‘good health’, and ‘moder- ate’ or ‘bad’ were grouped into ‘moderate–poor health’. To assess spousal retirement, employees first indicated whether they currently have a partner (yes/no). Employees who indicated that they currently have a partner were asked to indicate whether their partner was currently retired (yes/ no). These items were recoded into two categories: a ‘retired spouse’ category, and a ‘non-retired spouse’ category that Beside full LFE, it is possible to leave the labour force gradually, for instance by phased retirement combined with part-time employment. Therefore, in the analyses investigat- ing the specific types of LFE, employees were also consid- ered to have exited the labour force early if they indicated reasons for current non-employment and reasons for being partly (self)employed simultaneously. Employees were clas- sified as ‘retired early’ if they indicated they were fully or partly retired during any follow-up period. Employees who indicated receiving any type of disability insurance during follow-up were classified as being disabled for work, and employees who indicated receiving any type of unemploy- ment benefits during follow-up were classified as being unemployed. Retirement intentions and ability to prolong working life Items on intentions to prolong working life were first assessed at wave 2012 and were based on a study by van Dam et al. (2009). The first item measured an employee’s intention to prolong working life until reaching the man- datory retirement age: ‘It is my intention to keep working until I reach the mandatory retirement age’. A second item, measuring an employee’s intention to prolong working life beyond the mandatory retirement age, was assessed with a similar statement. The ability of employees to prolong work- ing life was assessed with two items based on a Koppes et al. (2011) study. The first item assessed the physical ability to prolong working life: ‘I think I am physically able to work in my current job until reaching the mandatory retirement age’. The second item assessed mental ability with a similar statement. All four items were scored on a five-point Likert scale, with response options ranging from ‘strongly agree’ to ‘strongly disagree’. These five response categories were recoded into two categories; a ‘(strongly) agree’ category and a ‘neutral–(strongly) disagree’ category. Early labour force exit schemes Access to LFE schemes was first assessed at wave 2012. Employees were asked to indicate ‘whether within the com- pany at which you are currently employed, schemes exist which facilitate labour force exit before reaching the manda- tory retirement age’. Response options were ‘no’, ‘yes’, ‘not applicable’ and ‘don’t know’. Employees indicating that such schemes exist within their company were asked to indicate at which age they were eligible to make use of these schemes. Need for recovery (2002), the response options ‘excellent’, ‘very good’ and ‘good’ were grouped into ‘good health’, and ‘moder- ate’ or ‘bad’ were grouped into ‘moderate–poor health’. To assess spousal retirement, employees first indicated whether they currently have a partner (yes/no). Employees who indicated that they currently have a partner were asked to indicate whether their partner was currently retired (yes/ no). These items were recoded into two categories: a ‘retired spouse’ category, and a ‘non-retired spouse’ category that included employees with a currently employed partner and employees without a partner. Attachment to work for finan- cial resources was assessed with a self-formulated item: ‘There are several reasons for employment. Could you indi- cate to which extent financial resources determine your level 2012), all confounders for these analyses were assessed at that wave. If possible, for descriptive and stratifica- tion purposes, factors were further recoded into categori- cal variables. Education level was measured at wave 1998 and recoded into three levels: low (primary school, lower vocational education), medium (lower secondary school, intermediate vocational school, upper secondary school) or high (higher vocational school, university). Our meas- ure for education level therefore reflects educational attain- ment at labour market entry. The number of working hours per week was measured by one item that included response options > 40, 36–40, 26–35, 16–25 or < 16 h per week, which were recoded into three categories: ≥ 36, 26–35 or ≤ 25 h per week. To assess work schedule, employees first indicated whether they were engaged in day or shift work. Next, employees performing shift work could indicate the specific type of shift in which they were engaged. Employ- ees who indicated they were engaged in shift work and/or indicated a specific shift type were classified as shift workers and all other employees were classified as day workers. For the purpose of stratification, age was also recoded in three categories: 45–49, 50–54 and 55–59 years. Psychological job demands were measured with a scale from the validated Dutch version of the Job Content Questionnaire (JCQ) (Kar- asek 1985) (Cronbach’s alpha of 0.71). To measure decision latitude, two subscales from the JCQ were combined: skill discretion and decision authority (Cronbach’s alpha of 0.80). Need for recovery Need for recovery was measured at wave 2008 and was assessed using a subscale from the Dutch Questionnaire 1 3 International Archives of Occupational and Environmental Health (2019) 92:683–697 686 2012), all confounders for these analyses were assessed at that wave. If possible, for descriptive and stratifica- tion purposes, factors were further recoded into categori- cal variables. Education level was measured at wave 1998 and recoded into three levels: low (primary school, lower vocational education), medium (lower secondary school, intermediate vocational school, upper secondary school) or high (higher vocational school, university). Our meas- ure for education level therefore reflects educational attain- ment at labour market entry. The number of working hours per week was measured by one item that included response options > 40, 36–40, 26–35, 16–25 or < 16 h per week, which were recoded into three categories: ≥ 36, 26–35 or ≤ 25 h per week. To assess work schedule, employees first indicated whether they were engaged in day or shift work. Next, employees performing shift work could indicate the specific type of shift in which they were engaged. Employ- ees who indicated they were engaged in shift work and/or indicated a specific shift type were classified as shift workers and all other employees were classified as day workers. For the purpose of stratification, age was also recoded in three categories: 45–49, 50–54 and 55–59 years. Psychological job demands were measured with a scale from the validated Dutch version of the Job Content Questionnaire (JCQ) (Kar- asek 1985) (Cronbach’s alpha of 0.71). To measure decision latitude, two subscales from the JCQ were combined: skill discretion and decision authority (Cronbach’s alpha of 0.80). The total scores for the psychological job demands (range 12–48) and decision latitude (range 24–96) subscales were grouped into tertiles of low, medium and high levels of psy- chological job demands and decision latitude for the strati- fied analyses. Supervisor social support was measured with a subscale of the JCQ, resulting in a range of 4–16 (Cron- bach’s alpha of 0.86). The total score of this subscale was dichotomized at the median, resulting in low and high super- visor social support for the stratified analyses. Self-perceived health was measured with one item from the SF-36 Health Survey (Aaronson et al. 1998): ‘How would you rate your health in general?’. In line with an earlier study by Dalstra et al. Statistical analysis First, the prevalence of the baseline demographic, work environment and personal characteristics and the prevalence of NFR caseness in the study population at study baseline were described. The distribution of NFR was skewed to the right, so Poisson regression analyses were conducted and pairwise comparisons were investigated by means of the Wald Chi square test to test for differences in the mean NFR scores for all characteristics separately. p y The relationship between NFR and overall LFE was investigated by means of Cox regression analyses. In all analyses, both NFR caseness and the continuous NFR score were investigated. Four models were investigated: a crude model (model 1), a model adjusting for demographic con- founders (model 2), a model additionally adjusting for con- founders of the work environment (model 3) and a model additionally adjusting for confounders from the personal domain (model 4). To investigate the role of these factors as effect-modifiers, results were stratified for each characteristic from these three domains. Hazard ratios (HR) and 95% con- fidence intervals (CI) were reported. The potential moderat- ing role of all characteristics was tested stepwise by adding the interaction term—based on the raw scores—between a characteristic and NFR scores or NFR caseness to the Cox regression analysis, respectively, whenever the HR was sig- nificant in at least one of the strata under consideration, for all models.The role of access to early retirement schemes in the risk of overall LFE was investigated with Cox regres- sion analysis. The starting point for this analysis was the 2012 wave, which is when such schemes were first assessed. Here, the same inclusion and exclusion criteria as mentioned earlier were used, except that the information of the 2012 wave was used and criteria applied to the 2012 respondents and only employees aged 60–63 years were included. The investigation of this age category was justified because the majority of employees in the Netherlands (within our study population, this majority was 89%) have to be 60 years or older to be eligible for LFE schemes (Euwals et al. 2011). Employees who indicated ‘not applicable’ or ‘don’t know’ on the item regarding the availability of LFE schemes were excluded. This resulted in a study population of 204 employ- ees. The four models mentioned above were investigated, and all confounders for this analysis were assessed at wave 2012. Confounding and modifying factors All factors were measured at wave 2008, except for edu- cation level. When analyses concern another baseline (i.e. 1 3 International Archives of Occupational and Environmental Health (2019) 92:683–697 687 of Cox regression. For all three specific types of LFE, the four models were investigated. of attachment to work?’ Response options included: ‘very attached’, ‘attached’, ‘somewhat attached’ and ‘not attached’ to work. The two latter response options were recoded into one category: ‘not to somewhat attached’. g For all Cox regression analyses performed, time-to-event was modelled monthly, based on the month and year an employee indicated that an event (e.g. losing employment) had occurred for the first time. Employees who reported that an event had occurred before or after the investigated follow-up period (e.g. a forthcoming early retirement date) were excluded for that particular analysis. The proportional hazard assumption was investigated for all Cox regression models and was met in the large majority of the models. As the intention and ability to prolong working life were first assessed at wave 2012, this wave was considered the starting point for these analyses. The same inclusion and exclusion criteria as mentioned earlier were used, except that these criteria were based on the 2012 respondents, inevitably resulting in a somewhat smaller study population (n = 1607). Cross-sectional associations between NFR and intention and ability to prolong working life were investigated by means of logistic regression analyses. Odds ratios (OR) and 95% CI were reported. For all items, the odds of agreeing with the statement were investigated: a larger OR implies higher odds of agreeing with the statement. To analyse the data, IBM SPSS Statistics 23.0 was used, and a p value of < 0.05 was considered statistically significant. Significant coefficients (HR and OR) have been marked in bold in all tables. If the lower limit of a 95% CI in bold contains 1.00, the lower limit is ≥ 1.001 and the corresponding p value is < 0.05. If the upper limit of a 95% CI in bold contains 1.00, the upper limit is ≤ 0.999 and the corresponding p value is < 0.05. Descriptives The baseline characteristics of the study population are presented in Table 1. The table demonstrates statistically significant differences in mean NFR scores among different demographic, work environment and personal factors. Statistical analysis Next, associations between NFR and specific types of LFE (risk of early retirement, becoming disabled for work and unemployment) were separately investigated by means Risk of early labour force exit In Table 2, the results on the association of NFR with overall risk of LFE are presented. In models 1 and 2, NFR case- ness was found to be associated with a higher risk of LFE (in model 2: HR 1.29, 95% CI 1.01–1.65; p < 0.05). The NFR continuous scores remained statistically significantly associated with a higher risk of LFE throughout all models (in model 4: HR 1.01, 95% CI 1.00–1.01; p < 0.05). The effect modification on these associations by significant demographic, personal and work environment factors was tested, but no significant interactions were found. However, 1 International Archives of Occupational and Environmental Health (2019) 92:683–697 688 Table 1 Description of and mean NFR scores according to demographics, work and personal characteristics of the study population October 2008 (n = 2312) N/A not applicable (%) NFR mean score (0–100) p value Demographics Gender Male 72.7 32.57 0.860 Female 27.3 32.29 Age 45–49 years 36.2 29.51 0.016 (45–49 vs. 50–54) 50–54 years 36.8 33.51 0.387 (50–54 vs. 55–59) 55–59 years 27.1 35.14 0.002 (45–49 vs. 55–59) Education level Low 15.2 36.17 0.014 (low vs. medium) Medium 43.9 30.67 0.121 (medium vs. high) High 40.8 33.10 0.177 (high vs. low) Work environment Number of working hours per week ≥ 36 71.0 32.78 0.457 (≥ 36 vs. 26–35) 26–35 17.6 34.26 0.005 (26–35 vs. ≤25) ≤ 25 11.5 26.78 0.005 (≥ 36 vs. ≤25) Work schedule Shift work 17.9 36.70 0.009 Day work 82.1 31.48 Psychological job demands Low 43.2 22.13 < 0.0001 (low vs. medium) Medium 30.8 33.92 < 0.0001 (medium vs. high) High 26.0 48.14 < 0.0001 (high vs. low) Decision latitude Low 29.6 37.57 0.002 (low vs. medium) Medium 36.2 31.96 0.048 (medium vs. high) High 34.1 28.67 < 0.0001 (high vs. low) Supervisor support Low 41.5 38.74 < 0.0001 High 58.5 28.18 Personal and health Self-perceived health Moderate-poor 12.9 57.94 < 0.0001 Good 87.1 28.72 Spousal retirement Yes 2.2 32.17 0.433 No 97.8 36.26 Attachment to work for financial resources Very attached 37.3 36.79 < 0.0001 (very attached vs. attached) Attached 56.0 30.45 0.058 (attached vs. not-somewhat attache Not–somewhat attached 6.6 25.46 < 0.0001 (very attached vs. Risk of early labour force exit In case the lower limit of a bold marked 95% CI contains 1.00, the lower limit is ≥ 1.001 and the corresponding p value is < 0.05 Model 1: crude Model 2: adjusted for gender, age and education level Model 3: additionally adjusted for number of working hours per week, work schedule, psychological job demands, decision latitude and supervi- sor social support Model 4: additionally adjusted for self-perceived health, spousal retirement and attachment to work for financial resources a The variable of stratification was not adjusted for in the models Table 2 (continued) Model 1 Model 2 Model 3 Model 4 HR 95% CI HR 95% CI HR 95% CI HR 95% CI NFR caseness 0.75 0.16–3.54 0.45 0.08–2.50 0.08 0.00–1.56 0.36 0.01–13.84 NFR continuous score 1.00 0.98–1.02 1.00 0.98–1.02 0.98 0.95–1.02 1.03 0.98–1.09 No (n = 2199) NFR caseness 1.39 1.08–1.79 1.27 0.98–1.64 1.21 0.92–1.61 1.23 0.92–1.64 NFR continuous score 1.01 1.00–1.01 1.01 1.00–1.01 1.00 1.00–1.01 1.01 1.00–1.01 Statistically significant HRs + 95% CIs are marked in bold. In case the lower limit of a bold marked 95% CI contains 1.00, the lower limit is ≥ 1.001 and the corresponding p value is < 0.05 Model 1: crude M d l 2 dj d f d d d i l l Model 3: additionally adjusted for number of working hours per week, work schedule, psychological job demands, de or social supporti Model 4: additionally adjusted for self-perceived health, spousal retirement and attachment to work for financial resources a The variable of stratification was not adjusted for in the models associations between NFR caseness and early retirement were not observed. Both NFR caseness and a higher NFR continuous score were statistically significantly associ- ated with a higher chance of work disability in all four models (in model 4: for NFR caseness: HR 2.76, 95% CI 1.08–7.05; p < 0.05; for NFR continuous score: HR 1.02, 95% CI 1.00–1.03; p < 0.05). However, no statistically sig- nificant associations between NFR caseness, or NFR con- tinuous score and unemployment were observed in any of the models. when stratifying for different demographic characteristics, different associations between NFR and LFE were observed. The NFR was a statistically significant risk factor for LFE among men, but not among women. Different associations between NFR and LFE were also observed among the dif- ferent work environment characteristics. Risk of early labour force exit Among employees reporting low levels of supervisor social support, NFR was statistically significantly associated with LFE, whereas such findings were not observed among employees with high lev- els of supervisor social support. In the personal domain, NFR was a statistically significant risk factor for LFE among employees who did not have a retired spouse, whereas such associations were not observed among employees who did have a retired spouse. Table 5 presents cross-sectional associations between NFR and the intention and ability to prolong working life. In model 1, NFR caseness was associated with lower odds of agreeing with the statement ‘It is my intention to keep working until I reach the mandatory retirement age’ (OR 0.77, 95% CI 0.60–0.99; p < 0.05). For the NFR continuous score, the odds were also statistically significant in models 1, 2 and 4 (OR 1.00. 95% CI 0.99–1.00; p < 0.05), but not in model 3. Both NFR caseness and a higher NFR continuous score were statistically significantly associated with lower odds of agreeing with the statement, ‘I think I am physically able to work in my current job until reaching the manda- tory retirement age’ in all four models (in model 4: for NFR caseness: OR 0.50. 95% CI 0.37–0.68; p < 0.05; for NFR continuous score: OR 0.99, 95% CI 0.98–0.99; p < 0.05). In all four models, both NFR caseness and a higher NFR con- tinuous score were statistically significantly associated with a lower odds of agreeing with the statement ‘I think I am mentally able to work in my current job until reaching the mandatory retirement age’ (in model 4: for NFR caseness: OR 0.49, 95% CI 0.37–0.65; p < 0.05; for NFR continuous score: OR 0.99, 95% CI 0.98–0.99; p < 0.05). No statisti- cally significant associations between NFR and intentions to Results regarding the role of availability of LFE schemes in the association between NFR and overall LFE are pre- sented in Table 3. No significant effect modification was found for the availability of LFE schemes in the relationship between NFR and overall LFE. Risk of early labour force exit not-somewha Need for recovery caseness No 74.6 N/A Yes 25.4 Table 1 Description of and mean NFR scores according to demographics, work and personal characteristics of the study population at baseline October 2008 (n = 2312) (%) NFR mean score p value Table 1 Description of and mean NFR scores according to demographics, work and personal characteristics of t October 2008 (n = 2312) 1 International Archives of Occupational and Environmental Health (2019) 92:683–697 689 1 3 Table 2 Hazard ratios and 95% confidence intervals for the chance of early labour force exit according to NFR caseness and NFR continuous score (October 2008–October 2014) Model 1 Model 2 Model 3 Model 4 HR 95% CI HR 95% CI HR 95% CI HR 95% CI Total study population (n = 2292) NFR caseness 1.39 1.09–1.78 1.29 1.01–1.65 1.24 0.94–1.62 1.22 0.92–1.62 NFR continuous score 1.01 1.00–1.01 1.01 1.00–1.01 1.00 1.00–1.01 1.01 1.00–1.01 Demographics Gendera Men (n = 1651) NFR caseness 1.46 1.11–1.93 1.38 1.04–1.81 1.33 0.98–1.79 1.30 0.94–1.78 NFR continuous score 1.01 1.00–1.01 1.01 1.00–1.01 1.01 1.00–1.01 1.01 1.00–1.01 Women (n = 623) NFR caseness 1.10 0.64–1.91 0.96 0.55–1.67 0.81 0.43–1.51 0.78 0.40–1.56 NFR continuous score 1.00 1.00–1.01 1.00 0.99–1.01 1.00 0.99–1.01 1.00 0.99–1.01 Age categorya 45–49 years (n = 831) NFR caseness 1.79 1.01–3.17 1.78 1.00–3.16 1.73 0.93–3.20 1.67 0.88–3.17 NFR continuous score 1.00 1.00–1.02 1.01 1.00–1.02 1.01 1.00–1.02 1.01 1.00–1.02 50–54 years (n = 843) NFR caseness 1.12 0.66–1.88 1.15 0.68–1.91 1.05 0.58–1.88 1.10 0.60–2.02 NFR continuous score 1.00 0.99–1.01 1.00 1.00–1.01 1.00 0.99–1.01 1.00 0.99–1.01 55–59 years (n = 618) NFR caseness 1.21 0.88–1.67 1.20 0.87–1.66 1.16 0.82–1.65 1.13 0.78–1.65 NFR continuous score 1.00 1.00–1.01 1.00 1.00–1.01 1.00 1.00–1.01 1.00 1.00–1.01 Education levela Low (n = 336) NFR caseness 1.38 0.76–2.51 1.47 0.81–2.67 1.89 0.90–3.98 1.95 0.88–4.33 NFR continuous score 1.00 0.99–1.01 1.00 1.00–1.01 1.01 1.00–1.02 1.01 1.00–1.02 Medium (n = 987) NFR caseness 1.51 1.04–2.20 1.35 0.93–1.97 1.26 0.84–1.90 1.29 0.83–1.99 NFR continuous score 1.01 1.00–1.01 1.01 1.00–1.01 1.01 1.00–1.01 1.01 1.00–1.01 High (n = 921) NFR caseness 1.31 0.89–1.93 1.11 0.75–1.65 1.06 0.70–1.61 0.99 0.63–1.55 NFR continuous score 1.01 1.00–1.01 1.00 1.00–1.01 1.00 1.00–1.01 1.00 1.00–1.01 Work environment Number of working hours per weeka ≥36 (n = 1584) NFR caseness 1.45 1.09–1.92 1.34 1.00–1.78 1.30 0.96–1.78 1.23 0.88–1.72 NFR continuous score 1.01 1.00–1.01 1.01 1.00–1.01 1.01 1.00–1.01 1.00 1.00–1.01 26–35 (n = 390) NFR caseness 1.58 0.93–2.69 1.34 0.77–2.32 1.31 0.73–2.36 1.48 0.80–2.74 NFR continuous score 1.01 1.00–1.01 1.00 1.00–1.01 1.00 0.99–1.01 1.00 1.00–1.01 ≤ 25 (n = 256) NFR caseness 0.23 0.03–1.69 0.29 0.04–2.22 0.21 0.02–1.85 0.24 0.03–2.06 NFR continuous score 1.00 0.99–1.02 1.01 0.99–1.02 1.01 0.99–1.03 1.01 0.99–1.03 Work schedulea Shift work (n = 396) NFR caseness 1.26 0.70–2.26 1.17 0.65–2.11 1.13 0.58–2.20 1.06 0.51–2.19 NFR continuous score 1.00 1.00–1.01 1.00 1.00–1.01 1.00 1.00–1.01 1.00 0.99–1.01 Day work (n = 1839) NFR caseness 1.47 1.12–1.92 1.34 1.02–1.76 1.30 0.96–1.74 1.28 0.94–1.76 Table 2 Hazard ratios and 95% confidence intervals for the chance of early labour force exit according to NFR score (October 2008–October 2014) ratios and 95% confidence intervals for the chance of early labour force exit according to NFR caseness and NFR 008–October 2014) HR Total study population (n = 2292) NFR caseness 1.39 NFR continuous score 1.01 Demographics Gendera Men (n = 1651) NFR caseness 1.46 NFR continuous score 1.01 Women (n = 623) NFR caseness 1.10 NFR continuous score 1.00 Age categorya 45–49 years (n = 831) NFR caseness 1.79 NFR continuous score 1.00 50–54 years (n = 843) NFR caseness 1.12 NFR continuous score 1.00 55–59 years (n = 618) NFR caseness 1.21 NFR continuous score 1.00 Education levela Low (n = 336) NFR caseness 1.38 NFR continuous score 1.00 Medium (n = 987) NFR caseness 1.51 NFR continuous score 1.01 High (n = 921) NFR caseness 1.31 NFR continuous score 1.01 Work environment Number of working hours per weeka ≥36 (n = 1584) NFR caseness 1.45 NFR continuous score 1.01 26–35 (n = 390) NFR caseness 1.58 NFR continuous score 1.01 ≤ 25 (n = 256) NFR caseness 0.23 NFR continuous score 1.00 Work schedulea Shift work (n = 396) NFR caseness 1.26 NFR continuous score 1.00 Day work (n = 1839) NFR caseness 1.47 1 International Archives of Occupational and Environmental Health (2019) 92:683–697 690 Table 2 (continued) Model 1 Model 2 Model 3 Model 4 HR 95% CI HR 95% CI HR 95% CI HR 95% CI NFR continuous score 1.01 1.00–1.01 1.01 1.00–1.01 1.01 1.00–1.01 1.01 1.00–1.01 Psychological job demandsa Low (n = 973) NFR caseness 1.42 0.93–2.18 1.30 0.84–2.01 1.22 0.78–1.90 1.21 0.76–1.94 NFR continuous score 1.01 1.00–1.01 1.01 1.00–1.01 1.00 1.00–1.01 1.00 1.00–1.01 Medium (n = 690) NFR caseness 1.50 0.93–2.40 1.38 0.85–2.22 1.34 0.82–2.19 1.41 0.83–2.38 NFR continuous score 1.01 1.00–1.02 1.01 1.00–1.02 1.01 1.00–1.01 1.01 1.00–1.02 High (n = 584) NFR caseness 1.62 1.03–2.54 1.34 0.85–2.13 1.19 0.73–1.94 1.13 0.67–1.92 NFR continuous score 1.01 1.00–1.01 1.01 1.00–1.01 1.00 1.00–1.01 1.00 0.99–1.01 Decision latitudea Low (n = 668) NFR caseness 1.28 0.86–1.91 1.20 0.80–1.80 1.24 0.79–1.94 1.21 0.75–1.96 NFR continuous score 1.00 1.00–1.01 1.00 1.00–1.01 1.00 1.00–1.01 1.00 1.00–1.01 Medium (n = 819) NFR caseness 1.46 0.99–2.16 1.24 0.84–1.84 1.28 0.84–1.95 1.20 0.76–1.88 NFR continuous score 1.01 1.00–1.01 1.01 1.00–1.01 1.01 1.00–1.01 1.01 1.00–1.01 High (n = 773) NFR caseness 1.29 0.76–2.19 1.24 0.73–2.11 1.27 0.72–2.23 1.32 0.73–2.39 NFR continuous score 1.01 1.00–1.01 1.01 1.00–1.01 1.01 1.00–1.02 1.01 1.00–1.02 Supervisor social supporta Low (n = 944) NFR caseness 1.54 1.07–2.19 1.38 0.96–1.97 1.32 0.90–1.96 1.34 0.88–2.02 NFR continuous score 1.01 1.00–1.01 1.01 1.00–1.01 1.01 1.00–1.01 1.01 1.00–1.01 High (n = 1330) NFR caseness 1.28 0.91–1.81 1.24 0.88–1.75 1.16 0.79–1.68 1.13 0.75–1.69 NFR continuous score 1.00 1.00–1.01 1.00 1.00–1.01 1.00 1.00–1.01 1.00 1.00–1.01 Personal characteristics Self-perceived healtha Moderate–poor (n = 293) NFR caseness 1.06 0.59–1.91 1.03 0.56–1.87 1.03 0.53–2.00 0.80 0.40–1.63 NFR continuous score 1.00 0.99–1.01 1.00 0.99–1.01 1.00 0.99–1.01 1.00 0.99–1.01 Good (n = 1995) NFR caseness 1.43 1.09–1.89 1.33 1.01–1.76 1.27 0.93–1.72 1.31 0.96–1.79 NFR continuous score 1.01 1.00–1.01 1.01 1.00–1.01 1.01 1.00–1.01 1.01 1.00–1.01 Attachment to work for financial resourcesa Not-somewhat attached (n = 153) NFR caseness 0.91 0.35–2.38 1.00 0.37–2.69 1.28 0.45–3.67 1.28 0.36–4.53 NFR continuous score 1.00 0.99–1.01 1.00 0.99–1.01 1.00 0.99–1.02 1.00 0.99–1.02 Attached (n = 1281) NFR caseness 1.60 1.14–2.24 1.47 1.05–2.06 1.31 0.90–1.91 1.34 0.90–1.98 NFR continuous score 1.01 1.00–1.01 1.01 1.00–1.01 1.01 1.00–1.01 1.01 1.00–1.01 Very attached (n = 854) NFR caseness 1.27 0.87–1.88 1.16 0.79–1.72 1.19 0.78–1.82 1.07 0.68–1.68 NFR continuous score 1.00 1.00–1.01 1.00 1.00–1.01 1.00 1.00–1.01 1.00 1.00–1.01 Spousal retirementa Yes (n = 50) Table 2 (continued) Model 1 HR NFR continuous score 1.01 Psychological job demandsa Low (n = 973) NFR caseness 1.42 NFR continuous score 1.01 Medium (n = 690) NFR caseness 1.50 NFR continuous score 1.01 High (n = 584) NFR caseness 1.62 NFR continuous score 1.01 Decision latitudea Low (n = 668) NFR caseness 1.28 NFR continuous score 1.00 Medium (n = 819) NFR caseness 1.46 NFR continuous score 1.01 High (n = 773) NFR caseness 1.29 NFR continuous score 1.01 Supervisor social supporta Low (n = 944) NFR caseness 1.54 NFR continuous score 1.01 High (n = 1330) NFR caseness 1.28 NFR continuous score 1.00 Personal characteristics Self-perceived healtha Moderate–poor (n = 293) NFR caseness 1.06 NFR continuous score 1.00 Good (n = 1995) NFR caseness 1.43 NFR continuous score 1.01 Attachment to work for financial reso Not-somewhat attached (n = 153) NFR caseness 0.91 NFR continuous score 1.00 Attached (n = 1281) NFR caseness 1.60 NFR continuous score 1.01 Very attached (n = 854) NFR caseness 1.27 NFR continuous score 1.00 Spousal retirementa Yes (n = 50) 1 3 International Archives of Occupational and Environmental Health (2019) 92:683–697 International Archives of Occupational and Environmental Health (2019) 92:683–697 691 Table 2 (continued) Model 1 Model 2 Model 3 Model 4 HR 95% CI HR 95% CI HR 95% CI HR 95% CI NFR caseness 0.75 0.16–3.54 0.45 0.08–2.50 0.08 0.00–1.56 0.36 0.01–13.84 NFR continuous score 1.00 0.98–1.02 1.00 0.98–1.02 0.98 0.95–1.02 1.03 0.98–1.09 No (n = 2199) NFR caseness 1.39 1.08–1.79 1.27 0.98–1.64 1.21 0.92–1.61 1.23 0.92–1.64 NFR continuous score 1.01 1.00–1.01 1.01 1.00–1.01 1.00 1.00–1.01 1.01 1.00–1.01 Statistically significant HRs + 95% CIs are marked in bold. Risk of early labour force exit However, stratified analy- ses showed that among employees who indicated that LFE schemes were available, both NFR caseness and a higher NFR continuous score were associated with a statistically significantly higher risk for LFE in models 1 and 2 (for NFR caseness: HR 3.11, 95% CI 1.42–6.80; p < 0.05; for NFR continuous score: HR 1.02, 95% CI 1.00–1.03; p < 0.05), whereas among employees who indicated that LFE schemes were not available, no statistically significant associations between NFR and LFE were observed in any of the models.i In Table 4, associations between NFR and specific types of LFE are presented. A higher NFR continuous score was statistically significantly associated with a higher chance of early retirement in all models (in model 4: HR 1.01, 95% CI 1.00–1.01; p < 0.05), whereas statistically significant 1 3 692 International Archives of Occupational and Environmental Health (2019) 92:683–697 prolong working life beyond the mandatory retirement age were observed in any of the models. Table 3 Hazard ratios and 95% confidence intervals for the chance of LFE according to the availability of schemes for early labour force exit: among employees aged 60–63, according to NFR caseness and NFR continuous score (October 2012–October 2014) Statistically significant HRs + 95% CIs are marked in bold. In case the lower limit of a bold marked 95% CI contains 1.00 the lower limit is ≥ 1.001 and the corresponding p value is < 0.05 Model 1: crude Model 2: adjusted for gender, age and education level Model 3: additionally adjusted for number of working hours per week, work schedule, psychological job demands, decision latitude and supervisor social support Model 4: additionally adjusted for self-perceived health, spousal retirement and attachment to work for financial resources Schemes for LFE available Model 1 Model 2 Model 3 Model 4 No (n = 97) Yes (n = 107) No Yes No Yes No Yes HR 95% CI HR 95% CI HR 95% CI HR 95% CI HR 95% CI HR 95% CI HR 95% CI HR 95% CI Total study population (n = 204) NFR caseness 1.31 0.55–3.14 2.79 1.29–6.02 1.63 0.62–4.26 3.11 1.42–6.80 1.31 0.39–4.35 1.73 0.70–4.28 1.64 0.45–6.03 2.30 0.80–6.5 NFR continuous score 1.00 0.99–1.02 1.01 1.00–1.02 1.00 0.99–1.02 1.02 1.00–1.03 1.00 0.98–1.02 1.00 1.00–1.02 1.00 0.98–1.02 1.01 1.00–1.0 Discussion The aim of this study was to investigate the possibility that NFR is a precursor of older workers’ labour partici- pation. Longitudinal relationships of NFR with (types of) LFE and cross-sectional associations between NFR with intentions and ability to prolong working life were studied. Key factors from the demographic, work environ- ment and personal domains were considered when study- ing these associations. The main finding of this study was that NFR is associated with an increased probability of overall LFE over time in workers aged 45 years and older. Higher levels of NFR (as a continuous measure) remained significantly related with LFE when adjusted for factors from all domains. However, NFR caseness was not sig- nificantly related with LFE when adjustments were made for factors from the work environment and the personal domain. In line with the work load–work capacity model of van Dijk et al. (1990), this finding suggests that long- term effects like LFE may occur via a pathway of short- term effects involving insufficient recovery from work (van Veldhoven and Broersen 2003). In this sense, NFR can be seen as a ‘precursor’ of LFE. Personal, demographic and work environment factors were studied to further enhance insight into the factors that may play a role in the relationship between NFR and LFE. Effect modifica- tion could not be formally established. However, from a descriptive statistics perspective, stratified analyses ren- dered an indication of differences according to some of the moderators. First, associations between NFR and over- all LFE were only observed among men, but not among women. Second, findings suggest significant associations between NFR and LFE when older workers are exposed to low supervisor support. However, these associations were only significant when they were not adjusted for addi- tional confounding factors. These findings suggest that the strength of the relationship between NFR and LFE may depend more upon static demographic characteristics like gender, as well as on unfavourable working conditions. Third, although the direction of the association between NFR and overall LFE was similar among employees with and without organizational schemes for LFE, only statis- tically significant associations between NFR and overall LFE were found only among employees who had such opportunities. Therefore, it is likely that the choice for LFE is at least partially embedded in the broader organi- zational context. Discussion In case the upper limit of a bold marked 95% CI contains 1.00 the upper limit is ≤ 0.999 and the corresponding p value is < 0.05 Model 1: crude Model 2: adjusted for gender, age and education level Model 3: additionally adjusted for number of working hours per week, work schedule, psychological job demands, decision latitude and supervisor social support Model 4: additionally adjusted for self-perceived health, spousal retirement and attachment to work for financial resources a OR < 1 implies less agreement with the statement Model 1 Model 2 Model 3 Model 4 ORa 95% CI OR 95% CI OR 95% CI OR 95% CI It is my intention to keep working until I reach the mandatory retirement age NFR caseness 0.77 0.60–0.99 0.80 0.62–1.02 0.84 0.64–1.10 0.76 0.58–1.01 NFR continuous score 1.00 0.99–1.00 1.00 0.99–1.00 1.00 0.99–1.00 1.00 0.99–1.00 I think I am physically able to work in my current job until reaching the mandatory retirement age NFR caseness 0.36 0.28–0.46 0.36 0.28–0.47 0.45 0.34–0.61 0.50 0.37–0.68 NFR continuous score 0.98 0.98–0.99 0.98 0.98–0.99 0.99 0.98–0.99 0.99 0.98–0.99 I think I am mentally able to work in my current job until reaching the mandatory retirement age NFR caseness 0.36 0.28–0.46 0.37 0.28–0.47 0.45 0.34–0.59 0.49 0.37–0.65 NFR continuous score 0.98 0.98–0.99 0.98 0.98–0.99 0.99 0.98–0.99 0.99 0.98–0.99 It is my intention to keep working beyond the mandatory retirement age NFR caseness 0.90 0.61–1.33 0.90 0.61–1.34 0.95 0.61–1.47 0.91 0.57–1.43 NFR continuous score 1.00 1.00–1.01 1.00 1.00–1.01 1.00 1.00–1.01 1.00 1.00–1.01 Table 4 Hazard ratios and 95% confidence intervals for specific types of early labour force exit: early retirement, work disability and unemployment, according to NFR caseness and NFR continuous score (October 2008–October 2014) Statistically significant HRs + 95% CIs are marked in bold. Table 5 Odds ratios and 95% confidence intervals for the different items on retirement intentions and ability to prolong working life according to NFR caseness and NFR continuous score (October 2012) (n = 1607) Discussion In case the lower limit of a bold marked 95% CI contains 1.00 the lower limit is ≥ 1.001 and the corresponding p value is < 0.05 Specific type of labour force exit Model 1 Model 2 Model 3 Model 4 HR 95% CI HR 95% CI HR 95% CI HR 95% CI Early retirement NFR caseness 1.37 0.94–2.00 1.29 0.88–1.89 1.31 0.86–1.98 1.22 0.78–1.90 NFR continuous score 1.01 1.00–1.01 1.01 1.00–1.01 1.01 1.00–1.01 1.01 1.00–1.01 Work disability NFR caseness 2.44 1.14–5.22 2.39 1.11–5.11 3.04 1.26–7.33 2.76 1.08–7.05 NFR continuous score 1.01 1.00–1.03 1.01 1.00–1.03 1.02 1.00–1.03 1.02 1.00–1.03 Unemployment NFR caseness 1.16 0.80–1.67 1.11 0.77–1.61 1.01 0.68–1.51 1.01 0.66–1.54 NFR continuous score 1.00 1.00–1.01 1.00 1.00–1.01 1.00 0.99–1.01 1.00 0.99–1.01 Model 1: crude Model 2: adjusted for gender, age and education level Model 3: additionally adjusted for number of working hours per week, work schedule, psychological job demands, decision latitude and supervisor social support Model 4: additionally adjusted for self-perceived health, spousal retirement and attachment to work for financial resources Model 3: additionally adjusted for number of working hours per week, work schedule, psychological job demands, decision latitude and supervisor social support Statistically significant ORs + 95% CIs are marked in bold. Table 4 Hazard ratios and 95% confidence intervals for specific types of early labour force exit: early retirement, work disability and unemployment, according to NFR caseness and NFR continuous score (October 2008–October 2014) Discussion In case the upper limit of a bold marked 95% CI contains 1.00 the upper limit is ≤ 0.999 and the corresponding p value is < 0.05 Model 1: crude Model 2: adjusted for gender, age and education level Model 3: additionally adjusted for number of working hours per week, work schedule, psychological job demands, decision latitude and supervisor social support Model 4: additionally adjusted for self perceived health spousal retirement and attachment to work for Model 1 Model 2 Model 3 Model 4 ORa 95% CI OR 95% CI OR 95% CI OR 95% CI It is my intention to keep working until I reach the mandatory retirement age NFR caseness 0.77 0.60–0.99 0.80 0.62–1.02 0.84 0.64–1.10 0.76 0.58–1.01 NFR continuous score 1.00 0.99–1.00 1.00 0.99–1.00 1.00 0.99–1.00 1.00 0.99–1.00 I think I am physically able to work in my current job until reaching the mandatory retirement age NFR caseness 0.36 0.28–0.46 0.36 0.28–0.47 0.45 0.34–0.61 0.50 0.37–0.68 NFR continuous score 0.98 0.98–0.99 0.98 0.98–0.99 0.99 0.98–0.99 0.99 0.98–0.99 I think I am mentally able to work in my current job until reaching the mandatory retirement age NFR caseness 0.36 0.28–0.46 0.37 0.28–0.47 0.45 0.34–0.59 0.49 0.37–0.65 NFR continuous score 0.98 0.98–0.99 0.98 0.98–0.99 0.99 0.98–0.99 0.99 0.98–0.99 It is my intention to keep working beyond the mandatory retirement age NFR caseness 0.90 0.61–1.33 0.90 0.61–1.34 0.95 0.61–1.47 0.91 0.57–1.43 NFR continuous score 1.00 1.00–1.01 1.00 1.00–1.01 1.00 1.00–1.01 1.00 1.00–1.01 Table 5 Odds ratios and 95% confidence intervals for the different items on retirement intentions and ability to prolong working life according to NFR caseness and NFR continuous score (October 2012) (n = 1607) Statistically significant ORs + 95% CIs are marked in bold. Discussion Similar findings in Vickerstaff et al.’s (2004) study demonstrated the influence of organizational Third, although the direction of the association between NFR and overall LFE was similar among employees with and without organizational schemes for LFE, only statis- tically significant associations between NFR and overall LFE were found only among employees who had such opportunities. Therefore, it is likely that the choice for LFE is at least partially embedded in the broader organi- zational context. Similar findings in Vickerstaff et al.’s (2004) study demonstrated the influence of organizational policies and practices on the decision to retire early. The 1 3 nternational Archives of Occupational and Environmental Health (2019) 92:683–697 International Archives of Occupational and Environmental Health (2019) 92:683–697 693 ssociations between NFR and LFE varied actors from various domains suggests that mated the true association between NFR and overall LFE; for instance, among employees with an elevated NFR but without opportunities for LFE, actual LFE might not have been feasible It should be noted that atios and 95% als for specific our force exit: work disability nt, according and NFR (October 14) Statistically significant HRs + 95% CIs are marked in bold. In case the lower limit of a bold marked 95% CI contains 1.00 the lower limit is ≥ 1.001 and the corresponding p value is < 0.05 Model 1: crude Model 2: adjusted for gender, age and education level Model 3: additionally adjusted for number of working hours per week, work schedule, psychological job demands, decision latitude and supervisor social support Model 4: additionally adjusted for self-perceived health, spousal retirement and attachment to work for financial resources Specific type of labour force exit Model 1 Model 2 Model 3 Model 4 HR 95% CI HR 95% CI HR 95% CI HR 95% CI Early retirement NFR caseness 1.37 0.94–2.00 1.29 0.88–1.89 1.31 0.86–1.98 1.22 0.78–1.90 NFR continuous score 1.01 1.00–1.01 1.01 1.00–1.01 1.01 1.00–1.01 1.01 1.00–1.01 Work disability NFR caseness 2.44 1.14–5.22 2.39 1.11–5.11 3.04 1.26–7.33 2.76 1.08–7.05 NFR continuous score 1.01 1.00–1.03 1.01 1.00–1.03 1.02 1.00–1.03 1.02 1.00–1.03 Unemployment NFR caseness 1.16 0.80–1.67 1.11 0.77–1.61 1.01 0.68–1.51 1.01 0.66–1.54 NFR continuous score 1.00 1.00–1.01 1.00 1.00–1.01 1.00 0.99–1.01 1.00 0.99–1.01 ios and 95% als for the n retirement ility to prolong rding to NFR R continuous 012) (n = 1607) Statistically significant ORs + 95% CIs are marked in bold. Discussion The results of the adjusted models might be somewhat over- corrected, which may have led to an underestimation of the strength of the relationships. Although stratification has the advantage of unambiguous interpretation and clearly indi- cated the strength of association between NFR and LFE in specific subgroups, statistically significant differences across strata could not be established. Despite the factors we took into account in this study, residual confounding cannot be ruled out, as it is possible that not all relevant confound- ers have been included, and some confounders might not have been optimally operationalized. For instance, education level was assessed as the highest education degree workers had obtained as of May 1998. Although educational attain- ment earlier in life (e.g. at labour market entry) remains an important determinant of labour participation and oppor- tunities (like training) in later life (Edgerton et al. 2012; Fouarge et al. 2013), workers may have gained additional education in later life, which may underestimate the impor- tance of education level in the association between NFR and LFE. In addition, the factor ‘attachment to work for financial resources’ was considered, but it does not fully grasp the employees’ or families’ financial situation in rela- tion to LFE opportunities. In addition, we investigated the role of spousal retirement, but the reference group com- prised both employees with a currently employed partner as well as a relatively small group of employees without a partner. Also, the influence of partnership itself on LFE was not examined in this study. Bias may also stem from the multi-level nature of variables like the availability of h f l LFE h ti b i il the findings might be affected by the uneven distribution across various strata, and that effect modification could not be formally established in terms of statistically significant differences between strata, warranting a cautious interpre- tation of these findings. i Furthermore, NFR was found to be differently associated with specific types of LFE. That is, NFR was associated with a higher risk of early retirement and work disability, but not with a higher chance of unemployment. The finding that NFR was associated with work disability is consistent with earlier studies, where NFR was associated with occupational diseases (Otten et al. 2012) and sickness absence (de Croon et al. 2003), which were both found to be associated with a higher risk of disability pension (Kivimäki et al. 2004). Discussion In case the upper limit of a bold marked 95% CI contains 1.00 the upper limit is ≤ 0.999 and the corresponding p value is < 0.05 Model 2: adjusted for gender, age and education level Model 3: additionally adjusted for number of working hours per week, work schedule, psychological job demands, decision latitude and supervisor social support Model 4: additionally adjusted for self-perceived health, spousal retirement and attachment to work for financial resources finding that associations between NFR and LFE varied according to factors from various domains suggests that we underestimated the true association between NFR finding that associations between NFR and LFE varied according to factors from various domains suggests that we underestimated the true association between NFR and overall LFE; for instance, among employees with an elevated NFR but without opportunities for LFE, actual LFE might not have been feasible. It should be noted that and overall LFE; for instance, among employees with an elevated NFR but without opportunities for LFE, actual LFE might not have been feasible. It should be noted that 1 3 International Archives of Occupational and Environmental Health (2019) 92:683–697 694 increase in the risk of LFE. It was useful to distinguish the main types of LFE in terms of early (obligatory) retirement, work disability and (in)voluntary unemployment, as the results for these specific types of LFE differed. Whereas LFE was approached as a dichotomous outcome in this study, this is not necessarily always the case, as gradual types and multiple combinations of types of LFE also exist. Employees can leave the labour force gradually via bridge employment or may become partially disabled for work. A suggestion for future research would be to further differenti- ate between gradual and full types of LFE when investigat- ing the impact of NFR. Also, possible return to the labour market after LFE was not taken into consideration in this study. Another strength of this study is that various potential confounders and effect-modifying factors were considered to explore the role of these factors in the association between NFR and LFE via adjusted and stratified models. However, it remains uncertain whether adjusting these associations is required and/or desirable, especially since NFR is consid- ered a comprehensive concept in which numerous effects from the psychosocial work environment are accumulated. Discussion However, to our knowledge, the finding that NFR can be regarded as an immediate precursor of types of LFE like early retirement and work disability among older workers is novel in the literature on NFR and retirement. The different associations between NFR and specific types of LFE might also be at least partly ascribed to the voluntariness of the type of exit. Robroek et al. (2013) found that determinants such as poor health were differently associated with invol- untary (e.g. unemployment) as compared to more voluntary types of LFE (e.g. early retirement). Also, contextual fac- tors (e.g. firm closure) might play a larger role in the risk of becoming unemployed, which might (slightly) conceal the association between NFR and unemployment. Furthermore, elevated NFR was also associated with reduced intentions to prolong working life, and employees with elevated NFR considered themselves less physically and mentally able to work until the mandatory retirement age. Although not explicitly tested, NFR might be associated with long-term outcomes such as LFE through an indirect pathway of reduced intention and ability to prolong work- ing life, which is in line with the work load–work capacity model. It should be noted, however, that these analyses are cross-sectional, and a longitudinal time-window may yield different findings. Strengths and limitations An important strength of this study is the large cohort, which allowed for an investigation of longitudinal associations between NFR and specific types of LFE that have not been previously investigated. Concerning NFR, we investigated both continuous NFR scores and NFR caseness. A cutoff score to define cases of elevated NFR may be helpful in determining which workers are at risk for LFE and could be useful to direct interventions. The HRs for continuous NFR levels were often low, with narrow confidence inter- vals. However, it should be noted that they were measured on a 100-point scale, meaning that with an HR of 1.01, any 10-point increase on the NFR scale is associated with a 10% 1 3 International Archives of Occupational and Environmental Health (2019) 92:683–697 695 between NFR and known determinants of retirement, such as lowered intention and ability to prolong working life, sug- gesting that NFR plays a role in the LFE decision process. Besides NFR, LFE also seemed to vary based on opportuni- ties to make use of LFE schemes, which might imply that LFE is a result of individual and/or contextual factors. This implication was further strengthened by the finding that fac- tors like gender and unfavourable work conditions seem to affect the strength of the association between NFR and LFE. As such, monitoring NFR among older workers may be used as an early warning system to identify workers with an ele- vated NFR and offer interventions to reduce NFR and early LFE. Such monitoring should also take into account fac- tors from the demographic, work environment and personal domains. Although such an approach has the potential to facilitate extending labour participation, it should be noted that the effectiveness of measures like worksite social and physical environment interventions or health interventions addressing elevated NFR have yet not been demonstrated (Coffeng et al. 2014; van Berkel et al. 2014). number of organizations (second order) in the analyses on the role of early exit schemes (Table 3), we cannot account statistically for this possible bias. Overall, this study relies only on self-reports, which may have artificially inflated the strength of the observed relationships (i.e. common method bias), in particular with respect to the cross-sectional analy- ses (Podsakoff et al. 2012). Strengths and limitations For descriptive and stratifica- tion purposes and for reasons of parsimony and statistical power, categorical and continuous variables were occasion- ally recoded into categorical variables with fewer categories, which may have resulted in loss of information. An equal set of confounders was investigated for all types of LFE. It is possible, however, that different confounders play a role in the associations between NFR and different types of LFE.f f yp To investigate possible short- and long-term effects of NFR on multiple outcomes, different time-windows, study populations and data-analysis techniques were required. For each type of outcome, the time-window, study popula- tion and technique were carefully considered, although this might have hindered direct comparisons between the study findings. As the present study excluded all employees aged 59 years and older at study baseline, the association between NFR and (specific types of) LFE has not been investigated among employees who were close to reaching the mandatory retirement age. Therefore, the findings of this study might not be fully applicable and/or generalized to the group of oldest employees. As this approach might have been some- what conservative, a suggestion for future studies would be to investigate the association between NFR and LFE in a sample of employees close to reaching their mandatory retirement age, as being close to reaching this age might result in different associations between NFR and LFE. Statement The presented results are based on data of the Maastricht Cohort Study, and are a unique contribution to our understanding on the determinants and consequences of NFR throughout the life span. All earlier studies on this topic which are based on data of the Maastricht Cohort Study have been listed in a supplementary electronic docu- ment to this manuscript. Acknowledgements The authors would like to thank Huub Hoofs for his help in the use of statistics. The authors acknowledge the contribu- tion of Fleur Gommans to earlier versions of this manuscript. f As all companies included in the cohort fall under Dutch legislation, the findings of this study should be considered in the Dutch context. Many European countries try to discour- age early retirement and minimize work disability as much as possible, whereas no general policies on unemployment exist. Social policies for the specific types of LFE and retire- ment regulations vary across countries (Siegrist et al. 2007), possibly affecting the employees’ opportunities for LFE. Compliance with ethical standards Conflict of interest The authors declare that they have no conflict of interest. Informed consent Informed consent was obtained from all individual participants included in the study. Ethical approval All procedures performed in studies involving human participants were in accordance with the ethical standards of the insti- tutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. Strengths and limitations Furthermore, varying definitions of (specific types of) LFE and mandatory retirement age might be observed among countries, which suggests that LFE in this study should be considered in the national context. Funding This study was financially supported by Instituut Gak, Grant No. 2011-089 WR and by School CAPHRI (School for Public Health and Primary Care), Maastricht, the Netherlands. 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https://openalex.org/W2140137811	https://dash.harvard.edu/bitstream/1/8231691/1/2981565.pdf	English	null	Analysis of Mice Lacking DNaseI Hypersensitive Sites at the 5′ End of the IgH Locus	PloS one	2,010	cc-by	9,515	Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of-use#LAA Permanent link http://nrs.harvard.edu/urn-3:HUL.InstRepos:8231691 Citation Perlot, Thomas, Inka Pawlitzky, John P. Manis, Ali A. Zarrin, Peter H. Brodeur, and Frederick W. Alt. 2010. Analysis of Mice Lacking DNaseI Hypersensitive Sites at the 5' End of the IgH Locus. PLoS ONE 5(11): e13992. Perlot, Thomas, Inka Pawlitzky, John P. Manis, Ali A. Zarrin, Peter H. Brodeur, and Frederick W. Alt. 2010. Analysis of Mice Lacking DNaseI Hypersensitive Sites at the 5' End of the IgH Locus. PLoS ONE 5(11): e13992. Abstract The 59 end of the IgH locus contains a cluster of DNaseI hypersensitive sites, one of which (HS1) was shown to be pro-B cell specific and to contain binding sites for the transcription factors PU.1, E2A, and Pax5. These data as well as the location of the hypersensitive sites at the 59 border of the IgH locus suggested a possible regulatory function for these elements with respect to the IgH locus. To test this notion, we generated mice carrying targeted deletions of either the pro-B cell specific site HS1 or the whole cluster of DNaseI hypersensitive sites. Lymphocytes carrying these deletions appear to undergo normal development, and mutant B cells do not exhibit any obvious defects in V(D)J recombination, allelic exclusion, or class switch recombination. We conclude that deletion of these DNaseI hypersensitive sites does not have an obvious impact on the IgH locus or B cell development. Citation: Perlot T, Pawlitzky I, Manis JP, Zarrin AA, Brodeur PH, et al. (2010) Analysis of Mice Lacking DNaseI Hypersensitive Sites at the 59 End of the IgH Locus. PLoS ONE 5(11): e13992. doi:10.1371/journal.pone.0013992 Editor: Sebastian D. Fugmann, National Institute on Aging, United States of America Editor: Sebastian D. Fugmann, National Institute on Aging, United States of America Received June 28, 2010; Accepted October 26, 2010; Published November 15, 2010 Received June 28, 2010; Accepted October 26, 2010; Published November 15, 2010 Received June 28, 2010; Accepted October 26, 2010; Published November 15, 2010 Copyright: 2010 Perlot 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. nding: This work was funded by National Institutes of Health R01 AI020047 (to FWA) and GM36064 (to PHB). FWA is an Investigator dical Institute. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manu Competing Interests: The authors have declared that no competing interests exist. Competing Interests: The authors have declared that no competing interests exist. * E-mail: alt@enders.tch.harvard.edu . These authors contributed equally to this work. Thomas Perlot1.¤a, Inka Pawlitzky2.¤b, John P. Manis3, Ali A. Zarrin1¤c, Peter H. Brodeur2, Frederick W. Alt1* Thomas Perlot1.¤a, Inka Pawlitzky2.¤b, John P. Manis3, Ali A. Zarrin1¤c, Peter H. Bro Alt1* nka Pawlitzky2.¤b, John P. Manis3, Ali A. Zarrin1¤c, Peter H. Brodeur2, Frederick W 1 The Howard Hughes Medical Institute, The Children’s Hospital, Immune Disease Institute, and Department of Genetics, Harvard Medical School, Boston, Massachusetts, United States of America, 2 Immunology Program, Sackler School of Graduate Biomedical Sciences, Tufts University School of Medicine, Boston, Massachusetts, United States of America, 3 Children’s Hospital Boston and Joint Program in Transfusion Medicine, Harvard Medical School, Boston, Massachusetts, United States of America Abstract ¤a Current address: IMBA, Institute of Molecular Biotechnology of the Austrian Academy of Sciences, Vienna, Austria ¤b Current address: Division of Molecular Genetics, Netherlands Cancer Institute, Amsterdam, The Netherlands ¤c Current address: Department of Immunology, Genentech, San Francisco, California, United States of America A large effort has been made to elucidate mechanisms of IgH locus regulation and a number of cis-regulatory elements have been described and characterized. The IgH intronic enhancer (Em) resides in the JH – CH intron and was shown to be necessary for efficient V(D)J recombination by promoting both D to JH and VH to DJH recombination [6,7]. Downstream of the CH genes at the very 39 end of the IgH locus a cluster of DNaseI hypersensitive sites was described, termed 39 IgH regulatory region (39IgH RR). So far two main functions have been assigned to this regulatory region: the 39IgH RR plays an important role in promoting CSR to most IgH isotypes, and the 39IgH RR was shown to be necessary for high level expression of the functionally assembled HC gene from the promoter 59 of the VHDJH exon [8]. Share Your Story The Harvard community has made this article openly available. Please share how this access benefits you. Submit a story . Accessibility PLoS ONE \| www.plosone.org November 2010 \| Volume 5 \| Issue 11 \| e13992 Introduction The variable region of an immunoglobulin heavy chain (IgH) is assembled from V (variable), D (diversity), and J (joining) gene segments that lie upstream of several IgH constant (C) region exons in a process called V(D)J recombination [1]. The mouse IgH locus contains large numbers of VH segments and multiple D and JH segments but an individual IgH V(D)J exon is assembled from only one VH, one D, and one JH segment. V(D)J recombination of the IgH locus takes place in pro-B cells in an ordered way such that D to JH recombination precedes VH to DJH recombination [2]. In this regard, activation of the IgH locus is thought to progress in a stepwise manner [3]. D to JH rearrangement efficiently occurs on both alleles, however, allelic exclusion ensures that VH to DJH recombination results in expression of a functional heavy chain (HC) from only one of the two alleles [4]. An additional potential regulatory region was identified at the 59 end of the IgH locus, consisting of four DNaseI hypersensitive sites [9]. One of these sites, HS1, was shown to be pro-B cell specific, the stage during which IgH V(D)J recombination takes place, and was suggested to include binding sites for the transcription factors PU.1, Pax5 and E2A [9]. These observations led to the suggestion that this region might represent a new regulatory region for IgH rearrangements. In this regard, the 59 end of the IgH locus is an attractive location for a regulatory element because it would not be deleted during the course of V(D)J recombination, and it might explain control of several unresolved phenomena in the IgH locus. Among these is the regulation of VH germline transcripts as so far Mature B-cells can undergo further alterations of their HCs. IgH class switch recombination (CSR) causes expression of different immunoglobulin isotypes which confer different effector functions. During this recombination process one of several sets of downstream CH exons replaces the Cm exons and the intervening sequence is deleted from the chromosome, which results in expression of a new C region without changing the specificity of the IgH variable region [5]. PLoS ONE \| www.plosone.org November 2010 \| Volume 5 \| Issue 11 \| e13992 1 59IgH Enhancer no cis-regulatory element has been identified that controls activity of the bulk of unrearranged VH promoters. IgH class switch recombination assay CD43- splenocytes were isolated by MACS, cultured with LPS or IL4/aCD40, and analyzed by flow cytometry as described previously [13]. Introduction Furthermore, it is not known how it is achieved that proximal and distal VH segments are activated independently or why usage of distal versus proximal VH gene families varies significantly. PCR assay for V(D)J rearrangements hi Pro-B cells (IgM- B220+ CD43hi), pre B-cells (IgM- B220+ CD43lo), and double positive T-cells (B220- CD4+ CD8+) were isolated by FACS on a FACSAria (BD Biosciences) and genomic DNA was extracted. 50 ng DNA or 5-fold dilutions were analyzed by PCR for DH–JH, VH-DJH, Vk–Jk, and Vl–Jl rearrangements with primers listed in Table S1. Input DNA amounts were normalized upon PCR amplification within DLG5. PCR was performed at 95uC for 49, 30 cycles of 95uC for 300, 60uC for 900, and 72uC for 29, followed by 72uC for 59. PCR products were transferred from ethidium bromide gels to nylon membranes and visualized with end labeled oligonucleotide probes (Table S1). CDR 3 lengths were generated from IgH VDH rearrangements from mature B cells using oligonucleotides for V558 and JH4 rearrangements. PCR fragments were amplified using iProof (Bio- Rad) polymerase and cloned into Zero Blunt Topo vectors (Invitrogen), and sequenced. Here we report the targeted deletion of the pro-B cell specific 59IgH HS1 as well as combined deletion of HS1, HS2, HS3a,b in mice. We analyzed potential implications on B cell development, V(D)J recombination, and IgH CSR. Flow cytometry and cell sorting Single cell suspensions from spleen, thymus, or bone marrow were stained in PBS 2% FBS with various antibodies: FITC-aIgM, PE-Cy5-aB220, PE-aCD8a, PE-aCD43, FITC-aLy9.1, APC- aIgM, APC-Cy7-aB220 (BD Pharmingen), PE-aAA4.1, FITC- aCD4 (eBioscience). FACS analysis was performed on a FACSCalibur (BD Biosciences) and a FACSAria (BD Biosciences) apparatus. Cell sorts were performed on a FACSAria (BD Biosciences) apparatus. EF1 heterozygous IgHa/b embryonic stem (ES) cells, generated in the Alt laboratory, were transfected with PvuI linearized RHS1 targeting vector to obtain RHS1/+ ES cells. To obtain DHS1/+ ES cells, the PGK-NeoR cassette was deleted by applying a Cre – expressing adenovirus vector. DHS1/+ ES cells were transfected with PvuI linearized R39HSs targeting vector to obtain R39HSs/+ ES cells. R39HSs/+ ES cells were selected for homozygocity of the targeted allele through increasing concentration of G418 to obtain R39HSs/R39HSs ES cells. Cre – loxP mediated deletion of the PGK-NeoR cassette resulted in DHSs/DHSs ES cells. Targeted ES cells were injected into Rag2-/- blastocysts to obtain RDBC chimeras [11] or into C57BL/6 blastocysts to obtain chimeras that could be crossed to 129Sv mice to achieve germline transmission of the targeted allele. Generation of mice with targeted deletion of 59IgH DNaseI hypersensitive sites To determine the in vivo function of the cluster of DNaseI hypersensitive sites described at the 59 end of the IgH locus [9] we first replaced a ,340 bp BccI – AatII fragment, harboring HS1, with a loxP flanked PGK-NeoR cassette. All targeting experiments were performed in heterozygous IgHa/b EF1 ES cells which have the advantage that IgHa (129 strain) and IgHb (C57BL/6 strain) alleles can be distinguished by antibodies against the different allotypes or by detection of restriction fragment length polymor- phisms (RFLP). Targeting vector homology arms were cloned from 129 strain genomic DNA, resulting in correct targeting events only on the IgHa allele. In heterozygous targeted ES cells, the IgHb allele always remained in the untargeted wildtype configuration. RT-PCR analysis RNA was extracted using TriPure Isolation Reagent (Roche). 200 ng–1 mg of total RNA was reverse transcribed for one hour at 50uC using random hexamers (Roche) and Superscript III (Invitrogen) reverse transcriptase. PCR was performed at 94uC for 49, 30–39 cycles of 94uC for 300, annealing temperature (Table S1) for 300, 72uC for 300, followed by 72uC for 59. cDNA input amount was normalized upon PCR amplification of b-actin cDNA. PCR products were visualized on ethidium bromide gels and/or subsequently transferred to nylon membranes and visualized with end labeled oligonucleotide probes (Table S1). Targeted deletion of 59IgH DNaseI hypersensitive sites in ES cells and generation of mutant mice All mouse were handled in strict accordance with good animal practice as defined by the relevant national and/or local animal welfare bodies, and all animal work was approved by Animal Research of Children’s Hospital Boston (Protocol # 08 11 1253R). The RHS1 targeting vector was assembled in pLNTK [10]. As a 59 homology arm a 2.2 kb PCR product was generated with primers 59 GTCGACGGATTTAGGAGGATACACAAC 39 and 59 GTCGACCTTGGATAACACAGAACTCTG 39 containing a SalI site at their 59 ends, which facilitate cloning of the PCR product into the SalI site of pLNTK. As a 39 homology arm a 7.3 kb AatII – ApaI fragment was blunt end cloned into the XhoI site of pLNTK. The R39HSs targeting vector was generated by blunt end cloning a 4.4 kb EcoRI fragment into the SalI site of pLNTK as a 59 homology arm, and a 7.0 kb KpnI fragment into the XhoI site as the 39 homology arm. Correct targeting events and cre – loxP deletion events were confirmed by Southern blotting (Fig 1). Probe 1 is a 830 bp PCR product amplified with primers 59 GCTCATGTACCAATCTGCACTCAC 39 and 59 CACTGT- GACCTCCATCTTATGTCTG 39. Probe 2 is a 1.2 kb PstI – EcoRI fragment 59 of HS2. Probe 3 is a 0.8 kb PstI – XbaI fragment about 11 kb 39 of HS3b. To confirm single integration of the targeting vectors a 525 bp NeoR probe was used, amplified with primers 59 GCAGCCATATGGGATCGGC 39 and 59 GTTCGGCTGGCGCGAGCCCC 39. November 2010 \| Volume 5 \| Issue 11 \| e13992 B cell hybridomas DHS1 ES cells exhibit a 15.7 kb DHS1 band and a 26.3 kb band for the wildtype IgHb allele (lane 9), R39HSs ES cells show a 11.1 kb R39H and a 26.3 kb band for the wildtype IgHb allele (lane 11), DHSs ES cells are identified by the presence of a 14.1 kb DHSs band in additio 26.3 kb wildtype IgHb allele (lane 10). All targeting events occured on the IgHa allele, whereas the IgHb allele remained in wildtype config Drawings not to scale. B - BglII; S - SacI; P - SphI. Figure 1. Targeting strategy for the generation of the RHS1, DHS1, and DHSs alleles. (A) the wildtype (wt) IgH locus and its 59 flanking region are shown. VH, DH, JH indicate representative IgH V, D, and J segments. Exons 1, 2, and 3 of Zfp386 are shown as grey rectangles, DNaseI hypersensitive sites HS1, HS2, HS3a, and HS3b are shown as black ovals. Correct targeting events of the RHS1 targeting vector were identified by Southern blotting on BglII digested ES cell DNA using probe 1, which results in a 15.8 kb band (lane 1) in addition to the 14.2 kb wildtype band (lane 2). Cre – loxP (black triangles) mediated deletion of the PGK-NeoR cassette (NeoR) from the RHS1 allele results in the DHS1 allele. Deletions were identified by Southern analysis of SacI digested DNA utilizing probe 2. A targeted clone before Cre – mediated deletion exhibits a 6.7 kb RHS1 band and a 5.1 kb wildtype band (lane 3). Upon deletion of the PGK-NeoR cassette, a 4.8 kb DHS1 band and a 5.1 kb wildtype band are visible (lane 4). Lane 5 shows untargeted wildtype DNA. (B) The DHS1 allele was targeted with the R39HSs targeting vector to introduce a PGK-NeoR cassette flanked by loxP sites. Correct targeting events were confirmed by Southern blotting on SphI digested ES cell DNA with probe 1, resulting in a 2.6 kb band for the wildtype IgHb allele and a 7.2 kb band for R39HSs, the targeted IgHa allele (lane 6). Cre – mediated recombination between the first and the third loxP site generates the DHSs allele (14.1 kb, lane 8). Homozygous R39HSs ES cells were generated under increasing concentrations of G418, resulting in a single 7.2 kb R39HSs band (lane 7). Southern analysis on SphI digested DNA with probe 3 confirms correct targeting events of the R39HSs targeting vector. B cell hybridomas CD43- splenocytes were isolated by MACS, stimulated with LPS (20 mg/ml), and fused to NS-1 plasmacytoma cells (TIB-18, ATCC) as described previously [12]. IgH V(D)J rearrangement status was analyzed by Southern blotting of EcoRI digested genomic DNA of clonal hybridomas with three different probes, a 1.6 kb HindIII - EcoRI fragment 39 of JH4, a 0.38 kb SacI - ApaI fragment 39 of DHQ52, and a 0.75 kb PCR product 59 of DHFL16.1 generated with oligonucleotides 59 GAACAG- CAACCCTTGACTGACTCTG 39 and 59 GATTGGTTCT- TATGGAATGGGTGG 39. Targetings were performed with the RHS1 targeting vector (Fig. 1A) to obtain the RHS1 allele and, upon cre/loxP deletion, PLoS ONE \| www.plosone.org November 2010 \| Volume 5 \| Issue 11 \| e13992 November 2010 \| Volume 5 \| Issue 11 \| e13992 2 59IgH Enhancer Figure 1. Targeting strategy for the generation of the RHS1, DHS1, and DHSs alleles. (A) the wildtype (wt) IgH locus and region are shown. VH, DH, JH indicate representative IgH V, D, and J segments. Exons 1, 2, and 3 of Zfp386 are shown as grey recta hypersensitive sites HS1, HS2, HS3a, and HS3b are shown as black ovals. Correct targeting events of the RHS1 targeting vector were Southern blotting on BglII digested ES cell DNA using probe 1, which results in a 15.8 kb band (lane 1) in addition to the 14.2 kb wildty 2). Cre – loxP (black triangles) mediated deletion of the PGK-NeoR cassette (NeoR) from the RHS1 allele results in the DHS1 allele. D identified by Southern analysis of SacI digested DNA utilizing probe 2. A targeted clone before Cre – mediated deletion exhibits a 6.7 and a 5.1 kb wildtype band (lane 3). Upon deletion of the PGK-NeoR cassette, a 4.8 kb DHS1 band and a 5.1 kb wildtype band are visible 5 shows untargeted wildtype DNA. (B) The DHS1 allele was targeted with the R39HSs targeting vector to introduce a PGK-NeoR casse loxP sites. Correct targeting events were confirmed by Southern blotting on SphI digested ES cell DNA with probe 1, resulting in a 2.6 k wildtype IgHb allele and a 7.2 kb band for R39HSs, the targeted IgHa allele (lane 6). Cre – mediated recombination between the first and site generates the DHSs allele (14.1 kb, lane 8). Homozygous R39HSs ES cells were generated under increasing concentrations of G418 single 7.2 kb R39HSs band (lane 7). B cell hybridomas Southern analysis on SphI digested DNA with probe 3 confirms correct targeting events of the R3 vector. DHS1 ES cells exhibit a 15.7 kb DHS1 band and a 26.3 kb band for the wildtype IgHb allele (lane 9), R39HSs ES cells show a 11.1 k and a 26.3 kb band for the wildtype IgHb allele (lane 11), DHSs ES cells are identified by the presence of a 14.1 kb DHSs band in a 26 3 kb wildtype IgHb allele (lane 10) All targeting events occured on the IgHa allele whereas the IgHb allele remained in wildtype 5 Figure 1. Targeting strategy for the generation of the RHS1, DHS1, and DHSs alleles. (A) the wildtype (wt) IgH locus and its 59 region are shown. VH, DH, JH indicate representative IgH V, D, and J segments. Exons 1, 2, and 3 of Zfp386 are shown as grey rectangles hypersensitive sites HS1, HS2, HS3a, and HS3b are shown as black ovals. Correct targeting events of the RHS1 targeting vector were iden Southern blotting on BglII digested ES cell DNA using probe 1, which results in a 15.8 kb band (lane 1) in addition to the 14.2 kb wildtype ba 2). Cre – loxP (black triangles) mediated deletion of the PGK-NeoR cassette (NeoR) from the RHS1 allele results in the DHS1 allele. Deletio identified by Southern analysis of SacI digested DNA utilizing probe 2. A targeted clone before Cre – mediated deletion exhibits a 6.7 kb RH and a 5.1 kb wildtype band (lane 3). Upon deletion of the PGK-NeoR cassette, a 4.8 kb DHS1 band and a 5.1 kb wildtype band are visible (lane 5 shows untargeted wildtype DNA. (B) The DHS1 allele was targeted with the R39HSs targeting vector to introduce a PGK-NeoR cassette fla loxP sites. Correct targeting events were confirmed by Southern blotting on SphI digested ES cell DNA with probe 1, resulting in a 2.6 kb band wildtype IgHb allele and a 7.2 kb band for R39HSs, the targeted IgHa allele (lane 6). Cre – mediated recombination between the first and the th site generates the DHSs allele (14.1 kb, lane 8). Homozygous R39HSs ES cells were generated under increasing concentrations of G418, resul single 7.2 kb R39HSs band (lane 7). Southern analysis on SphI digested DNA with probe 3 confirms correct targeting events of the R39HSs t vector. Development of homozygous RHS1, DHS1, and DHSs lymphocytes Development of homozygous RHS1, DHS1, and DHSs lymphocytes y p y Lymphocytes of different developmental stages can be identified by FACS analysis of cells from lymphoid tissues such as bone marrow, thymus, or spleen. We analyzed 8 week old wildtype mice, homozygous RHS1, and homozygous DHS1 mice that carry the mutant alleles in their germline, as well as lymphocytes from RDBC chimeras generated from homozygous DHSs ES cells (Fig. 2). In wildtype bone marrow, pro-B cells can be identified as IgM- B220+ CD43hi and pre-B cells as IgM- B220+ CD43lo cells, respectively. Defects in B-cell development can be revealed by the increase or decrease of certain lymphocyte populations. In this regard, impaired IgH V(D)J recombination leads to an accumu- lation of pro-B cells and to reduced numbers of pre-B cells [7]. We performed FACS analyses of bone marrow from three mice of each genotype to measure the percentage of pro- and pre-B cells in the lymphocyte gate. These analyses revealed the average percentage (6 standard deviation) of pro-B and pre-B cells, respectively of B220+/CD43+ events in the total lymphocyte gate were 1462 and 50620 for wildtype, 963 and 5666 for DHS1, and 963 and 42611 for RHS1 mice (Fig. 2A). Thus, there were no obvious differences in early B-cell development in wildtype and mutant mice. However we cannot exclude minor developmental defects not readily detectable by such analyses. Homozygous mutant DHSs bone marrow cells were analyzed in a similar fashion, but only Ly9.1+ cells were included in the analysis. Ly9.1 is exclusively expressed on cells derived from the DHSs ES cells but not on cells derived from the Rag2-/- blastocyst. The presence of a large compartment of blastocyst derived Rag-deficient pro-B cells in the bone marrow can interfere with development of ES cell It was speculated that HS1 might regulate the differential activation of distal versus proximal VH families [9]; therefore, we analyzed the rearrangement efficiencies of the proximal VH7183 family (Fig. 3C), the distal VHJ558 familiy (Fig. 3D), and the distal most VH segment VHJ558.55 (Fig. 3E). We found that pro-B cells and pre-B cells from wildtype, homozygous RHS1, and homozy- gous DHS1 mice rearrange the proximal VH7183 family at similar levels (Fig. 3C). Also, the distal family VHJ558 (Fig. 3D) as well as the distal most VH segment VHJ558.55 (Fig. 3E) rearranged at comparable efficiencies in pro-B cells and pre-B cells from the three different genotypes. B cell hybridomas Subsequently, targeted ES cells were injected into Rag2-/- blastocysts to obtain Rag-deficient blastocyst complementation (RDBC) chimeras, and into wildtype blastocyts to generate mice that carry the RHS1 or DHS1 allele in their germline. In order to delete all four hypersensitivity sites (HS1, HS2, HS3a, and HS3b), ES cells containing the DHS1 allele were targeted with the R39HSs targeting vector to obtain the R39HSs allele (Fig. 1B). Cre/loxP recombination between the loxP site originating from the DHS1 allele and the loxP site 39 of the PGK- NeoR cassette results in the replacement of a 8.9 kb region, harboring all described 59IgH DNaseI hypersensitive sites, with a single loxP site, referred to as the DHSs allele. Germline transmission could not be achieved for either of the R39HSs or DHSs heterozygous ES cell lines. Therefore, we placed ES cells containing the R39HSs allele under increasing concentrations of G418 to select for homozygous mutant ES cells. The homozygous mutant ES cells were subsequently subjected to cre/loxP recombination to delete the Neor gene and generate ES cells homozygous for the DHSs allele. The homozygous mutant DHSs ES cells were injected into Rag2-/- blastocysts, and chimeras generated by RDBC and lymphocytes were analyzed. and did not suggest a block in B-cell development (Fig. 2A). Next we analyzed spleens for IgM+ B220+ AA4.1+ transitional B-cells and IgM+ B220+ AA4.1- mature B-cells (Fig. 2B). In homozygous RHS1, and homozygous DHS1 mice transitional (19.7%–27.5%) and mature (65.3%–72.7%) B-cell compartments similar to wildtype were identified; whereas, in spleens from RDBC chimeras generated from homozygous DHSs ES cells strongly reduced numbers of transitional B-cells were observed (6.44%). This reduction in the transitional B-cell compartment compared to the mature B-cell compartment (75%) might be due to overall reduced numbers of developing B cells in the obtained RDBC chimeras and to the accumulation of mature B-cells in the periphery of these mice and not to a defect in B cell development. Finally, we observed normal development of T-lymphocytes in the thymi of wildtype, homozygous RHS1, and homozygous DHS1 mice as well as RDBC chimeras generated from homozygous DHSs ES cells (Fig. 2C). The DHS1, RHS1, and DHSs alleles show no significant defect in V(D)J recombination The data indicating that HS1 is pro-B cell specific and contains binding sites for the transcription factors PU.1, Pax5, and E2A led to the suggestion that HS1 could be involved in regulation of V(D)J recombination at the IgH locus [9]. We utilized a PCR based assay to assess V(D)J recombination efficiencies in developing lymphocytes from mice with homozygous deletion of HS1. FACS-sorted pro-B cells (IgM- B220+ CD43hi) and pre-B cells (IgM- B220+ CD43lo) from bone marrow and double positive (DP) T-cells (B220- CD4+ CD8+) from thymus were analyzed for D to JH, VH to DJH, Vk to Jk and Vl to Jl rearrangements. Intensities of PCR bands for DHQ52 to JH (Fig. 3A) and DSP to JH rearrangements (Fig. 3B) were comparable in pro-B cells, pre-B cells, and DP T-cells from wildtype, homozygous RHS1, and homozygous DHS1 mice indicating that deletion of the pro-B cell specific HS1 site does not detectably affect the D to JH recombination step. DNA input amounts were normalized to the presence of a genomic sequence within the murine DLG5 gene (Fig. 3H). B cell hybridomas DHS1 ES cells exhibit a 15.7 kb DHS1 band and a 26.3 kb band for the wildtype IgHb allele (lane 9), R39HSs ES cells show a 11.1 kb R39HSs band and a 26.3 kb band for the wildtype IgHb allele (lane 11), DHSs ES cells are identified by the presence of a 14.1 kb DHSs band in addition to the 26.3 kb wildtype IgHb allele (lane 10). All targeting events occured on the IgHa allele, whereas the IgHb allele remained in wildtype configuration. Drawings not to scale. B - BglII; S - SacI; P - SphI. doi:10.1371/journal.pone.0013992.g001 PLo November 2010 \| Volume 5 \| Issue 11 \| e13992 PLoS ONE \| www.plosone.org 3 59IgH Enhancer 59IgH Enhancer g PLoS ONE \| www.plosone.org 4 November 2010 \| Volume 5 \| Issue 11 \| e13992 PLoS ONE \| www.plosone.org PLoS ONE \| www.plosone.org November 2010 \| Volume 5 \| Issue 11 \| e13992 PLoS ONE \| www.plosone.org 59IgH Enhancer Figure 2. Development of homozygous DHS1, RHS1, and DHSs lymphocytes. (A) Bone marrow from wildtype (wt), homozygous DHS1, homozygous RHS1 mice, and RDBC chimeras generated from homozygous DHSs ES cells was subjected to FACS analysis. Gates were set on the lymphocyte population, Ly9.1 positive population, and on IgM negative population (upper three blots, left to right) to analyze pro-B cell (IgM- B220+ CD43hi) and pre-B cell (IgM- B220+ CD43lo) populations (lower blots). (B) FACS analysis of splenocytes from wildtype (wt), homozygous DHS1, homozygous RHS1 mice, and RDBC chimeras generated from homozygous DHSs ES cells. Gates were set on the lymphocyte population and on the IgM positive population (upper two blots, left to right) to analyze transitional B-cell (IgM+ B220+ AA4.1+) and mature B-cell (IgM+ B220+ AA4.1-) populations (lower blots). (C) FACS analysis of thymocytes gated on the lymphocyte population (upper blot) from wildtype (wt), homozygous DHS1, homozygous RHS1 mice, and RDBC chimeras generated from homozygous DHSs ES cells (lower blots). doi:10.1371/journal.pone.0013992.g002 derived B-lymphocytes. However, FACS analysis of DHSs bone marrow B cells indicated the presence of both pro- and pre-B cells and did not suggest a block in B-cell development (Fig. 2A). Next we analyzed spleens for IgM+ B220+ AA4.1+ transitional B-cells and IgM+ B220+ AA4.1- mature B-cells (Fig. 2B). B cell hybridomas In homozygous RHS1, and homozygous DHS1 mice transitional (19.7%–27.5%) and mature (65.3%–72.7%) B-cell compartments similar to wildtype were identified; whereas, in spleens from RDBC chimeras generated from homozygous DHSs ES cells strongly reduced numbers of transitional B-cells were observed (6.44%). This reduction in the transitional B-cell compartment compared to the mature B-cell compartment (75%) might be due to overall reduced numbers of developing B cells in the obtained RDBC chimeras and to the accumulation of mature B-cells in the periphery of these mice and not to a defect in B cell development. Finally, we observed normal development of T-lymphocytes in the thymi of wildtype, homozygous RHS1, and homozygous DHS1 mice as well as RDBC chimeras generated from homozygous DHSs ES cells (Fig. 2C). the DHS1 allele. Correct targeting events (Fig1) and single integration of the targeting vector (Figure S1) were confirmed by Southern blotting. Subsequently, targeted ES cells were injected into Rag2-/- blastocysts to obtain Rag-deficient blastocyst complementation (RDBC) chimeras, and into wildtype blastocyts to generate mice that carry the RHS1 or DHS1 allele in their germline. In order to delete all four hypersensitivity sites (HS1, HS2, HS3a, and HS3b), ES cells containing the DHS1 allele were targeted with the R39HSs targeting vector to obtain the R39HSs allele (Fig. 1B). Cre/loxP recombination between the loxP site originating from the DHS1 allele and the loxP site 39 of the PGK- NeoR cassette results in the replacement of a 8.9 kb region, harboring all described 59IgH DNaseI hypersensitive sites, with a single loxP site, referred to as the DHSs allele. Germline transmission could not be achieved for either of the R39HSs or DHSs heterozygous ES cell lines. Therefore, we placed ES cells containing the R39HSs allele under increasing concentrations of G418 to select for homozygous mutant ES cells. The homozygous mutant ES cells were subsequently subjected to cre/loxP recombination to delete the Neor gene and generate ES cells homozygous for the DHSs allele. The homozygous mutant DHSs ES cells were injected into Rag2-/- blastocysts, and chimeras generated by RDBC and lymphocytes were analyzed. derived B-lymphocytes. However, FACS analysis of DHSs bone marrow B cells indicated the presence of both pro- and pre-B cells and did not suggest a block in B-cell development (Fig. 2A). the DHS1 allele. Correct targeting events (Fig1) and single integration of the targeting vector (Figure S1) were confirmed by Southern blotting. Development of homozygous RHS1, DHS1, and DHSs lymphocytes VH to DJH recombination was absent in DP T-cells from wildtype, homozygous RHS1, and homozygous DHS1 mice as the VH to DJH recombination step is restricted to the B-lineage (Fig 3C, D, E). These data show that HS1 is not PLoS ONE \| www.plosone.org PLoS November 2010 \| Volume 5 \| Issue 11 \| e13992 PLoS ONE \| www.plosone.org 5 59IgH Enhancer Figure 3. V(D)J recombination in DHS1 and RHS1 mice. Pro-B cells, pre-B cells and double positive (DP) T-cells from wildtype 129 mice, from homozygous DHS1, and homozygous RHS1 mice were sorted by FACS. 5-fold dilutions of genomic DNA were subjected to PCR analysis. IgH V(D)J recombination efficiencies were assessed using a reverse primer downstream of JH4 and a forward primer recognizing DQ52 (A), DSPs (B), the VH7183 family (C), the VHJ558 family (D), or the VHJ558.55 segment (E). Rearrangements can occur to JH1, JH2, JH3, or JH4 as indicated. GL indicates PCR product from germline configuration. Igk rearrangements were quantified (F), rearrangements can occur to Jk1, Jk2, Jk4, or Jk5 as indicated. Igl rearrangement efficiency was analyzed (G). Bands correspond to Vl2 - Jl2, Vl1 - Jl3, or Vl1 - Jl1 rearrangements as indicated. DNA input was normalized to DLG5 PCR products (H). doi:10.1371/journal.pone.0013992.g003 Figure 3. V(D)J recombination in DHS1 and RHS1 mice. Pro-B cells, pre-B cells and double positive (DP) T-cells from wildtype 129 mice, from homozygous DHS1, and homozygous RHS1 mice were sorted by FACS. 5-fold dilutions of genomic DNA were subjected to PCR analysis. IgH V(D)J recombination efficiencies were assessed using a reverse primer downstream of JH4 and a forward primer recognizing DQ52 (A), DSPs (B), the VH7183 family (C), the VHJ558 family (D), or the VHJ558.55 segment (E). Rearrangements can occur to JH1, JH2, JH3, or JH4 as indicated. GL indicates PCR product from germline configuration. Igk rearrangements were quantified (F), rearrangements can occur to Jk1, Jk2, Jk4, or Jk5 as indicated. Igl rearrangement efficiency was analyzed (G). Bands correspond to Vl2 - Jl2, Vl1 - Jl3, or Vl1 - Jl1 rearrangements as indicated. DNA input was normalized to DLG5 PCR products (H). Development of homozygous RHS1, DHS1, and DHSs lymphocytes Numbers for D to JH rearranged alleles (DJ) and nonproductive VH to DJH rearranged alleles (VDJ-) are shown. doi:10.1371/journal.pone.0013992.t001 Wildtype B cells undergo D to JH rearrangements on both alleles; but still, consistent with earlier studies, about 5% of hybridomas harbor an IgH allele in germline configuration which presumably originates from tripartite fusions involving non B-cells [12] (not shown). The number of mutant alleles in germline configuration was not increased compared to wildtype indicating that RHS1, DHS1, and DHSs alleles can undergo efficient D to JH recombination (not shown). In 50–60% of wildtype B-cells the nonproductive allele is in DJH configuration; whereas in 40–50% the nonproductive allele is in VHDJH configuration [15]. An increased percentage of DJH alleles could indicate less efficient VH to DJH recombination: in contrast, an increased percentage of VHDJH alleles might indicate a break in allelic exclusion. IgMa expressing hybridomas generated from B-cells heterozygous for RHS1, DHS1, and DHSs were analyzed for their rearrangement status of the wildtype IgMb allele and show ratios of DJH (56%– 61%), and VHDJH alleles (39%–44%) in the expected range (Table 1). IgMb expressing hybridomas were analyzed for the necessary for rendering the distal part of the VH cluster accessible and, therefore, suggest that HS1 does not play a major role in regulation of usage or accessibility of distal versus proximal VH families. Recently, it has been shown that IgH and Igk loci can colocalize during B-cell development, mainly at the pre-B cell stage, and it was suggested that this colocalization induces decontraction of the IgH locus [14]. We therefore performed an assay to evaluate Igk (Fig. 3F) and Igl (Fig. 3G) V(D)J recombination efficiencies. Both Igk and Igl loci show similar V(D)J recombination levels in the analyzed developing B cells from wildtype, homozygous RHS1, and homozygous DHS1 mice, while light chain rearrangements were absent in DP T-cells from the three different genotypes. Therefore, we conclude that deletion of HS1 does not markedly affect Ig light chain gene rearrangements. Figure 4. IgMa versus IgMb expression in DHS1, RHS1, and DHSs B cells. Heterozygous IgMa/b B-cells from spleen (A) and bone marrow (B) of 129 wildtype (wt) or DHS1, RHS1, and DHSs RDBC chimeras were analyzed IgMa and IgMb expression. In DHS1, RHS1, and DHSs B-cells the IgMb allele is in wildtype configuration whereas the IgMa allele is the mutant allele. doi:10.1371/journal.pone.0013992.g004 Figure 4. Development of homozygous RHS1, DHS1, and DHSs lymphocytes doi:10.1371/journal.pone.0013992.g003 November 2010 \| Volume 5 \| Issue 11 \| e13992 PLoS ONE \| www.plosone.org 6 59IgH Enhancer As an independent method to evaluate D to JH and VH to DJH recombination efficiencies, we generated clonal hybridoma lines from splenic B-cells of IgHa/b heterozygous RHS1, DHS1 mice carrying the mutant allele in their germline and of RDBC chimeras generated from heterozygous DHSs ES cells (Table 1). In each case the IgHa allele was the mutant allele while the IgHb allele was the wildtype allele. In splenic B-cells, one allele exists as a functional VHDJH rearrangement, while the second allele can either be in germline configuration, or it exists as a DJH or an nonproductive VHDJH rearrangement. The rearrangement status of the second IgH allele was assessed by Southern blot analysis. Consequently, hybridomas expressing the mutant IgHa allele can be analyzed for rearrangement efficiency of the wildtype IgHb allele, and vice versa, in hybridomas expressing the wildtype IgHb allele, the rearrangement status of the mutant IgHa allele can be assessed. Table 1. DHS1, RHS1, and DHSs hybridoma analysis. DJ VDJ- DHS1 IgMa+ 66 (61%) 43 (39%) DHS1 IgMb+ 52 (57%) 39 (43%) RHS1 IgMa+ 62 (56%) 49 (44%) RHS1 IgMb+ 45 (52%) 41 (48%) DHSs IgMa+ 51 (61%) 32 (39%) DHSs IgMb+ 55 (69%) 25 (31%) Hybridomas were generated from heterozygous IgMa/b DHS1, RHS1, and DHSs splenic B-cells. In each case, IgMb is the wildtype allele and IgMa is the mutant allele. IgMa expressing hybridomas (IgMa+) and IgMb expressing hybridomas (IgMb+) of each genotype were analyzed for the rearrangement status of their nonproductive allele. Numbers for D to JH rearranged alleles (DJ) and nonproductive VH to DJH rearranged alleles (VDJ-) are shown. doi:10.1371/journal.pone.0013992.t001 Hybridomas were generated from heterozygous IgMa/b DHS1, RHS1, and DHSs splenic B-cells. In each case, IgMb is the wildtype allele and IgMa is the mutant allele. IgMa expressing hybridomas (IgMa+) and IgMb expressing hybridomas (IgMb+) of each genotype were analyzed for the rearrangement status of their nonproductive allele. Numbers for D to JH rearranged alleles (DJ) and nonproductive VH to DJH rearranged alleles (VDJ-) are shown. doi:10.1371/journal.pone.0013992.t001 Hybridomas were generated from heterozygous IgMa/b DHS1, RHS1, and DHSs splenic B-cells. In each case, IgMb is the wildtype allele and IgMa is the mutant allele. IgMa expressing hybridomas (IgMa+) and IgMb expressing hybridomas (IgMb+) of each genotype were analyzed for the rearrangement status of their nonproductive allele. The DHS1, RHS1, and DHSs alleles do not affect allelic exclusion FACS analysis of wt B cells from spleen (Fig. 4A) and bone marrow (Fig. 4B) shows distinct populations of similar size for B cells that are single positive for either IgHa or IgHb, but intact allelic exclusion prevents the appearance of an obvious IgHa, IgHb double producing population. Similarly, RDBC chimeras gener- ated from heterozygous RHS1, DHS1, and DHSs ES cells exhibited IgHa or IgHb single positive B-cell populations of similar size in spleen (Fig. 4A) and bone marrow (Fig. 4B) but no IgHa, IgHb double producing population. These data indicate that the deleted sequences of the targeted alleles do not contain a regulatory element that is necessary for implementation of allelic exclusion. Development of homozygous RHS1, DHS1, and DHSs lymphocytes IgMa versus IgMb expression in DHS1, RHS1, and DHSs B cells. Heterozygous IgMa/b B-cells from spleen (A) and bone marrow (B) of 129 wildtype (wt) or DHS1, RHS1, and DHSs RDBC chimeras were analyzed IgMa and IgMb expression. In DHS1, RHS1, and DHSs B-cells the IgMb allele is in wildtype configuration whereas the IgMa allele is the mutant allele. doi:10.1371/journal.pone.0013992.g004 November 2010 \| Volume 5 \| Issue 11 \| e13992 PLoS ONE \| www.plosone.org 7 59IgH Enhancer rearrangement status of their mutant IgMa allele. RHS1, DHS1, and DHSs alleles do not show significantly increased or decreased (Fisher’s exact test) rearrangement ratios compared to wt alleles, as 52%–69% of mutant alleles were in DJH configuration while 31%–48% were in VHDJH configuration. Furthermore, data from hybridoma analysis (Tab. 1) support this notion as in the case of a break in allelic exclusion increased numbers of hybridomas with VH to DJH rearrangements on both alleles would be expected. Such an increase compared to wildtype alleles could not be observed (Tab. 1), which indicates intact allelic exclusion of RHS1, DHS1, and DHSs alleles. FACS analysis was performed on B-cells from spleens (Fig. 4A) and bone marrow (Fig. 4B) of RDBC chimeras generated from heterozygous RHS1, DHS1, and DHSs ES cells. IgMa expressing populations, representing the targeted allele, and IgMb expressing populations, representing the wildtype allele, were of similar size both in bone marrow and in spleen from RHS1, DHS1, and DHSs chimeras, suggesting that the RHS1, DHS1, and DHSs alleles can undergo V(D)J recombination at the IgH locus at similar efficiencies as wildtype alleles. The 59IgH DNaseI hypersensitive sites are not required for efficient class switch recombination To assess a potential effect of the 59IgH DNaseI hypersensitive sites on CSR, B-cells were stimulated to undergo CSR and analyzed by FACS (Fig. 5). Stimulation with LPS induces IgH isotype switching to c3, while stimulation with IL4+ aCD40 promotes switching to c1. B-cells from AID-/- mice served as negative controls, while wildtype B-cells represented a positive control and therefore switched to the appropriate isotypes under LPS or IL4+ aCD40 stimulation. CSR in homozygous DHSs B- cells occurs at similar levels as in wildtype B-cells implying that the cluster of 59IgH DNaseI hypersensitive sites is not required for efficient CSR to c1 (Fig. 5A) and c3 (Fig. 5B). The DHS1, RHS1, and DHSs alleles do not affect allelic exclusion Complex phenotypes without an obvious relation to the IgH locus in DHS1 mice Currently, we do not know what causes these phenotypes, but we exclude that this phenotype is caused by a second integration of the targeting vector at an undefined site in the genome (Figure S1). The deletion in the DHS1 allele deletes 340 bp within intron 1 of Zfp386. Therefore, age, likely due to a lack of food intake. These mice exhibit an abnormal limp grasping phenotype, i.e. mice clasp their front and hind feet almost immediately upon being lifted by their tail (Fig. 6A, B). Furthermore these mice develop a hydrocephalus, which is already visible at about one week of age and is enlarged over the following weeks (Fig. 6C, D). Histological analysis confirmed the presence of a hydrocephalus, revealed abnormal hindbrain development, and revealed retinal abnormalities (Fig. 6E, F, G). The wildtype retina is organized in a delicate layer system (Fig. 6E): stratum opticum and ganglionic layer (1), inner plexiform layer (2), inner nuclear layer (3), outer plexiform layer (4), outer nuclear layer (5), layer of rods and cones (6), pigment layer (7). In the DHS1 mutant mice, the organization of retinal layers is impaired in such a way that nuclei from the outer nuclear layer are aberrantly located in the layer of rods and cones (Fig. 6F). In some more severe cases rosette formation in the outer nuclear layer is evident (Fig. 6G). Currently, we do not know what causes these phenotypes, but we exclude that this phenotype is caused by a second integration of the targeting vector at an undefined site in the genome (Figure S1). The deletion in the DHS1 allele deletes 340 bp within intron 1 of Zfp386. Therefore, Complex phenotypes without an obvious relation to the IgH locus in DHS1 mice We performed targeted deletion experiments of the 59IgH DNaseI hypersensitive sites to test their suggested function in IgH locus regulation. So far no major IgH related phenotype was identified. However, about 20% of homozygous DHS1 mice develop a complex neurological phenotype and die at 3–5 weeks of Figure 5. Ig class switch recombination in absence of the 59IgH DNaseI hypersensitive sites. MACS purified splenic B-cells were stimulated in culture with LPS or IL4+ aCD40 as indicated. FACS analysis shows B-cells that underwent CSR as B220+ IgG1+ or B220+ IgG3+ cells, respectively. AID-/- B-cells served as negative controls, wildtype (wt) 129 B-cells as positive controls. Homozygous DHSs B-cells were isolated from RDBC chimeras. doi:10.1371/journal.pone.0013992.g005 Figure 5. Ig class switch recombination in absence of the 59IgH DNaseI hypersensitive sites. MACS purified splenic B-cells were stimulated in culture with LPS or IL4+ aCD40 as indicated. FACS analysis shows B-cells that underwent CSR as B220+ IgG1+ or B220+ IgG3+ cells, respectively. AID-/- B-cells served as negative controls, wildtype (wt) 129 B-cells as positive controls. Homozygous DHSs B-cells were isolated from RDBC chimeras. doi:10.1371/journal.pone.0013992.g005 PLoS ONE \| www.plosone.org November 2010 \| Volume 5 \| Issue 11 \| e13992 8 59IgH Enhancer 59IgH Enhancer misregulation of that poorly described gene might cause the described phenotypes although other possibilities are conceivable. age, likely due to a lack of food intake. These mice exhibit an abnormal limp grasping phenotype, i.e. mice clasp their front and hind feet almost immediately upon being lifted by their tail (Fig. 6A, B). Furthermore these mice develop a hydrocephalus, which is already visible at about one week of age and is enlarged over the following weeks (Fig. 6C, D). Histological analysis confirmed the presence of a hydrocephalus, revealed abnormal hindbrain development, and revealed retinal abnormalities (Fig. 6E, F, G). The wildtype retina is organized in a delicate layer system (Fig. 6E): stratum opticum and ganglionic layer (1), inner plexiform layer (2), inner nuclear layer (3), outer plexiform layer (4), outer nuclear layer (5), layer of rods and cones (6), pigment layer (7). In the DHS1 mutant mice, the organization of retinal layers is impaired in such a way that nuclei from the outer nuclear layer are aberrantly located in the layer of rods and cones (Fig. 6F). In some more severe cases rosette formation in the outer nuclear layer is evident (Fig. 6G). Discussion This study aimed for elucidating the potential regulatory functions of a cluster of recently described DNaseI hypersensitive sites at the 59 end of the IgH locus [9]. We performed targeted deletion of either the pro-B cell specific site HS1 (DHS1) or deletion of the entire cluster of hypersensitive sites (DHSs) in mice or in their lymphocytes, respectively. A potential regulatory element at the 59end of the IgH locus was speculated to regulate processes such as IgH allelic exclusion, VH germline transcription, differential accessibility or usage of distal versus proximal VH gene families. Furthermore, it was suggested that the 59end of the IgH locus might play a role in positioning the IgH locus in distinct subnuclear compartments [16,17,18], and it was suggested to harbor insulator or boundary capacity [19]. B- and T-lymphocytes homozygous for the DHS1, RHS1, and DHSs alleles appear to proceed through lymphocyte development in an unimpaired way. Data from RDBC chimeras generated from Figure 6. Complex phenotypes of homozygous DHS1 mice. Homozygous DHS1 mice exhibit an abnormal limp grasping phenotype (B) whereas wildtype (wt) mice do not (A). DHS1 mice can develop severe hydrocephalus as indicated by arrows in (C) and (D). A wildtype mouse without hydrocephalus is shown in (C). The wildtype retina is organized in distinct layers (E): Stratum opticum and ganglionic layer (1), inner plexiform layer (2), inner nuclear layer (3), outer plexiform layer (4), outer nuclear layer (5), layer of rods and cones (6), pigment layer (7). The retina of homozygous DHS1 mice shows external nuclei from the outer nuclear layer (5) in the layer of rods and cones (6) indicated by arrows in (F), or rosette formation of the outer nuclear layer (5) indicated by arrows in (G). doi:10.1371/journal.pone.0013992.g006 Figure 6. Complex phenotypes of homozygous DHS1 mice. Homozygous DHS1 mice exhibit an abnormal limp grasping phenotype (B) whereas wildtype (wt) mice do not (A). DHS1 mice can develop severe hydrocephalus as indicated by arrows in (C) and (D). A wildtype mouse without hydrocephalus is shown in (C). The wildtype retina is organized in distinct layers (E): Stratum opticum and ganglionic layer (1), inner plexiform layer (2), inner nuclear layer (3), outer plexiform layer (4), outer nuclear layer (5), layer of rods and cones (6), pigment layer (7). Acknowledgments We thank Drs. Meinrad Busslinger, Thomas Decker, Andrew Franklin, Cosmas Giallourakis, Gang Li, Raul Mostoslavsky, Sheila Ranganath, Jing Wang, and Shan Zha for helpful discussions, Lisa Acquaviva and Aimee Williams for mouse work, and Natasha Barteneva for cell sorting. Overall, our analysis of the deletion of the pro-B cell specific site HS1 or the whole cluster of 59IgH DNaseI hypersensitive sites did not support the existence of a cis-regulatory function of these elements regarding the IgH locus. References 11. Chen J, Lansford R, Stewart V, Young F, Alt FW (1993) RAG-2-deficient blastocyst complementation: an assay of gene function in lymphocyte development. Proc Natl Acad Sci U S A 90: 4528–4532. 1. Perlot T, Alt FW (2008) Cis-regulatory elements and epigenetic changes control genomic rearrangements of the IgH locus. Adv Immunol 99: 1–32. 1. Perlot T, Alt FW (2008) Cis-regulatory elements and epigenetic changes control genomic rearrangements of the IgH locus. Adv Immunol 99: 1–32. 2. Alt FW, Yancopoulos GD, Blackwell TK, Wood C, Thomas E, et al. (1984) Ordered rearrangement of immunoglobulin heavy chain variable region segments. EMBO J 3: 1209–1219. 12. Dudley DD, Sekiguchi J, Zhu C, Sadofsky MJ, Whitlow S, et al. (2003) Impaired V(D)J recombination and lymphocyte development in core RAG1-expressing mice. J Exp Med 198: 1439–1450. 3. Chowdhury D, Sen R (2001) Stepwise activation of the immunoglobulin mu heavy chain gene locus. EMBO J 20: 6394–6403. 13. Franco S, Gostissa M, Zha S, Lombard DB, Murphy MM, et al. (2006) H2AX prevents DNA breaks from progressing to chromosome breaks and transloca- tions. Mol Cell 21: 201–214. 4. Jung D, Giallourakis C, Mostoslavsky R, Alt FW (2006) Mechanism and control of V(D)J recombination at the immunoglobulin heavy chain locus. Annu Rev Immunol 24: 541–570. 14. Hewitt SL, Farmer D, Marszalek K, Cadera E, Liang HE, et al. (2008) Association between the Igk and Igh immunoglobulin loci mediated by the 39 Igk enhancer induces ‘decontraction’ of the Igh locus in pre-B cells. Nat Immunol 9: 396–404. 5. Chaudhuri J, Basu U, Zarrin A, Yan C, Franco S, et al. (2007) Evolution of the immunoglobulin heavy chain class switch recombination mechanism. Adv Immunol 94: 157–214. 6. Sakai E, Bottaro A, Davidson L, Sleckman BP, Alt FW (1999) Recombination and transcription of the endogenous Ig heavy chain locus is effected by the Ig heavy chain intronic enhancer core region in the absence of the matrix attachment regions. Proc Natl Acad Sci U S A 96: 1526–1531. 15. Mostoslavsky R, Alt FW, Rajewsky K (2004) The lingering enigma of the allelic exclusion mechanism. Cell 118: 539–544. 16. Kosak ST, Skok JA, Medina KL, Riblet R, Le Beau MM, et al. (2002) Subnuclear compartmentalization of immunoglobulin loci during lymphocyte development. Science 296: 158–162. 7. Perlot T, Alt FW, Bassing CH, Suh H, Pinaud E (2005) Elucidation of IgH intronic enhancer functions via germ-line deletion. Table S1 Found at: doi:10.1371/journal.pone.0013992.s003 (0.08 MB DOCX) The only observed phenotypes so far occurred in homozygous DHS1 mice and seem to be independent of the IgH locus. DHS1 mice show abnormal limp grasping indicating a neurological abnormality, DHS1 mice can develop severe hydrocephalus and exhibit retinal impairments. A possible explanation for these phenotypes is a potential defect in regulation of the zinc finger protein Zfp386. DHS1 deletes a 340 bp region from intron 1 of Zfp386 which might result in different splice forms, impaired expression levels, or expression patterns of this gene. Discussion heterozygous DHS1, RHS1, and DHSs ES cells indicated that allelic exclusion is not affected in mutant B-cells and that mutant IgH alleles can undergo efficient V(D)J recombination of their IgH locus. Furthermore, data from PCR assays to analyze V(D)J recombination efficiency in mice with HS1 deleted on both alleles supports the notion that HS1 is not necessary for neither the D to JH nor the VH to DJH recombination step. Both proximal and distal VH families as well as the distal most VH segment VHJ558.55 rearrange as efficiently as on wildtype alleles. Similarly, IgL loci in HS1 deleted B-cells rearrange at the same efficiency as wildtype IgL loci. Analysis of IgH V(D)J rearrangement status in hybridomas generated from heterozygous DHS1, RHS1, and DHSs B-cells also strengthens the idea that the deleted DNAseI hypersensitive sites would not regulate IgH V(D)J recombination. We tested for potential alterations associated with DNA end processing during V(D)J recombination by examining the CDR3 sequence obtained from homozygous DHS1 B cells and found a distribution in length that was similar to wildtype B cells [20] (Figure S2). Author Contributions Conceived and designed the experiments: TP IP PB FWA. Performed the experiments: TP IP. Analyzed the data: TP IP JPM AZ PB. Wrote the paper: TP IP FWA. Conceived and designed the experiments: TP IP PB FWA. Performed the experiments: TP IP. Analyzed the data: TP IP JPM AZ PB. Wrote the paper: TP IP FWA. Supporting Information Figure S1 Single integration of the RHS1 targeting vector. The targeting vector (targeting vector RHS1), the targeted locus (RHS1), and the wildtype (wt) IgH locus with its 59 flanking region are shown. VH, DH, JH indicate representative IgH V, D, and J segments. Exons 1, 2, and 3 of Zfp386 are shown as grey rectangles, DNaseI hypersensitive sites HS1, HS2, HS3a, and HS3b are shown as black ovals, the NeoR specific Southern probe as a black rectangle. X - XbaI. Southern analysis of XbaI digested genomic DNA from the targeted RHS1 clones 5 (lane 1) and 23 (lane 2) utilizing the NeoR specific probe shows a single 16.0 kb band. No bands are visible from untargeted wildtype ES cell DNA (lane 3). M - Fermentas 1 kb ladder. Found at: doi:10.1371/journal.pone.0013992.s001 (0.17 MB TIF) Figure S2 CDR3 length in DHS1 B cells. Peripheral B cells were isolated from one DHS1 mouse and a wildtype mouse and amplified for V558-JH4 rearrangements. Heavy Chain CDR3 lengths were calculated as the number of nucleotides between the consensus Cys residue and the Trp residue. 23 individual sequences were analyzed from DHS1 B cells and 10 from wildtype. Found at: doi:10.1371/journal.pone.0013992.s002 (0.05 MB PDF) Figure S2 CDR3 length in DHS1 B cells. Peripheral B cells were isolated from one DHS1 mouse and a wildtype mouse and amplified for V558-JH4 rearrangements. Heavy Chain CDR3 lengths were calculated as the number of nucleotides between the consensus Cys residue and the Trp residue. 23 individual sequences were analyzed from DHS1 B cells and 10 from wildtype. Found at: doi:10.1371/journal.pone.0013992.s002 (0.05 MB PDF) We tested a potential effect of the cluster of DNaseI hypersensitive site on the process of IgH CSR. Assaying class switching upon different in vitro stimulations in wildtype and homozygous DHSs B-cells let us conclude that the cluster of 59IgH DNaseI hypersensitive sites does not play a crucial role in CSR. Discussion The retina of homozygous DHS1 mice shows external nuclei from the outer nuclear layer (5) in the layer of rods and cones (6) indicated by arrows in (F), or rosette formation of the outer nuclear layer (5) indicated by arrows in (G). doi:10.1371/journal.pone.0013992.g006 Figure 6. Complex phenotypes of homozygous DHS1 mice. Homozygous DHS1 mice exhibit an abnormal limp grasping phenotype (B) whereas wildtype (wt) mice do not (A). DHS1 mice can develop severe hydrocephalus as indicated by arrows in (C) and (D). A wildtype mouse without hydrocephalus is shown in (C). The wildtype retina is organized in distinct layers (E): Stratum opticum and ganglionic layer (1), inner plexiform layer (2), inner nuclear layer (3), outer plexiform layer (4), outer nuclear layer (5), layer of rods and cones (6), pigment layer (7). The retina of homozygous DHS1 mice shows external nuclei from the outer nuclear layer (5) in the layer of rods and cones (6) indicated by arrows in (F), or rosette formation of the outer nuclear layer (5) indicated by arrows in (G). doi:10.1371/journal.pone.0013992.g006 November 2010 \| Volume 5 \| Issue 11 \| e13992 PLoS ONE \| www.plosone.org 9 59IgH Enhancer heterozygous DHS1, RHS1, and DHSs ES cells indicated that allelic exclusion is not affected in mutant B-cells and that mutant IgH alleles can undergo efficient V(D)J recombination of their IgH locus. Furthermore, data from PCR assays to analyze V(D)J recombination efficiency in mice with HS1 deleted on both alleles supports the notion that HS1 is not necessary for neither the D to JH nor the VH to DJH recombination step. Both proximal and distal VH families as well as the distal most VH segment VHJ558.55 rearrange as efficiently as on wildtype alleles. Similarly, IgL loci in HS1 deleted B-cells rearrange at the same efficiency as wildtype IgL loci. Analysis of IgH V(D)J rearrangement status in hybridomas generated from heterozygous DHS1, RHS1, and DHSs B-cells also strengthens the idea that the deleted DNAseI hypersensitive sites would not regulate IgH V(D)J recombination. We tested for potential alterations associated with DNA end processing during V(D)J recombination by examining the CDR3 sequence obtained from homozygous DHS1 B cells and found a distribution in length that was similar to wildtype B cells [20] (Figure S2). References Proc Natl Acad Sci U S A 102: 14362–14367. 17. Fuxa M, Skok J, Souabni A, Salvagiotto G, Roldan E, et al. (2004) Pax5 induces V-to-DJ rearrangements and locus contraction of the immunoglobulin heavy- chain gene. Genes Dev 18: 411–422. 8. Khamlichi AA, Pinaud E, Decourt C, Chauveau C, Cogne M (2000) The 39 IgH regulatory region: a complex structure in a search for a function. Adv Immunol 75: 317–345. 18. Yang Q, Riblet R, Schildkraut CL (2005) Sites that direct nuclear compartmentalization are near the 59 end of the mouse immunoglobulin heavy-chain locus. Mol Cell Biol 25: 6021–6030. 9. Pawlitzky I, Angeles CV, Siegel AM, Stanton ML, Riblet R, et al. (2006) Identification of a candidate regulatory element within the 59 flanking region of the mouse Igh locus defined by pro-B cell-specific hypersensitivity associated with binding of PU.1, Pax5, and E2A. J Immunol 176: 6839–6851. 19. Reddy KL, Zullo JM, Bertolino E, Singh H (2008) Transcriptional repression mediated by repositioning of genes to the nuclear lamina. Nature 452: 243–247. 20. Jolly CJ, Klix N, Neuberger MS (1997) Rapid methods for the analysis of immunoglobulin gene hypermutation: application to transgenic and gene targeted mice. Nucleic Acids Res 25(10): 1913–9. 10. Sleckman BP, Bardon CG, Ferrini R, Davidson L, Alt FW (1997) Function of the TCR alpha enhancer in alphabeta and gammadelta T cells. Immunity 7: 505–515. PLoS ONE \| www.plosone.org PLoS ONE \| www.plosone.org November 2010 \| Volume 5 \| Issue 11 \| e13992 10
https://openalex.org/W2772493264	https://europepmc.org/articles/pmc6036440?pdf=render	English	null	How well can we simulate complex hydro‐geomorphic process chains? The 2012 multi‐lake outburst flood in the Santa Cruz Valley (Cordillera Blanca, Perú)	Earth surface processes and landforms	2,018	cc-by	15,803	EARTH SURFACE PROCESSES AND LANDFORMS Earth Surf. Process. Landforms 43, 1373–1389 (2018) © 2017 The Authors. Earth Surface Processes and Landforms published by John Wiley & Sons Ltd. Published online 15 January 2018 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/esp.4318 EARTH SURFACE PROCESSES AND LANDFORMS Earth Surf. Process. Landforms 43, 1373–1389 (2018) © 2017 The Authors. Earth Surface Processes and Landforms published by John Wiley & Sons Ltd. Published online 15 January 2018 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/esp.4318 EARTH SURFACE PROCESSES AND LANDFORMS Earth Surf. Process. Landforms 43, 1373–1389 (2018) © 2017 The Authors. Earth Surface Processes and Landforms published by John Wiley & Sons Ltd. Published online 15 January 2018 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/esp.4318 Received 31 October 2016; Revised 4 December 2017; Accepted 7 December 2017 Received 31 October 2016; Revised 4 December 2017; Accepted 7 December 2017 Correspondence to: M. Mergili, Institute of Applied Geology, University of Natural Resources and Life Sciences (BOKU), Peter-Jordan-Straße 82, 1190 Vienna, Austria. E-mail: martin.mergili@boku.ac.at e under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, is properly cited. ABSTRACT: Changing high-mountain environments are characterized by destabilizing ice, rock or debris slopes connected to evolving glacial lakes. Such configurations may lead to potentially devastating sequences of mass movements (process chains or cascades). Computer simulations are supposed to assist in anticipating the possible consequences of such phenomena in order to reduce the losses. The present study explores the potential of the novel computational tool r.avaflow for simulating complex process chains. r.avaflow employs an enhanced version of the Pudasaini (2012) general two-phase mass flow model, allowing consideration of the interactions between solid and fluid components of the flow. We back-calculate an event that occurred in 2012 when a landslide from a moraine slope triggered a multi-lake outburst flood in the Artizón and Santa Cruz valleys, Cordillera Blanca, Peru, involving four lakes and a substantial amount of entrained debris along the path. The documented and reconstructed flow patterns are reproduced in a largely satisfactory way in the sense of empirical adequacy. However, small variations in the uncertain parameters can fundamentally influence the behaviour of the process chain through threshold effects and positive feedbacks. Forward simulations of possible future cascading events will rely on more comprehensive case and parameter studies, but particularly on the development of appropriate strategies for decision-making based on uncertain simulation results. © 2017 The Authors. Earth Surface Processes and Landforms published by John Wiley & Sons Ltd. KEYWORDS: r.avaflow; GLOF; high-mountain lakes; process chain; two-phase mass flow model Correspondence to: M. Mergili, Institute of Applied Geology, University of Natural Resources and Life Sciences (BOKU), Peter-Jordan-Straße 82, 1190 Vienna, Austria. E-mail: martin.mergili@boku.ac.at This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. Received 31 October 2016; Revised 4 December 2017; Accepted 7 December 2017 How well can we simulate complex hydro- geomorphic process chains? The 2012 multi-lake outburst flood in the Santa Cruz Valley (Cordillera Blanca, Perú) Martin Mergili,1,2* Adam Emmer,3,4 Anna Juřicová,3,5 Alejo Cochachin,6 Jan-Thomas Fischer,7 Christian Huggel8 and Shiva P. How well can we simulate complex hydro- geomorphic process chains? The 2012 multi-lake outburst flood in the Santa Cruz Valley (Cordillera Blanca, Perú) Pudasaini1,9 1 1 Institute of Applied Geology, University of Natural Resources and Life Sciences (BOKU), Peter-Jordan-Straße 82, 1190 Vienna, Austria 2 Geomorphological Systems and Risk Research, Department of Geography and Regional Research, University of Vienna, Universitätsstraße 7, 1010 Vienna, Austria 1 Institute of Applied Geology, University of Natural Resources and Life Sciences (BOKU), Peter-Jordan-Straße 8 Institute of Applied Geology, University of Natural Resources and Life Sciences (BOKU), Peter Jordan Straße 82, 1190 Vienna, Austria 2 Geomorphological Systems and Risk Research, Department of Geography and Regional Research, University of Vienna, Universitätsstraße 7, 1010 Vienna, Austria , , ysical Geography and Geoecology, Faculty of Science, Charles University in Prague, Albertov 6, 128 43 Pragu c Department of Physical Geography and Geoecology, Faculty of Science, Charles University in Prague, Alberto 2, Czech Republic , p 4 Department of the Human Dimensions of Global Change, Global Change Research Institute, Academy of Sciences of the Czech Republic, Bělidla 986/4a, 603 00 Brno, Czech Republic , p 4 Department of the Human Dimensions of Global Change, Global Change Research Institute, Academy of Sciences of the Czech Republic, Bělidla 986/4a, 603 00 Brno, Czech Republic Department of the Human Dimensions of Global Change, Global Change Research Institute, Academy of Sc Republic, Bělidla 986/4a, 603 00 Brno, Czech Republic p , , , p 5 Department of Soil Survey, Research Institute for Soil and Water Conservation, Zabovreska 250, 156 27, Prague 5 – Zbraslav, Czech Republic p p 5 Department of Soil Survey, Research Institute for Soil and Water Conservation, Zabovreska 250, 156 27, Prague 5 – Zbraslav, Czech Republic 6 Unidad de Glaciología y Recursos Hidricos, Autoridad Nacional del Agua, Confraternidad Internacional 167, Huaráz, Perú 7 Department of Natural Hazards, Austrian Research Centre for Forests (BFW), Rennweg 1, 6020 Innsbruck, Austria 6 Unidad de Glaciología y Recursos Hidricos, Autoridad Nacional del Agua, Confraternidad Internacional 167, Huaráz, Perú 7 Department of Natural Hazards, Austrian Research Centre for Forests (BFW), Rennweg 1, 6020 Innsbruck, Austria p , , g , , 8 Glaciology and Geomorphodynamics Group, Division of Physical Geography, Department of Geography, University of Zürich, Winterthurerstrasse 190, 8057 Zürich, Switzerland 8 Glaciology and Geomorphodynamics Group, Division of Physical Geography, Department of Geography, University of Zürich, Winterthurerstrasse 190, 8057 Zürich, Switzerland Winterthurerstrasse 190, 8057 Zürich, Switzerland 9 Department of Geophysics, University of Bonn, Meckenheimer Allee 176, 53115 Bonn, Germany 9 Department of Geophysics, University of Bonn, Meckenheimer Allee 176, 53115 Bonn, Germany Introduction Software packages such as RAMMS (Christen et al., 2010) or FLO-2D have been applied to GLOF simulations (Mergili et al., 2011). Schneider et al. (2014) and Worni et al. (2014) have coupled different numerical models for simulating mass movement – GLOF process chains. Single-phase models, how- ever, cannot describe the solid and fluid phase interactions, nor the dynamic interaction between a landslide and a lake, and the consequences thereof (propagation of displacement wave) without major workarounds (Gabl et al., 2015). Worni et al. (2014) have therefore emphasized the need for more integrated concepts. Such approaches would have to rely on two-phase flow models considering separately the solid and the fluid phase, but also the strong interactions between the phases. Pudasaini (2012) proposed such a general two-phase flow model. This model has been applied to simulate generic exam- ples representing submarine landslides and particle transport in lakes (Kafle et al., 2016), and GLOFs (Kattel et al., 2016). The computational tool r.avaflow (Mergili et al., 2017) employs an enhanced version of the Pudasaini (2012) model. Appropriate, well-documented and well-understood case studies are needed to test the suitability of this novel model and its computational implementation for real-world events. In this sense, the present article aims to: Kolka–Karmadon ice-rock avalanche in the Russian Caucasus (Huggel et al., 2005) are only some examples. ( gg , ) y p Process chains often occur related to a changing cryosphere (Evans and Delaney, 2014; Haeberli and Whiteman, 2014). Climate-induced environmental changes such as glacier re- treat, the associated formation of hanging glaciers, the expo- sure of steep, debuttressed rock or moraine walls, and the degradation of mountain permafrost facilitates the release of initial mass movements (Alean, 1985; Haeberli, 1992; Harberli et al., 1997; Huggel et al., 2003, 2010, 2012; Noetzli et al., 2006; Gruber and Haeberli, 2007; Harris et al., 2009; Ravanel and Deline, 2011; Krautblatter et al., 2013; Haeberli et al., 2017). Also the formation and evolution of glacial lakes – most often associated with retreating or downwasting glaciers – may act as the basis for the occurrence of process chains. Introduction Plenty of literature on glacial lakes and related hazards is available for many high-mountain areas worldwide (Hewitt, 1982; Haeberli, 1983; Richardson and Reynolds, 2000; Huggel et al., 2003; Breien et al., 2008; Hewitt and Liu, 2010; Bolch et al., 2011; Gruber and Mergili, 2013; Mergili et al., 2013; Clague and O’Connor, 2014; Emmer et al., 2015). The current key research challenges include the understand- ing of processes at the boundary of individual mass flows as well as their physically based modelling. For instance, despite sub- stantial progress in research on landslide generated impact waves and tsunamis the understanding of wave generation in dependence of the characteristic impacting mass movement in high mountains is still limited (Westoby et al., 2014). Similarly, key flood hazard parameters such as lake overtopping water volume and discharge can currently be poorly constrained. As shown in recent studies a critical challenge is the downstream propagation of increasing uncertainties of the parameter space, i.e. the uncertainties of initial landslide characteristics substan- tially increase downstream because of accumulating uncer- tainties at each process boundary (Schaub et al., 2016). 1. evaluate the suitability, potential and challenges of the novel computational tool r.avaflow with regard to the simu- lation of multi-lake outburst floods, employing the 2012 Santa Cruz event as the case study; 2. based on the outcomes, identify the general key challenges process chain simulation efforts have to deal with. 2. based on the outcomes, identify the general key challenges process chain simulation efforts have to deal with. Thereby we rely on a detailed analysis of the hydro- geomorphic characteristics and processes of this multi-lake outburst flood, outlined in the following section. Then we introduce the computational framework for two-phase mass flows, r.avaflow. We outline the data employed for multi-lake outburst modelling and the parameterization and modelling strategy in the 4th section, before the sections presenting, and discussing our results. Finally, in the 7th section we conclude with the key messages of the work. Well documented cases of process chain events are therefore of great importance to advance the understanding. Well documented cases of process chain events are therefore of great importance to advance the understanding. Several cases of mass movement-induced glacial lake outburst floods (GLOFs) have been documented in the previous decades. This is particularly true for the Cordillera Blanca, Peru, where the communities are highly vulnerable to this type of event (Carey, 2005). Emmer et al. Introduction (2016a) have identified 882 high-mountain lakes there, some of which are potentially hazardous (Vilímek et al., 2005; Emmer and Vilímek, 2013, 2014; Iturrizaga, 2014). A failure of the moraine dam of Lake Palcacocha in 1941 – induced by an ice avalanche or glacier calving, but possibly also by piping – caused many fatalities (Broggi, 1942; Oppenheim, 1946; Concha, 1952). More recently, GLOFs have occurred at Lake Safuna Alta in 2002 (triggered by a rock avalanche; Hubbard et al., 2005), at Lake Palcacocha in 2003 (landslide-triggered dam overtopping; Vilímek et al., 2005), and at Lake 513 in 2010 (triggered by an ice avalanche; Carey et al., 2012). In 2012, a landslide from a steep moraine wall plunged into Lake Artizón Alto and triggered a process chain in- volving four lakes and substantial entrainment of material in the Artizón and Santa Cruz valleys (called 2012 Santa Cruz event). Introduction bodies such as high-mountain lakes, or through amplification by entrainment of basal material. Process chains have led to major disasters in recorded history: the 1963 Vajont landslide and flood disaster in the Italian Alps (Genevois and Tecca, 2013), the 1970 earthquake-triggered Huascarán avalanche in the Cordillera Blanca, Peru (Evans et al., 2009) or the 2002 High-mountain areas are often characterized by tightly coupled process domains. Gravitational mass movements starting in higher areas may impact distant lower areas not only directly, but also through cascading effects sometimes involving water 1374 M. MERGILI ET AL. Kolka–Karmadon ice-rock avalanche in the Russian Caucasus (Huggel et al., 2005) are only some examples. and more recently have also been increasingly implemented in computer codes (Voellmy, 1955; Savage and Hutter, 1989; Iverson, 1997; Takahashi et al., 1992; Pitman et al., 2003; McDougall and Hungr, 2004; Pudasaini and Hutter, 2007). Software packages such as RAMMS (Christen et al., 2010) or FLO-2D have been applied to GLOF simulations (Mergili et al., 2011). Schneider et al. (2014) and Worni et al. (2014) have coupled different numerical models for simulating mass movement – GLOF process chains. Single-phase models, how- ever, cannot describe the solid and fluid phase interactions, nor the dynamic interaction between a landslide and a lake, and the consequences thereof (propagation of displacement wave) without major workarounds (Gabl et al., 2015). Worni et al. (2014) have therefore emphasized the need for more integrated concepts. Such approaches would have to rely on two-phase flow models considering separately the solid and the fluid phase, but also the strong interactions between the phases. Pudasaini (2012) proposed such a general two-phase flow model. This model has been applied to simulate generic exam- ples representing submarine landslides and particle transport in lakes (Kafle et al., 2016), and GLOFs (Kattel et al., 2016). The computational tool r.avaflow (Mergili et al., 2017) employs an enhanced version of the Pudasaini (2012) model. Appropriate, well-documented and well-understood case studies are needed to test the suitability of this novel model and its computational implementation for real-world events. In this sense, the present article aims to: and more recently have also been increasingly implemented in computer codes (Voellmy, 1955; Savage and Hutter, 1989; Iverson, 1997; Takahashi et al., 1992; Pitman et al., 2003; McDougall and Hungr, 2004; Pudasaini and Hutter, 2007). © 2017 The Authors. Earth Surface Processes and Landforms published by John Wiley & Sons Ltd. The 2012 Santa Cruz event The Santa Cruz and Artizón catchments Earth Surf. Process. Landforms, Vol. 43, 1373–1389 (2018) The Santa Cruz and Artizón catchments (a) General location; (b) Santa Cruz asnd Artizón valleys before the 2012 event; (c) Santa Cruz and Artizón valleys after the 2012 event; (d) upper part of the Artizón valley before the 2012 event; (e) upper part of the Artizón valley after the 2012 event. [Colour figure can be viewed at wileyonlinelibrary.com] valley existed in 1948: an unnamed lake downstream from today’s Lake Artizón Bajo appeared on the 1962 and 1970 images, but disappeared later. The lakes Artizón Bajo and Artizón Alto first appeared on the 1970 image (Figure 2). On 20 May 1997, a debris flow deposit from the right bank blocked the outlet of Lake Artizón Bajo, resulting in an increase of the lake level. This temporary blockage breached immedi- ately, producing a GLOF-induced debris flow which deposited in the Santa Cruz valley close to Lake Jatuncocha. Part of the Santa Cruz trekking path was damaged (Zapata, 2002). Reflecting this event, Huaman (2001) considered the lakes in the Artizón valley as hazardous and suggested the implementa- tion of remediation measures. Cochachin and Torres (2011) described Lake Artizón Alto as hazardous and indicated that the recently deglaciated, very steep left lateral moraine alongside the lake would be unstable. Considering the condi- tions before 2012, Emmer and Vilímek (2014) retrospectively identified a high GLOF susceptibility for Lake Artizón Alto. The situation before 2012 is shown in Figure 1(b) and (d). part of the Santa Cruz valley (lakes Ichiccocha and Jatuncocha; Table I) and several mostly small glacial lakes have formed in hanging tributary valleys. valley existed in 1948: an unnamed lake downstream from today’s Lake Artizón Bajo appeared on the 1962 and 1970 images, but disappeared later. The lakes Artizón Bajo and Artizón Alto first appeared on the 1970 image (Figure 2). The Artizón valley (Figure 1) is a left-side tributary to the Santa Cruz valley. The confluence is located at 3985 m a.s.l., while the highest point of the Artizón catchment is Artesonraju (6025 m a.s.l.), so that the catchment extends over a vertical distance of more than 2000 m. The Artizón catchment has an area of 8.1 km2. An area of 3.0 km2 (37% of the catchment) was occupied by glaciers in 2015, while an area of 5.6 km2 (69.1% of the catchment) was occupied by glaciers back in 1948. In 2016, two lakes were situated in Artizón valley: Artizón Bajo and Artizón Alto (Table I). The Santa Cruz and Artizón catchments On 20 May 1997, a debris flow deposit from the right bank blocked the outlet of Lake Artizón Bajo, resulting in an increase of the lake level. This temporary blockage breached immedi- ately, producing a GLOF-induced debris flow which deposited in the Santa Cruz valley close to Lake Jatuncocha. Part of the Santa Cruz trekking path was damaged (Zapata, 2002). Reflecting this event, Huaman (2001) considered the lakes in the Artizón valley as hazardous and suggested the implementa- tion of remediation measures. Cochachin and Torres (2011) described Lake Artizón Alto as hazardous and indicated that the recently deglaciated, very steep left lateral moraine alongside the lake would be unstable. Considering the condi- tions before 2012, Emmer and Vilímek (2014) retrospectively identified a high GLOF susceptibility for Lake Artizón Alto. The situation before 2012 is shown in Figure 1(b) and (d). The Santa Cruz and Artizón catchments The Santa Cruz valley is located in the Río Santa catchment in the northern part of the Cordillera Blanca, Peru (Figure 1). It oc- cupies an area of 231 km2 and extends over a vertical distance of more than 4200 m between Nevado Santa Cruz (6241 m a.s. l.) and the confluence with the Santa river at Colcas (1965 m a. s.l.). Three other summits along the catchment boundary peak above 6000 m a.s.l.: Nevado Quitaraju (6036 m a.s.l.) and Pucajirca Oeste (6039 m a.s.l.) in the north and Artesonraju (6025 m a.s.l.) in the south. Most glaciers terminate at approxi- mately 4800 m a.s.l. while large Little Ice Age (LIA) moraines are found at approximately 4400 m a.s.l., such as the moraine dams of the lakes Quitacocha, Arhueycocha and Taullicocha. The zone between 4400 m and 4800 m a.s.l. has recently been deglaciated, so that steep slopes are considered particularly susceptible to different types of mass movements (in the sense of Haeberli, 2013; Haeberli et al., 2017). Two large debris cone-dammed lakes are situated in the gently-sloped middle Even though some of the lakes in the Cordillera Blanca have been equipped with technical and structural protection mea- sures (Oppenheim, 1946; Zapata 1978; Portocarrero, 1984; Carey, 2005; Portocarrero, 2014; Emmer et al., 2016b), manag- ing the risks associated with GLOFs in an appropriate way still remains a major challenge (Carey et al., 2014). The anticipation of high-mountain process chains – and therefore also hazard mapping and zoning – largely rely on computer simulations based on mass flow models. Effectively single-phase flow models, considering either solid or fluid, or a mixture of both, have been theoretically developed during the past decades, HOW WELL CAN WE SIMULATE COMPLEX HYDRO-GEOMORPHIC PROCESS CHAINS? 1375 Figure 1. Study area before and after the event under investigation. (a) General location; (b) Santa Cruz asnd Artizón valleys before the 2012 event; (c) Santa Cruz and Artizón valleys after the 2012 event; (d) upper part of the Artizón valley before the 2012 event; (e) upper part of the Artizón valley after the 2012 event. [Colour figure can be viewed at wileyonlinelibrary.com] Figure 1. Study area before and after the event under investigation. © 2017 The Authors. Earth Surface Processes and Landforms published by John Wiley & Sons Ltd. Earth Surf. Process. Landforms, Vol. 43, 1373–1389 (2018) The 2012 multi-lake outburst flood On 8 February 2012, the Artizón and Santa Cruz valleys were affected by a multi-lake outburst flood involving two lakes in the Artizón valley and two lakes in the Santa Cruz valley (Figures 1 and 3; Table I). The process chain was preliminarily described by Emmer et al. (2014) based on the analysis of re- motely sensed images. A first field survey focusing on the Santa Cruz event was performed by the Autoridad Nacional del Agua directly after the event, while a detailed geomorphological field survey was performed in July 2015. Despite this time lag, the dominant geomorphological processes (erosion–accumulation interactions) were still identifiable in the field, due to the high magnitude of the event. The dominant geomorphological phe- nomena were mapped, described and roughly quantified, using detailed topographical measurements (laser inclinometer and rangemeter Trupulse E 200 with a resolution of 1°/0.01 m and an effective measuring range 0.5–1000 m without reflecting tar- get; GPS /GLONASS positioning with +/- 5 m accuracy). 15 cross-profiles were measured all over the affected valleys (approximately four profiles per stream line km in the upper part and one profile per stream line km in the lower part; Figure 4). These cross-profiles reflect the dominant geomorpho- logical processes, and also show clearly recognizable flood marks. These data were subsequently used for the correction of the digital terrain model (DTM) and for the spatial control of remotely sensed data-based interpretation of dominant pro- cesses and delimitation of affected areas. The trigger of the entire chain of events was a landslide re- leased from the left lateral moraine into Lake Artizón Alto (Figure 1(e) and Figure 3(a)). The moraine slopes surrounding the lake are up to 200 m high and often very steep. Based on the analysis of remotely sensed images, the volume of the trig- gering landslide was estimated to 3–8×105 m3 (Emmer et al., 2014). This relatively broad range is related to the uncertainty of the change in the bathymetry of Lake Artizón Alto after the event. Part of the landslide mass accumulated on the slopes above the lake (Figures 1(e) and 3 (a)) and on the lake shore. However, a significant mass of debris rushed into the shallower upper (southern) basin of Lake Artizón Alto. This part of the lake displayed a maximum depth up to 20 m before the event (Cochachin and Torres, 2011). Situation before 2012 Analyzing three sets of historical aerial photographs (1948, 1962 and 1970) reveals that none of the lakes in the Artizón 1376 M. MERGILI ET AL. Table I. Basic characteristics of the lakes involved in the 2012 event Longitude Latitude Dam type Catchment area (km2) Lake level elevation (m a.s.l.) Surface area (103 m2) Volume (106 m3) Max. depth (m) Reference Artizón Alto 77.6165 W 8.9482 S Bedrock dam 3.6 4639 138 1.42 24.6 Huaman (2001); Cochachin and Torres (2011) Artizón Bajo 77.6152 W 8.9438 S Moraine dam covered by debris cone accumulation 4.8 4477 32 0.36 23.3 Cochachin and Torres (2011) Jatuncocha 77.6585 W 8.9287 S Debris cone dam with artificial outlet 71.8 3870 501 7.22 23 Zapata (1978) Ichiccocha 77.6781 W 8.9376 S Debris cone dam 82.2 3828 243 Not measured (partial marshland) Not measured (partial marshland) Zapata (1978) Earth Surf. Process. Landforms, Vol. 43, 1373–1389 (2018) esses and Landforms published by John Wiley & Sons Ltd. Earth Surf. Process. Landforms, Vol. 43, 1373–1389 (2018) © 2017 The Authors. Earth Surface Processes and Landforms published by John Wiley & Sons Ltd. Earth Surf. Process. Landforms, Vol. 43, 1373–1389 (2018) The 2012 multi-lake outburst flood The landslide impact produced a water wave overtopping the bedrock dam of Lake Artizón Alto, washing away the thin moraine cover into Lake Artizón Bajo downstream. Table I. Basic characteristics of the lakes involved in the 2012 event Lake Artizón Bajo is situated directly beneath the approxi- mately 170 m high bedrock step impounding Lake Artizón Alto (Figure 3(b)). Dam overflow and sudden increase of discharge initiated a process of progressive dam erosion which eventually resulted in a release of approx. 300 000 m3 of water and caused the lake level to drop by 10 m (corresponding to the breach depth). However, lake Artizón Bajo did not empty completely, instead leaving behind a small remnant lake (Figure 3(c)), prob- ably because of decreasing shear stresses after peak discharge, decreasing slope of the outflow channel, and bedrock at the bottom of the breach inhibiting further erosion (see also Worni et al., 2012). The bedrock bottom of the Artizón valley was exposed during the event at several particularly steep locations. A large amount of moraine and colluvium material was eroded from the middle section of the Artizón valley between Lake Artizón Bajo and the outwash plain in the lower part of the val- ley (mean slope of this section >15°; Figure 1(c), (e), Figure 3(d) and Figure 4). Large boulders (up to 1 m in diameter) were first accumu- lated in the relatively gentle outwash plain (mean slope approx. 5°; Figure 3(e)). The flood wave overtopped the 20 m high banks of the outwash plain during the event, resulting in Earth Surf. Process. Landforms, Vol. 43, 1373–1389 (2018) 1377 HOW WELL CAN WE SIMULATE COMPLEX HYDRO-GEOMORPHIC PROCESS CHAINS? HOW WELL CAN WE SIMULATE COMPLEX HYDRO-GEOMORPHIC PROCESS CHAINS? Figure 2. Deglaciation of the Artizón valley between 1948 and 1970 and formation of the lakes Artizón Alto and Artizón Bajo. Source: Servicio Aerofotogramétrico Nacional. [Colour figure can be viewed at wileyonlinelibrary.com] Figure 2. Deglaciation of the Artizón valley between 1948 and 1970 and formation of the lakes Artizón Alto and Artizón Bajo. Source: Servicio Aerofotogramétrico Nacional. [Colour figure can be viewed at wileyonlinelibrary.com] Figure 3. The multi-lake outburst flood of 8 February 2012. © 2017 The Authors. Earth Surface Processes and Landforms published by John Wiley & Sons Ltd. Earth Surf. Process. Landforms, Vol. 43, 1373–1389 (2018) th Surface Processes and Landforms published by John Wiley & Sons Ltd. Earth Surf. Process. Landforms, Vol. 43, 1373–1389 (201 The 2012 multi-lake outburst flood (a) Steep moraine slopes surrounding Lake Artizón Alto with the 2012 l rock step between the lakes Artizón Alto sand Artizón Bajo, impounding lake Artizón Alto; (c) failed dam of Lake Artizón Bajo; (d) m Artizón valley leading into an outwash plain; (e) outwash plain; (f) confluence of Artizón and Santa Cruz streams; (g) Santa Cruz val by fine-grained deposits; (h) damaged outlet of Lake Jatuncocha. Photos: Adam Emmer, 7 and 8 July 2015. [Colour figure c wileyonlinelibrary.com] h h h f d df bl h d b h l d h f df l Figure 3. The multi-lake outburst flood of 8 February 2012. (a) Steep moraine slopes surrounding Lake Artizón Alto with the 2012 landslide; (b) bed- rock step between the lakes Artizón Alto sand Artizón Bajo, impounding lake Artizón Alto; (c) failed dam of Lake Artizón Bajo; (d) middle part of the Artizón valley leading into an outwash plain; (e) outwash plain; (f) confluence of Artizón and Santa Cruz streams; (g) Santa Cruz valley floor covered by fine-grained deposits; (h) damaged outlet of Lake Jatuncocha. Photos: Adam Emmer, 7 and 8 July 2015. [Colour figure can be viewed at wileyonlinelibrary.com] © 2017 The Authors. Earth Surface Processes and Landforms published by John Wiley & Sons Ltd. Earth Surf. Process. Landforms, Vol. 43, 1373–1389 (2018) © 2017 The Authors. Earth Surface Processes and Landforms published by John Wiley & Sons Ltd. 1378 M. MERGILI ET AL. Figure 4. Longitudinal profile through the Artizón and Santa Cruz valleys with the dominant processes and the locations of the cross-profiles be- tween Lake Artizón Alto and Lake Ichiccocha. [Colour figure can be viewed at wileyonlinelibrary.com] Figure 4. Longitudinal profile through the Artizón and Santa Cruz valleys with the dominant processes and the locations of the cross-profiles be- tween Lake Artizón Alto and Lake Ichiccocha. [Colour figure can be viewed at wileyonlinelibrary.com] (Figure 3(h)). A marginal amount of material, compared with the amount transported upstream, was eroded downstream from Lake Jatuncocha and deposited in Lake Ichiccocha. The increased discharge also caused bank erosion and associated accumulation processes downstream in the Santa Cruz valley. damage to the prevailing shrub vegetation. The 2012 multi-lake outburst flood In the lower part of the outwash plain, the smooth Last Glacial Maximum (LGM) moraine ridge dividing the Artizón valley and the Santa Cruz valley was overtopped, and the flood wave reached the Santa Cruz stream 1 km upstream of the confluence (Figure 3(e)), washing away soil and vegetation (shrub) cover in the area affected. Part of the Artizón stream between the outwash plain and the confluence with the Santa Cruz stream is relatively steep (mean slope >10°). Erosion again dominated in this part of the valley, exposing the bedrock (Figure 4). © 2017 The Authors. Earth Surface Processes and Landforms published by John Wiley & Sons Ltd. h Surface Processes and Landforms published by John Wiley & Sons Ltd. Earth Surf. Process. Landforms, Vol. 43, 1373–1389 (2018 HOW WELL CAN WE SIMULATE COMPLEX HYDRO-GEOMORPHIC PROCESS CHAINS? 1379 areas with observed mass flow impact are referred to as ob- served positives (OP), the ones without observed mass flow impact as observed negatives (ON). All areas with simulated mass flow impact are considered as predicted positives (PP), the ones without simulated mass flow impact as predicted neg- atives (PN). In the present work we relate the true positive (TP), true negative (TN), false positive (FP) and false negative (FN) predictions in order to derive the performance indicators criti- cal success index (CSI), distance to perfect classification (D2PC), and factor of conservativeness (FoC) (Formetta et al., 2015; Mergili et al., 2017; Table II). While CSI and D2PC indi- cate the degree of overlay between modelled and observed im- pact areas, FoC measures whether the simulation overestimates (conservative result), or underestimates the observed impact area (non-conservative result). The Pudasaini (2012) two-phase mass flow model is employed for routing mass flows from one or more defined re- lease areas through the mountain topography given by a digital terrain model (DTM). Material can be entrained from the basal surface. Arbitrary assemblages of solid and fluid release heights and entrainable heights may be defined. r.avaflow further relies on a set of flow parameters (see Mergili et al., 2017 for a de- tailed list of input parameters). The output of r.avaflow essen- tially consists of raster maps – maximum and for all time steps – of solid and fluid flow heights H (m), velocities, pressures, kinetic energies, and entrained heights E (m). Optionally, solid and fluid output hydrographs are computed. areas with observed mass flow impact are referred to as ob- served positives (OP), the ones without observed mass flow impact as observed negatives (ON). All areas with simulated mass flow impact are considered as predicted positives (PP), the ones without simulated mass flow impact as predicted neg- atives (PN). In the present work we relate the true positive (TP), true negative (TN), false positive (FP) and false negative (FN) predictions in order to derive the performance indicators criti- cal success index (CSI), distance to perfect classification (D2PC), and factor of conservativeness (FoC) (Formetta et al., 2015; Mergili et al., 2017; Table II). While CSI and D2PC indi- cate the degree of overlay between modelled and observed im- pact areas, FoC measures whether the simulation overestimates (conservative result), or underestimates the observed impact area (non-conservative result). Simulation input We use a TanDEM-X Digital Elevation Model (DEM) from 2013 (after the 2012 event). The DEM is available with a 10 m post- ing from TanDEM-X acquisitions performed along ascending and descending orbits on 10 October 2013 and 24 January 2013, respectively, combined to reduce problems of areas masked by layover and shadow which affect differently oppo- site slopes along north–south oriented valleys in mountainous regions (Wegmüller et al., 2014). The DEM is corrected for entrainment that has occurred during the event, based on a de- tailed field survey, the comparison of pre-event and post-event Landsat 7/8 and Google Earth Pro® v. 7.1.5.1557 high resolu- tion scenes (CNES/Astrium images), and topographic plausibil- ity. Further, the lakes Artizón Alto, Artizón Bajo and Jatuncocha are removed by using bathymetric data (Zapata 1978; Cochachin and Torres, 2011) in order to derive a digital terrain model (DTM) displaying the basal surface of the lakes. This DTM suffers from low quality particularly in the upper section of the Artizón Valley (surrounding the lakes Artizón Alto and Artizón Bajo) so that further manual pre-processing has to be applied (Figure 6). This step builds mainly on the well- documented pre-event level of the two upper lakes (Table I), the same pre-event Google Earth Pro® scenes as used for the estimation of entrainment, and field evidence. Given the sound basis for pre-processing, we expect the resulting error to be manageable. However, the error cannot be quantified due to missing reference data. Optional complementary functions include diffusion control, conservation of volume, surface control, entrainment, and stopping (Mergili et al., 2017). Entrainment is computed through an empirical approach, multiplying a user-defined, conceptual entrainment coefficient CE (kg-1) with the momen- tum of the flow. Solid and fluid flow heights and momenta as well as the change of the basal topography are updated in time. r.avaflow operates on the basis of GIS raster cells. The analyt- ical model equations are solved with the high resolution total variation diminishing non-oscillatory central differencing (TVD-NOC) scheme (Nessyahu and Tadmor, 1990). This scheme is commonly applied to mass flow problems (Wang et al., 2004). It uses a staggered grid, whereby the system is moved half of the numerical cell size with every time step. HOW WELL CAN WE SIMULATE COMPLEX HYDRO-GEOMORPHIC PROCESS CHAINS? The propagation of the flow is computed through depth- averaged mass and momentum conservation equations for the solid and fluid components. Thereby the Mohr-Coulomb plas- ticity is employed for computing the solid stress, while a solid-volume-fraction-gradient-enhanced non-Newtonian vis- cous stress is applied to the fluid. Generalized viscous drag and virtual mass induced by relative acceleration between the phases are employed to account for the interfacial momentum transfer. Similarly, the model includes buoyancy. Strong cou- pling between the solid and the fluid momentum transfers leads to simultaneous deformation, mixing, and separation of the phases. The full details of the dynamical model are given by Pudasaini (2012). We refer to Mergili et al. (2017) for further details on the r.avaflow computational framework. The model codes, a user manual, a collection of test data and the associated start scripts are available at http://www.avaflow.org. The simulation framework r.avaflow The comprehensive GIS-based open source computational framework r.avaflow was designed for simulating the propaga- tion of various types of geomorphic mass flows. In contrast to most other related tools it employs a real-two-phase-flow model and considers entrainment and deposition of material along the path, and is therefore suitable for the simulation of process chains and interactions. r.avaflow is described in full detail by Mergili et al. (2017). Essentially, only those aspects relevant for the back-calculation of the 2012 Santa Cruz pro- cess chain are outlined in this section. The logical framework of r.avaflow is illustrated in Figure 5. At the confluence with the Santa Cruz stream (Figure 3(f)), the flood wave spread across the up to 450 m wide Santa Cruz valley with a gentle mean slope of <2°. Accumulation of large boulders occurred directly at the confluence, while fine-grained material was deposited along the entire 3.5 km long valley section between the confluence and Lake Jatuncocha (Figure 1(c), Figure 3(g), and Figure 4). Lake Jatuncocha retained the rest of the solid material transported. The outlet of Lake Jatuncocha equipped since 1969 with an open cut and an arti- ficial dam resisted the increased water discharge. Only minor damage was recorded at the downstream face of the dam Figure 5. Logical framework of r.avaflow. Only those elements relevant for the present study are shown. See Mergili et al. (2017) for a comprehen- sive logical framework. [Colour figure can be viewed at wileyonlinelibrary.com] © 2017 The Authors. Earth Surface Processes and Landforms published by John Wiley & Sons Ltd. Earth Surf. Process. Landforms, Vol. 43, 1373–1389 (2018) Figure 5. Logical framework of r.avaflow. Only those elements relevant for the present study are shown. See Mergili et al. (2017) for a comprehen- sive logical framework. [Colour figure can be viewed at wileyonlinelibrary.com] © 2017 The Authors. Earth Surface Processes and Landforms published by John Wiley & Sons Ltd. Earth Surf. Process. Landforms, Vol. 43, 1373–1389 (2018) © 2017 The Authors. Earth Surface Processes and Landforms published by John Wiley & Sons Ltd. Earth Surf. Process. Landforms, Vol. 43, 1373–1389 (2018) HOW WELL CAN WE SIMULATE COMPLEX HYDRO-GEOMORPHIC PROCESS CHAINS? Simulation input In the present work we use the distribution r.avaflow [EXPERT] (Mergili et al., 2017), implemented as a raster module of the open source software package GRASS GIS 7 (GRASS Development Team, 2017), and building on the Python and C programming languages and the R software environment for statistical computing and graphics (R Core Team, 2017). Paral- lel processing exploiting multiple cores is enabled, facilitating multiple model runs, the computation of impact and deposition indicator scores, and parameter sensitivity analysis and optimi- zation. r.avaflow was designed for UNIX operating systems and tested on Ubuntu 12.04, 14.04 and 16.04 LTS and Scientific Linux 6.6 (Red Hat). It is most efficiently managed on the shell script level. The extent and depth of the release mass of the moraine slide are approximated from comparison of the same Landsat and Google Earth Pro® scenes used for the estimation of entrain- ment, and from field estimates of the displaced debris volume. Reconstruction yields a release volume VR ≈5.3×105 m3 and is therefore well in the range estimated (section ‘The 2012 multi- The computational framework produces map layouts and animations of the main results. The model results are further validated against the observed impact area, if available. Those Table II. Performance indicators used in the present study (Formetta et al., 2015; Mergili et al., 2017) Name Definition Possible range Optimum Factor of conservativeness (FoC) FoC ¼ PP OP ¼ TPþFP TPþFN [0,∞] 1.0 Critical success index (CSI) CSI ¼ TP TPþFPþFN [0,1] 1.0 Distance to perfect classification (D2PC) D2PC ¼ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 1 rTP ð Þ2 þ r2 FP q [0,1] 0.0 rTP ¼ TP OP, rFP ¼ FP ON © 2017 The Authors. Earth Surface Processes and Landforms published by John Wiley & Sons Ltd. Earth Surf. Process. Landforms, Vol. 43, 1373–1389 (2018) Table II. Performance indicators used in the present study (Formetta et al., 2015; Mergili et al., 2017) © 2017 The Authors. Earth Surface Processes and Landforms published by John Wiley & Sons Ltd. Earth Surf. Process. Landforms, Vol. 43, 1373–1389 (2018) © 2017 The Authors. Earth Surface Processes and Landforms published by John Wiley & Sons Ltd. 1380 M. MERGILI ET AL. Figure 6. Spatial input and reference data for simulation of the 2012 Santa Cruz process chain with r.avaflow. The bold black lines separate the zones I–III. All raster maps are produced at a cell size of 5 m. Simulation input [Colour figure can be viewed at wileyonlinelibrary.com] Figure 6. Spatial input and reference data for simulation of the 2012 Santa Cruz process chain with r.avaflow. The bold black lines separate the zones I–III. All raster maps are produced at a cell size of 5 m. [Colour figure can be viewed at wileyonlinelibrary.com] lake outburst flood’ and Figure 6). The release height HR is adapted accordingly for each computational experiment in order to tune the release volumes to be tested (Table III). The lakes Artizón Alto, Artizón Bajo and Jatuncocha are considered as fluid release masses. However, they are triggered only when impacted by a moving mass. The distribution of the fluid release heights (i.e. of the lake depths) is derived from the bathymetric surveys available for all three lakes (Zapata, 1978; Cochachin and Torres, 2011). All release heights are imposed onto the DTM. TanDEM-X data only support a spatial resolution of 10 m, re- sampling to 5 m is considered useful for exploring the influence of the computational cell size on the results (section ‘Experi- ment E2: initial stage of process chain, sensitivity to cell size’). We perform six computational experiments (E1–E6) for the 2012 Santa Cruz event (Table III). Preliminary simulations have suggested that the results in terms of the travel distance, impact area, and entrained volumes are sensitive to the uncertain initial landslide volume VR, and to the uncertain basal friction angle δ and entrainment coefficient CE. While these parameters and the cell size employed for the simulations are varied within and between the experiments, all other parameter values are kept constant in accordance with Mergili et al. (2017). The in- ternal friction angle is φ = 35°. Further, all experiments build on the same set of basic assumptions: The distribution of the entrainable height of unconsolidated basal material is approximated from the reconstruction of the pre-event topography, according to the findings of the geomor- phologic field analysis and remotely sensed data. We arrive at a total entrained volume between Lake Artizón Bajo and the con- fluence of the Artizón and Santa Cruz valleys ERec ≈1 × 106 m3. Also the mass deposited in the Santa Cruz Valley down to Lake Jatuncocha is estimated with DRec ≈1 × 106 m3. Simulation input As- suming that much of the initial landslide release volume was deposited within and close to Lake Artizón Alto, and some of the solid material was deposited in Lake Jatuncocha, we con- sider these two estimates consistent and plausible. All raster maps serving as input for r.avaflow are prepared at a cell size of 5 m and are illustrated in Figure 6. Even though the original • Both the landslide release mass and the entrainable material are in a state near or at saturation. Precipitation had been re- corded at the stations nearby the days before, but the real state of saturation remains unknown and was most likely more complex. We apply rates of 70% solid and 30% fluid per volume – an estimate considered reasonable for the ma- terials involved – throughout all the experiments E1–E6. The lakes are considered 100% fluid. Table III. Computational experiments performed with r.avaflow for the Santa Cruz process chain. In Experiment 3,VR is varied by adapting the release height. δI, δII and δIII refer to the values of the basal friction angle of the zones I, II and III. The entrainment coefficient CE is assumed constant throughout the potential entrainment area (Figure 6). A full list of input parameters of r.avaflow is provided by Mergili et al. (2017) Table III. Computational experiments performed with r.avaflow for the Santa Cruz process chain. In Experiment 3,VR is varied by adapting the release height. δI, δII and δIII refer to the values of the basal friction angle of the zones I, II and III. The entrainment coefficient CE is assumed constant throughout the potential entrainment area (Figure 6). A full list of input parameters of r.avaflow is provided by Mergili et al. (2017) Experiment Description VR 105 m3 δI degree δII degree δIII degree CE kg-1 Cell size m E1 Reference parameters 8.0 10 20 0 10-6.45 10 E2* Sensitivity to cell size 8.0 10 20 0 10-6.45 5–10 E3 Sensitivity to VR and δI 3.0–8.0 10–18 20 0 10-6.45 10 E4 Sensitivity to δII 8.0 10 14–26 0 10-6.45 10 E5 Sensitivity to δIII 8.0 10 20 0–6 10-6.45 10 E6 Sensitivity to CE 8.0 10 20 0 10-6.55–10-6.35 10 Experiment E2 considers only the initial part of the process chain until t = 100 s. © 2017 The Authors. Earth Surface Processes and Landforms published by John Wiley & Sons Ltd. Earth Surf. Process. © 2017 The Authors. Earth Surface Processes and Landforms published by John Wiley & Sons Ltd. Earth Surf. Process. Lan Earth Surface Processes and Landforms published by John Wiley & Sons Ltd. Earth Surf. Process. Landforms, Vol. 43, 1373–1389 ( © 2017 The Authors. Earth Surface Processes and Landforms published by John Wiley & Sons Ltd. Experiment E1: reference simulation configuration We only consider the part of the process chain starting from the release of the initial landslide down to Lake Jatuncocha, disregarding the minor flood wave downstream from Lake Jatuncocha. Simulation times of t = 600 s (experiment E1), t = 100 s (Experiment E2), and t = 400 s (all other experiments) are applied. Experiment E1 builds on an ad hoc assumption of the key parameters (VR, δ, CE, cell size) in terms of qualitative and quantitative correspondence with the event documenta- tion and computational efficiency. In the experiments E2–E6 we vary each of the assumed parameters in order to explore the sensitivity of the model results and performance and to jus- tify the choice of the values used in experiment E1 (Table III). The observed impact area between Lake Artizón Bajo and Lake Jatuncocha (zones II and III) is used as reference for model val- idation (Figure 6). The impact area in zone I is not well defined through the event documentation and therefore neglected as The results in terms of the simulated impact areas (maximum flow height HMax ≥0.2 m) are shown in Figure 7 and corre- spond satisfactorily well to the event documentation with pa- rameter assumptions of δI = 10°, δII = 20°, and δIII = 0°, CE = 10-6.45 kg-1, and a cell size of 10 m (Table III and Table IV). However, we particularly note that: (i) lateral spreading of the flow is overestimated in the Artizón valley down to the outwash plain by locally up to approximately 180 m on one side; (ii) run-up on the slopes of the Santa Cruz valley is overestimated (locally up to 120 m, compared with very limited observed run-up); and (iii) the simulated flow, in contrast to the observed flow, does not impact the entire width of the Santa Cruz valley all the way down to Lake Jatuncocha. Figure 7 illus- trates the flow height evolution from t = 0 s to t = 450 s, and the Figure 7. Flow height maps derived in the computational experiment E1 for selected points in time, and for the maximum over the entire simulation (HMax). The dashed yellow line represents the main flow path (defined manually from the DTM and satellite imagery), the solid red line the observed impact area of the process chain down to Lake Jatuncocha. O1–O4 represent the output hydrographs (Figure 6 and Figure 8). Simulation input Landforms, Vol. 43, 1373–1389 (2018) © 2017 The Authors. Earth Surface Processes and Landforms published by John Wiley & Sons Ltd. Earth Surf. Process. Landforms, Vol. 43, 1373–1389 (2018) HOW WELL CAN WE SIMULATE COMPLEX HYDRO-GEOMORPHIC PROCESS CHAINS? 1381 • We divide the flow path into three zones I, II and III, differing in terms of δ and the possibility of entrainment. Zone I covers the initial part of the process chain down to Lake Artizón Bajo, zone II the potential entrainment area down to the confluence of the Artizón and Santa Cruz Valleys, and zone III the Santa Cruz Valley downstream from the conflu- ence (Figure 6). well as the lakes themselves and the area downstream from Lake Jatuncocha. Further, we do not evaluate the simulation results (or adapt the parameters respectively) in terms of flow velocities or travel times as no field observations of these parameters are available. r.avaflow simulation results • CE is constant throughout the potential entrainment area. • CE is constant throughout the potential entrainment area. © 2017 The Authors. Earth Surface Processes and Landforms published by John Wiley & Sons Ltd. th Surface Processes and Landforms published by John Wiley & Sons Ltd. Earth Surf. Process. Landforms, Vol. 43, 1373–1389 (201 Earth Surf. Process. Landforms, Vol. 43, 1373–1389 (2018) 7 The Authors. Earth Surface Processes and Landforms published by John Wiley & Sons Ltd. Earth Surf. Process. Landforms, Vol Experiment E1: reference simulation configuration [Colour figure can be viewed at wileyonlinelibrary.com] h h h f d df bl h d b h l d h f df l Figure 7. Flow height maps derived in the computational experiment E1 for selected points in time, and for the maximum over the entire simulation (HMax). The dashed yellow line represents the main flow path (defined manually from the DTM and satellite imagery), the solid red line the observed impact area of the process chain down to Lake Jatuncocha. O1–O4 represent the output hydrographs (Figure 6 and Figure 8). [Colour figure can be viewed at wileyonlinelibrary.com] © 2017 The Authors. Earth Surface Processes and Landforms published by John Wiley & Sons Ltd. Earth Surf. Process. Landforms, Vol. 43, 1373–1389 (2018) © 2017 The Authors. Earth Surface Processes and Landforms published by John Wiley & Sons Ltd. M. MERGILI ET AL. 1382 Table IV. Validation summary for the computational experiments E3–E6. Refer to Table 2 for the description of the performance indicators CSI, D2PC, and FoC. Bold letters refer to the optimized parameter set and scores (experiment E1) (Mergili et al., 2017), a generalization that possibly leads to an overestimate of the momentum. This effect is compensated by increasing the basal friction. On the other hand, a value of δIII very close to zero (approximated by δIII = 0°) is needed to prop- agate the flow down to Lake Jatuncocha. Such a low value can partly be justified by the interaction of the flow with a saturated valley bottom. However, in the context of this computational experiment δI, δII, and δIII cannot be considered as purely physical parameters, but rather as the result of an optimization procedure, aggregating various effects (experiments E3–E5; Table II; Discussion). Experiment E1: reference simulation configuration Experiment E3 – VR = 3 · 105 m3 δI (°) CE (kg-1) CSI D2PC FoC VE (106 m3) 10 10-6.45 0.47 0.47 0.66 0.15 15 10-6.45 0.02 0.98 0.02 0.01 20 10-6.45 0.02 0.98 0.02 0.00 Experiment E3 – VR = 8 · 105 m3 δI CE CSI D2PC FoC VE 10 10-6.45 0.59 0.20 1.21 1.09 15 10-6.45 0.34 0.60 0.60 0.32 20 10-6.45 0.04 0.96 0.05 0.02 Experiment E4 δII CE CSI D2PC FoC VE 14 10-6.45 0.56 0.15 1.56 1.44 20 10-6.45 0.59 0.20 1.21 1.09 26 10-6.45 0.40 0.48 0.85 0.70 Experiment E5 δIII CE CSI D2PC FoC VE 0 10-6.45 0.59 0.20 1.21 1.09 2 10-6.45 0.52 0.30 1.07 1.09 6 10-6.45 0.40 0.47 0.88 1.09 Experiment E6 δ CE CSI D2PC FoC VE Raster map 10-6.55 0.58 0.19 1.25 0.76 Raster map* 10-6.45 0.59 0.20 1.21 1.09 Raster map* 10-6.35 0.58 0.22 1.15 1.60 *δ is varied spatially, using the optimum values for the zones I, II, and III. The hydrographs O1–O3 (Figure 6 and Figure 8) clearly show a sudden peak in fluid and solid discharge at the point of time the flood wave passes through. The decrease of the dis- charge being slower than the increase reveals a strong frontal surge of solid and fluid. All three hydrographs suggest a rapid run-through of the main flood wave (few tens of seconds), with substantial residual discharge afterwards only observed at O1. O4 displays a second, slower, increase and still records a nota- ble simulated discharge at t = 600 s. Also the effects of entrainment are documented in the out- put hydrographs: while the solid content in O1 originates from the initial landslide, the increasing solid fraction from O2–O4 reflects the entrainment along the flow path. With CE = 10-6.45 kg-1 the simulated entrained volume is VE = 1.09×106 m3, corresponding well to the reconstructed entrained volume of 1×106 m3. Therefore we consider CE = 10-6.45 kg-1 a reasonable assumption. However, lowering of the dam level of Lake Artizón Bajo – and therefore also the degree of emptying of this lake – is underestimated, compared with field observations: the simulated total fluid discharge at the hydrograph O2 directly downstream from Lake Artizón Bajo sums up to approx. 250 000 m3, the same magnitude as the estimated 300 000 m3 release from the lake. © 2017 The Authors. Earth Surface Processes and Landforms published by John Wiley & Sons Ltd. Experiment E2: initial stage of process chain, sensitivity to cell size stage of the phenomenon, i.e. the release volume and δI. Figure 10 shows the simulated impact areas in terms of HMax ≥0.2 m, obtained with six combinations of VR and δI. With the combination of VR = 3.0×105 m3 and δI = 20°, r.avaflow does not simulate overtopping of the dam of Lake Artizón Alto and, consequently, no flood wave downstream. WithVR = 3.0×105 m3 and δI = 15°, and withVR = 8.0×105 m3 and δI = 20°, the flow alleviates in Lake Artizón Bajo. The simulated flood wave proceeds farther downstream with the other combinations listed in Table IV. Figure 11 illustrates the depen- dency of the occurrence of overtopping and the development of a process chain on the combination of VR and δI. The process chain reaches Lake Jatuncocha only with VR = 8.0×105 m3 and δI = 10°. Run-up against the slopes of the Santa Cruz valley is more pronounced with larger release volumes. With VR = 8.0×105 m3 and δI ≤15° excessive spreading of the flow is sim- ulated in the Artizón valley. stage of the phenomenon, i.e. the release volume and δI. Figure 10 shows the simulated impact areas in terms of HMax ≥0.2 m, obtained with six combinations of VR and δI. With the combination of VR = 3.0×105 m3 and δI = 20°, r.avaflow does not simulate overtopping of the dam of Lake Artizón Alto and, consequently, no flood wave downstream. WithVR = 3.0×105 m3 and δI = 15°, and withVR = 8.0×105 m3 and δI = 20°, the flow alleviates in Lake Artizón Bajo. The simulated flood wave proceeds farther downstream with the other combinations listed in Table IV. Figure 11 illustrates the depen- dency of the occurrence of overtopping and the development of a process chain on the combination of VR and δI. The process chain reaches Lake Jatuncocha only with VR = 8.0×105 m3 and δI = 10°. Run-up against the slopes of the Santa Cruz valley is more pronounced with larger release volumes. With VR = 8.0×105 m3 and δI ≤15° excessive spreading of the flow is sim- ulated in the Artizón valley. The flow should be a sufficient number of cells wide in order to ensure a proper numerical treatment. HOW WELL CAN WE SIMULATE COMPLEX HYDRO-GEOMORPHIC PROCESS CHAINS? 1383 Experiment E2: initial stage of process chain, sensitivity to cell size Even if we assume that 10 m DTM resolution is sufficient for capturing the topographic characteristics relevant for the flow, and despite a cell size of 5 m represents an oversampling, simulations at the finer resolu- tion assist in evaluating whether 10 m resolution are also suffi- cient from a numeric point of view. Experiment 2 considers in detail the initial stage of the process chain (until downstream from Lake Artizón Bajo) and thereby evaluates the effect of the cell size applied to the simulation on the patterns and evolution of the flow height (Figure 9; Table III). The landslide reaches Lake Artizón Alto after a few seconds and causes an impact wave running up the opposite slope to- wards NE (t = 20 s). Thereby, the maximum simulated value of H = 102 m (10 m cell size) and 114 m (5 m cell size), respec- tively. The wave moves back into Lake Artizón Alto and over- tops the bedrock dam of the lake. The resulting flood wave impacts Lake Artizón Bajo situated downstream (t = 70 s). The resulting wave in Lake Artizón Bajo overtops its moraine/debris cone dam, triggering erosion of the dam mate- rial and a debris flood propagating farther downstream (t = 100 s; Figure 7 and Figure 8). Except for the difference in the maximum value of H as well as some details in the flow patterns the simulations at 10 m and 5 m cell size yield fairly identical results. Consequently, we consider a cell size of 10 m acceptable for the purpose of the present study. VE is severely underestimated compared with the reconstruc- tion for all experiments except VR = 8.0×105 m3 and δI = 10° (Table IV). Both CSI and D2PC indicate the best model perfor- mance with VR = 8.0×105 m3 and δI = 10°. This combination expectedly also yields the most conservative result (highest value of FoC). Experiment E4: sensitivity to the basal friction in zone II Figure 12 illustrates the sensitivity of the distribution of HMax on δII (assumption of basal friction angle in zone II), leaving δI and δIII unchanged (Table IV). The flow reaches Lake Jatuncocha with δII ≤20°. Furthermore, lower values of δII lead to a higher degree of lateral spreading than higher values of δII, which is reasonable. With values of δII = 14° r.avaflow simulates an overflow of Lake Jatuncocha before t = 400 s. These Experiment E1: reference simulation configuration However, the simulated volume is supposed to in- clude also water from Lake Artizón Alto, and the rapid drop of the discharge after its peak to almost zero indicates a very limited degree of progressive entrainment and lowering of the dam (Figure 8(b)). maximum flow height throughout the simulation period. The period from t = 0 s until t = 100 s is considered in detail in experiment E2. The differentiation of δ most probably aggregates various aspects of the complex interaction of the flow with the basal surface. On the one hand, δII > δI > δIII results from entrainment: r.avaflow considers full momentum uptake through entrainment Figure 8. Output hydrographs O1–O4 generated in experiment E1 (see Figure 6 for the location of the hydrograph profiles). Columns represent the discharge, lines the flow height. The solid component is shown above, the fluid component below the horizontal line. Note that the fluid components are drawn in negative direction in order to enhance the readability of the figure. (a) Output hydrograph O1; (b) output hydrograph O2; (c) output hydrograph O3; (d) output hydrograph O4. [Colour figure can be viewed at wileyonlinelibrary.com] © 2017 The Authors. Earth Surface Processes and Landforms published by John Wiley & Sons Ltd. Earth Surf. Process. Landforms, Vol. 43, 1373–1389 (2018) Figure 8. Output hydrographs O1–O4 generated in experiment E1 (see Figure 6 for the location of the hydrograph profiles). Columns represent the discharge, lines the flow height. The solid component is shown above, the fluid component below the horizontal line. Note that the fluid components are drawn in negative direction in order to enhance the readability of the figure. (a) Output hydrograph O1; (b) output hydrograph O2; (c) output hydrograph O3; (d) output hydrograph O4. [Colour figure can be viewed at wileyonlinelibrary.com] 2017 The Authors. Earth Surface Processes and Landforms published by John Wiley & Sons Ltd. Earth Surf. Process. Landforms, Vol. 43, 1373–1389 (2018) © 2017 The Authors. Earth Surface Processes and Landforms published by John Wiley & Sons Ltd. Earth Surf. Process. Landforms, Vol. 43, 1373–1389 (2018) HOW WELL CAN WE SIMULATE COMPLEX HYDRO-GEOMORPHIC PROCESS CHAINS? Experiment E3: importance of the initial stage Experiment E3 (Table II) demonstrates the sensitivity of the pro- cess chain simulation to the assumptions concerning the initial e Authors. Earth Surface Processes and Landforms published by John Wiley & Sons Ltd. Earth Surf. Process. Landforms, Vol. 43, 1 © 2017 The Authors. Earth Surface Processes and Landforms published by John Wiley & Sons Ltd. Earth Surf. Process. Landforms, Vol. 43, 1373–1389 (2018) Within the tested range of assumptions, entrainment influences the general pattern of HMax ≥0.2 m to a relatively minor degree only, so that the validation scores – except for FoC – are poorly differentiated (Table IV). However, with CE = 10-6.35 kg-1 the flow stops approximately 150 m before reaching Lake Jatuncocha. Higher values of CE further result in less pro- nounced run-up and less lateral spreading in the Santa Cruz Valley. This pattern is most likely explained by the higher solid content induced by higher entrainment rates. Flows rich in solid are less diffusive in terms of spreading than those rich in fluid. Figure 11. Dependency of overtopping of the lakes Artizón Alto and Artizón Bajo on the combination of the release volume of the initial landslide VR and the basal friction angle δI applied to r.avaflow. [Colour figure can be viewed at wileyonlinelibrary.com] phenomena are reflected in FoC (Table IV) and are a conse- quence of the fact that more momentum is gained in zone II with lower values of δII. Lower values of δII further induce intense entrainment in the lower part of zone II (VE = 1.44×106 m3). CSI indicates the optimum model performance at 20°, D2PC at 14° (Table IV). The zone of simulated entrainment laterally extends beyond the zone of reconstructed entrainment, reflecting the overestimated degree of spreading of the simulated flow (Figure 14; experiment E1). More importantly, entrainment in the lowermost section of the Artizón valley between the outwash plain and the Santa Cruz valley is generally overestimated, compared with the dam of Lake Artizón Bajo and the valley section directly downstream (Figure 1 and Figure 6). This phenomenon is possibly explained by a spatial pattern in the mechanical properties of the entrainable material (higher level of compaction of the older sediments de- posited downstream). However, the event documentation does not support a spatial differentiation of CE. Figure 9. Flow height maps resulting from experiment E2 for selected points in time from t = 0 s to t = 100 s, representing the initial stage of the pro- cess chain. The dashed line represents the main flow path. (a) Simulation with 10 m cell size; (b) simulation with 5 m cell size. [Colour figure can be viewed at wileyonlinelibrary.com] Figure 9. Flow height maps resulting from experiment E2 for selected points in time from t = 0 s to t = 100 s, representing the initial stage of the pro- cess chain. The dashed line represents the main flow path. (a) Simulation with 10 m cell size; (b) simulation with 5 m cell size. [Colour figure can be viewed at wileyonlinelibrary.com] © 2017 The Authors. Earth Surface Processes and Landforms published by John Wiley & Sons Ltd. Earth Surf. Process. Landforms, Vol. 43, 1373–1389 (2018) 1384 M. MERGILI ET AL. Figure 10. Simulated impact area (HMax ≥0.2 m) derived with six combinations of VR and δI (experiment E3). The observed impact area is shown as reference. [Colour figure can be viewed at wileyonlinelibrary.com] 1384 M. MERGILI ET AL. Figure 10. Simulated impact area (HMax ≥0.2 m) derived with six combinations of VR and δI (experiment E3). The observed impact area is shown as reference. [Colour figure can be viewed at wileyonlinelibrary.com] Figure 11. Dependency of overtopping of the lakes Artizón Alto and Artizón Bajo on the combination of the release volume of the initial landslide VR and the basal friction angle δI applied to r.avaflow. [Colour figure can be viewed at wileyonlinelibrary.com] not influence the patterns of HMax in zone II, but strongly im- pacts HMax farther downstream in zone III. The flow only reaches Lake Jatuncocha with the assumption of δIII = 0°. Even with δIII = 2° it stops well before Lake Jatuncocha, and with δIII = 6° the flow proceeds less than half-way from the confluence towards Lake Jatuncocha. The relatively flat terrain only per- mits a longer runout in the case of very low values of δIII. This phenomenon is also reflected in the validation scores (Table IV): CSI and D2PC indicate a decrease of the model per- formance with increasing values of δIII, FoC indicates a decreas- ing degree of conservativeness. As prescribed by the definition of the potential entrainment zone, VE is not influenced by vari- ations in δIII. © 2017 The Authors. Earth Surface Processes and Landforms published by John Wiley & Sons Ltd. Earth Surf. Process. Landforms, Vol. 43, 1373–1389 (2018) Experiment E5: sensitivity to the basal friction in zone III Some of the patterns observed in experiment E4 are even more pronounced in experiment E5 (Figure 13). The variation of δIII (assumption of basal friction angle in zone III) obviously does Earth Surf. Process. Landforms, Vol. 43, 1373–1389 (2018) HOW WELL CAN WE SIMULATE COMPLEX HYDRO-GEOMORPHIC PROCESS CHAINS? 1385 Figure 12. Simulated impact area (HMax ≥0.2 m) derived with three assumptions of δII (experiment E4). The observed impact area is shown as ref- erence. [Colour figure can be viewed at wileyonlinelibrary.com] 1385 HOW WELL CAN WE SIMULATE COMPLEX HYDRO-GEOMORPHIC PROCESS CHAINS? Figure 12. Simulated impact area (HMax ≥0.2 m) derived with three assumptions of δII (experiment E4). The observed impact area is shown as ref- erence. [Colour figure can be viewed at wileyonlinelibrary.com] Figure 12. Simulated impact area (HMax ≥0.2 m) derived with three assumptions of δII (experiment E4). The observed impact area is shown as ref- erence. [Colour figure can be viewed at wileyonlinelibrary.com] Figure 13. Simulated impact area (HMax ≥0.2 m) derived with three assumptions of δIII (experiment E5). The observed impact area is shown as ref- erence. [Colour figure can be viewed at wileyonlinelibrary.com] pact area (HMax ≥0.2 m) derived with three assumptions of δIII (experiment E5). The observed impact area is shown as ref- n be viewed at wileyonlinelibrary.com] Figure 13. Simulated impact area (HMax ≥0.2 m) derived with three assumptions of δIII (experiment E5). The observed impact area is shown as ref- erence. [Colour figure can be viewed at wileyonlinelibrary.com] highlight some of the major challenges process chain simula- tions are facing, particularly with regard to the data they rely on: © 2017 The Authors. Earth Surface Processes and Landforms published by John Wiley & Sons Ltd. Earth Earth Surf. Process. Landforms, Vol. 43, 1373–1389 (2018) © 2017 The Authors. Earth Surface Processes and Landforms published by John Wiley & Sons Ltd. Earth Surf. Process. Landforms, Vol. 43, 1373–1389 (2018) cesses and Landforms published by John Wiley & Sons Ltd. Earth Surf. Process. Landforms, Vol. 43, 1373–1389 (2018) Discussion Increasing δIII from 0° to 2° and 4° leads to decreases in FoC of 0.14 and 0.19, respec- tively. These patterns demonstrate that the energy gained by a flow, and therefore also its travel distance and lateral spreading, show a near-linear relationship with the basal friction as long as no interactions with a reservoir occur. Likewise, increasing CE in Zone II from 10-6.55 to 10-6.45 and further to 10-6.35 leads to a relatively linear increase of VE of 0.33×106 and 0.51×106, re- spectively. However, these near-linear patterns have to be interpreted with caution as the processes involved may be non- linear: the empirical entrainment model applied with r.avaflow represents a rough approximation only, which will be complemented with a physically-based model (Pudasaini and Fischer, 2016) in the future. The extent to which a moraine dam is eroded strongly determines the availability of the solid fraction in the mixture downstream, and therefore the behav- iour of the flow. The resulting variations in the solid content and the flow volume can exert manifold impacts on the mobil- ity of the flow. Still, we do not expect threshold effects as strong as those inferred in Zone I. associated volumes. Basal friction values and entrainment coefficients are the products of back-calculations, without a direct mechanical basis. The implementation of a physically-based entrainment model (Pudasaini and Fischer, 2016) with r.avaflow is in progress. 3. No reference data are available to evaluate the simulated flow velocities or travel times. Therefore, parameters are op- timized on the basis of observed spatial, but not temporal patterns. Simulation results are particularly sensitive to variations of uncertain key input: initial conditions such as volume and solid content of the release and flow parameters such as the basal friction angle, or the entrainment coefficient. We note that vary- ing the conditions in Zone I (down to Lake Artizón Bajo) may lead to a highly nonlinear behaviour (Figure 11). The reason is the fact that process chains involve threshold phenomena and strong positive feedbacks: even moderate overtopping of a dam may induce entrainment and a powerful flood wave or debris flow downstream, while there is no process chain at all in the case of no overtopping. Discussion Multi-hazard contexts have been increasingly considered by the scientific communities in the past years, both on a concep- tual level (Kappes et al., 2012), and – particularly for process chains – on the level of event back-calculation by means of computer simulations (Schneider et al., 2014). Recent studies have analyzed and quantified the error range involved when simulating process chains due to limited physical under- standing, limited capabilities of current models at process boundaries, and associated uncertainties concerning the sur- rounding surface topography, material and mass flow parame- ters and characteristics (Westoby et al., 2014; Schaub et al., 2016). A particular challenge concerns the coupling of different simulation models, or the integration of different cascading mass flow processes in one integrated model. Here we follow the call of Worni et al. (2014) for more integrated approaches. Thereby, the 2012 Santa Cruz event serves to demonstrate and 1. Process chains often occur in remote areas characterized by steep topography. Even though modern remote sensing techniques help to capture the situation before and after events, the quality of such data is often limited. Further, highly demanding field work is necessary to: (i) improve the understanding of the individual and interacting pro- cesses; and (ii) build a sound basis for back-calculations. 2. As a consequence of (1), the detailed process understanding is limited and simulation input and reference data involve uncertainties. In the present study, the reconstruction of the topography was based on state-of-the-art radar remote sensing technology, bathymetric data and further field ob- servations. This is more than is typically available in remote high-mountain areas but still implies uncertainties with re- spect to release and entrained depths (Figure 6) and the 1386 M. MERGILI ET AL. Figure 14. Reconstructed and simulated entrained heights in the potential entrainment area (Figure 6). (a) Reconstructed entrained height (ERec); (b) simulated entrained height (ESim). [Colour figure can be viewed at wileyonlinelibrary.com] 1386 M. MERGILI ET AL. Figure 14. Reconstructed and simulated entrained heights in the potential entrainment area (Figure 6). (a) Reconstructed entrained height (ERec); (b) simulated entrained height (ESim). [Colour figure can be viewed at wileyonlinelibrary.com] VE. Increasing δII from 14° to 20° and 26° leads to decreases in FoC by 0.35 and 0.36, respectively. This would mean that the size of the simulated impact area would decrease by 35% and 36% of the observed impact area. © 2017 The Authors. Earth Surface Processes and Landforms published by John Wiley & Sons Ltd. Earth Surf. Process. Landforms, Vol. 43, 1373–1389 (2018) © 2017 The Authors. Earth Surface Processes and Landforms published by John Wiley & Sons Ltd. Earth Surf. Process. Landforms, Vo Conclusions Glacier retreat and the accompanying geomorphologic changes in the Artizón valley, Cordillera Blanca, Peru, have formed an environment favouring the development of a lake outburst cascade i.e. a steep and unstable lateral moraine slope geomorphologically connected to a system of glacial lakes. In 2012, a landslide from the left lateral moraine impacted the upper lake and induced a process chain proceeding kilometres downstream, entraining, transporting, and subsequently accumulating a significant amount of material. We have reconstructed the geomorphologic changes induced by the event from field observations and remotely sensed data, and attempted to back-calculate the process chain with the two-phase mass flow model employed within the com- putational framework r.avaflow. The design of r.avaflow enables continuous simulations across process boundaries, representing a major step forward. The results partly reveal the high sensitivity of process chain modelling to key inputs, such as release vol- ume, basal friction or the entrainment coefficient. Due to multi- ple feedbacks and dependencies, these effects may even be stronger than for ‘ordinary’ mass flow processes. This is particu- larly true for the upper part of the chain, where the threshold ef- fect of lake overtopping decides on the occurrence and characteristics of the flow downstream. With adequately chosen parameters, the documented and reconstructed patterns of the process chain are generally well reproduced by the r.avaflow calculations in terms of empirical adequacy, demonstrating the potential of computer simulations also for complex mass flow phenomena. However, our results indicate that the processes involved, and the input parameters and parameter combinations governing the simulation are not yet fully understood in their spatial distribution. Improved databases and thorough back-calculations and parameter studies with a larger number of well-documented events are needed to enable the anticipa- tion of future events and their possible consequences in terms of scenario analyses or probability measures. 1. Acknowledging that the uncertainties in the initial condi- tions (release mass, water content) and key flow parameters are to some extent aleatoric – particularly with regard to their spatial pattern – we depend on well-founded empirical adequacy. The limitations of one-at-a-time parameter studies, as applied in the present work, are well docu- mented (Saltelli and Annoni, 2010). More appropriate ap- proaches were proposed, e.g. by Formetta et al. (2015) or Fischer et al. (2015). Their application would be imperative, but represents a challenge due to the multi-dimensional parameter space, including the spatial pattern in some parameters. 2. Conclusions Considering what was said in (1), systematic back- calculations of multiple events may lead to a set of guiding parameter ranges. Independent test cases would be neces- sary to confirm the general findings and to make a success- ful forward calculation more probable. Such an effort would be beyond the scope of the present article. 3. Some of the key characteristics of process chains are rarely documented (e.g. velocities and travel times), so that empir- ical adequacy has to remain incomplete. Well-designed laboratory experiments could be employed in order to move as close as possible to an assessment of the physical adequacy of the simulation approach, e.g. with regard to the landslide–lake interactions. 4. The weakest link for forward predictions consists in the potentially inadequate assessment of the conditions above potentially impacted lakes, as even small misestimates of these conditions may lead to completely inappropriate results (threshold effects; Figure 11). Consequently, avail- able resources should be put particularly in investigations on the characteristics of potential landslide release masses, and the material friction parameters in the upper most flow region. Acknowledgements—The work was conducted as part of the interna- tional cooperation project ‘A GIS simulation model for avalanche and debris flows (avaflow)’ supported by the German Research Foundation (DFG, project number PU 386/3-1) and the Austrian Science Fund (FWF, project number I 1600-N30). While (1) to (3) apply to all efforts of spatial landslide model- ling and beyond, but are particularly important when consider- ing process chains, (4) is specific for process chains such as multi-lake outburst floods. Due to the highly sensitive behav- iour with regard to threshold effects such as lake overtopping, the assumption of fixed initial conditions and parameter values likely result in inadequate predictions. Consequently, forward predictions will, even more than for more common landslide processes, rely on: (i) the consideration of multiple scenarios; or (ii) the application of ranges of initial conditions and flow parameters instead of fixed values. The second of these results would result in likelihood measures such as impact indicator indices (Mergili et al., 2017) increasing the probability of The field work was supported by GAUK Project No. 70413 and GAUK Project No. 730216 awarded to Adam Emmer and by the Charles University Mobility Fund awarded to Anna Juřicová. Discussion Targeting various groups of stakeholders and the public in a well-adapted way will be essential for effective management of the risks associated with lake outburst floods and other types of process chains in the Cordillera Blanca and elsewhere in the world (Carey, 2005; Carey et al., 2012). combinations of parameters may provide comparable results; and (ii) different simplifying or imperfect assumptions concerning the analytical model, the numerical approach and the parameterization may cancel out. Even though we can link the trends in the response of the simulation to the key inputs to our understanding of the related physical pro- cesses, the chosen parameters are unlikely to be physically correct. Consequently, a concerted communication process with de- cision makers is necessary to appropriately deal with uncertain simulation results. Targeting various groups of stakeholders and the public in a well-adapted way will be essential for effective management of the risks associated with lake outburst floods and other types of process chains in the Cordillera Blanca and elsewhere in the world (Carey, 2005; Carey et al., 2012). Using r.avaflow allows back-calculating complex hydro- geomorphic process chains in an empirically adequate way. In contrast to most earlier approaches (Schneider et al., 2014; Schaub et al., 2016; Somos-Valenzuela et al., 2016), r.avaflow supports a continuous simulation across the boundaries of the individual components of the process chain. Considering this a major step forward, we note that based on the present study there still remain important limitations for the predictive appli- cation of the model to possible future process chains for the following reasons: Earth Surf. Process. Landforms, Vol. 43, 1373–1389 (2018) 2017 The Authors. Earth Surface Processes and Landforms published by John Wiley & Sons Ltd. © 2017 The Authors. Earth Surface Processes and Landforms published by John Wiley & Sons Ltd. Earth Surf. Process. Landforms, Vol. 43, 1373–1389 (2018) cesses and Landforms published by John Wiley & Sons Ltd. Earth Surf. Process. Landforms, Vol. 43, 1373–1389 (2018) Discussion An initial landslide of VR = 3×105 m3 requires a lower basal friction angle (δ <12–14°) to develop sufficient kinetic energy to result in overtopping of Lake Artizón Bajo than an initial landslide of VR = 8×105 m (δ <18–20°). While the general patterns illustrated in Figure 11 are most probably generally valid, the threshold values would shift when changing the initial conditions or numerical treat- ments. For input combinations with predicted overtopping of Lake Artizón Bajo, the effects downstream depend on the varied parameters in a near-linear way (Figure 10). Comparing the observed and the simulated impact area be- tween the lakes Artizón Bajo and Jatuncocha, the maximum CSI value does not reach beyond 0.6, and the D2PC not below 0.15 (Table IV). A better performance is impeded by (i) an over- estimate of lateral spreading and run-up of the flow in the lower portion of Zone II; and (ii) an underestimate of the flow width over much of Zone III (Figures 10–13). Even though the result obtained in experiment E1 appears plausible (Figure 7) we have to keep in mind that it relies on an ad hoc parameter assumption, chosen towards confirmation of the observed patterns. This does not mean that the simulation would necessarily be fully valid. Instead, we can only confirm a certain degree of empirical adequacy in the sense of Oreskes et al. (1994), keeping in mind that: (i) other Varying the initial conditions or flow parameters in the Zones II or III (i.e. after the interaction of the mass movement with the two upper lakes) generally leads to a rather linear (predictable) behaviour (Figures 12 and 13). The FoC (which is proportional to the simulated affected area) is most suitable to quantify this observation: varying the basal friction angle in the zones II or III leads to more or less linear changes of the FoC, and also of HOW WELL CAN WE SIMULATE COMPLEX HYDRO-GEOMORPHIC PROCESS CHAINS? 1387 successful forward simulations, and enabling the implicit con- sideration of uncertainties in the results (Krenn et al., 2016). successful forward simulations, and enabling the implicit con- sideration of uncertainties in the results (Krenn et al., 2016). Consequently, a concerted communication process with de- cision makers is necessary to appropriately deal with uncertain simulation results. 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https://openalex.org/W2092205936	https://journals.plos.org/plosntds/article/file?id=10.1371/journal.pntd.0002356&type=printable	English	null	Inter-epidemic Transmission of Rift Valley Fever in Livestock in the Kilombero River Valley, Tanzania: A Cross-Sectional Survey	PLoS neglected tropical diseases	2,013	cc-by	6,992	Abstract This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was funded by the generous support through PhD scholarship awarded to RDS by Directorate General for Development Cooperation (DGDC), Belgium, through Institute of Tropical Medicine, Antwerp. Part of the research work was also supported by Hassan Mshinda Fellowship of the Liverpool School of Tropical Medicine (LSTM), UK and Ifakara Health Institute (IHI), Tanzania. The funders of this study had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: rsumaye@ihi.or.tz, rsumaye@yahoo.com Robert D. Sumaye1,2,3, Eveline Geubbels1, Edgar Mbeyela1, Dirk Berkvens3 1 Ifakara Health Institute, Ifakara, Tanzania, 2 Department of Infectious and Parasitic Diseases, Faculty of Veterinary Medicine, University of Liege, Liege, Belgium, 3 Department of Biomedical Sciences, Institute of Tropical Medicine, Antwerp, Belgium Ifakara Health Institute, Ifakara, Tanzania, 2 Department of Infectious and Parasitic Diseases, Faculty of Veterinary Medicine, Univ Department of Biomedical Sciences, Institute of Tropical Medicine, Antwerp, Belgium Abstract Background: In recent years, evidence of Rift Valley fever (RVF) transmission during inter-epidemic periods in parts of Africa has increasingly been reported. The inter-epidemic transmissions generally pass undetected where there is no surveillance in the livestock or human populations. We studied the presence of and the determinants for inter-epidemic RVF transmission in an area experiencing annual flooding in southern Tanzania. Methodology: A cross-sectional sero-survey was conducted in randomly selected cattle, sheep and goats in the Kilombero river valley from May to August 2011, approximately four years after the 2006/07 RVF outbreak in Tanzania. The exposure status to RVF virus (RVFV) was determined using two commercial ELISA kits, detecting IgM and IgG antibodies in serum. Information about determinants was obtained through structured interviews with herd owners. Findings: An overall seroprevalence of 11.3% (n = 1680) was recorded; 5.5% in animals born after the 2006/07 RVF outbreak and 22.7% in animals present during the outbreak. There was a linear increase in prevalence in the post-epidemic annual cohorts. Nine inhibition-ELISA positive samples were also positive for RVFV IgM antibodies indicating a recent infection. The spatial distribution of seroprevalence exhibited a few hotspots. The sex difference in seroprevalence in animals born after the previous epidemic was not significant (6.1% vs. 4.6% for females and males respectively, p = 0.158) whereas it was significant in animals present during the outbreak (26.0% vs. 7.8% for females and males respectively, p,0.001). Animals living .15 km from the flood plain were more likely to have antibodies than those living ,5 km (OR 1.92; 95% CI 1.04– 3.56). Species, breed, herd composition, grazing practices and altitude were not associated with seropositivity. Conclusion: These findings indicate post-epidemic transmission of RVFV in the study area. The linear increase in seroprevalence in the post-epidemic annual cohorts implies a constant exposure and presence of active foci transmission preceding the survey. Citation: Sumaye RD, Geubbels E, Mbeyela E, Berkvens D (2013) Inter-epidemic Transmission of Rift Valley Fever in Livestock in the Kilombero River Valley, Tanzania: A Cross-Sectional Survey. PLoS Negl Trop Dis 7(8): e2356. doi:10.1371/journal.pntd.0002356 Editor: Brian Bird, Centers for Disease Control and Prevention, United States of America Received February 26, 2013; Accepted June 20, 2013; Published August 8, 2013 eceived February 26, 2013; Accepted June 20, 2013; Published Copyright: 2013 Sumaye et al. PLOS Neglected Tropical Diseases \| www.plosntds.org August 2013 \| Volume 7 \| Issue 8 \| e2356 Inter-epidemic Transmission of Rift Valley Fever in Livestock in the Kilombero River Valley, Tanzania: A Cross-Sectional Survey Robert D. Sumaye1,2,3, Eveline Geubbels1, Edgar Mbeyela1, Dirk Berkvens3 1 Ifakara Health Institute, Ifakara, Tanzania, 2 Department of Infectious and Parasitic Diseases, Faculty of Veterinary Medicine, University of Liege, Liege, Belgium, 3 Department of Biomedical Sciences, Institute of Tropical Medicine, Antwerp, Belgium Design, sampling and data collection hydroelectric power dams along Senegal river and thereafter established itself as endemic disease [14,15]. In the Arabian Peninsula RVF was introduced through trading of live animals with countries in the horn of African [16,17]. g , p g The sero-survey was done from May to August 2011, approximately four years after the end of the 2006/07 RVF outbreak in Tanzania. Blood samples were collected from three livestock populations, namely cattle, goats and sheep from 44 villages in both districts. These villages were selected based on their inclusion in a demographic surveillance system [35,36] and the proximity to the Kilombero river flood plains (figure 1). In each village four households that kept at least one of the three species, were randomly selected from the list of livestock keepers in that particular village. For each participating household a maximum of 18 samples were collected (i.e. maximum 10 cattle, maximum 4 goats and maximum 4 sheep, the actual numbers sampled depending on the number of a particular species present). The sampling strategy was based on selecting animals of all age groups in order to be able to characterize epidemic and inter- epidemic transmission. The blood samples were collected in 6 ml vacutainer tubes with clot activator, labeled and stored in a cooler box with ice packs while in the field. After coagulation, serum was eluted from the whole blood into a 1.8 ml cryovial tube, labeled and stored in a car fridge until transfer to the laboratory for laboratory analysis. Characteristics of individual animals together with the herd history were obtained through a structured interview with the herd owner. RVF maintenance in nature between epidemics both in the mammalian host and vector populations has not been fully explained [18,19]. This is partly due to the factors driving its maintenance at fine geographical scales that are yet to be described in detail. Such factors interact in diverse ways in different geographical regions of Africa or beyond and may play a crucial role in vector population dynamics and the disease transmission [20]. In the past two decades, RVF outbreaks have been reported twice in Tanzania, with the most recent outbreak occurring in 2006/07. Age determination Individual animal age was estimated using history taking, review of available records on date of birth and dentition. Records were available only from households keeping exotic dairy cattle. Dentition was used in determining age of cattle between 24 to 54 months only [37]. When the above two methods yielded no useful results, age was estimated by probing the head of the household, herd boys and other members of the household for the animal’s month/season and year of birth and for female animals also by taking into account number of births and average birth rate for the particular species in the valley. Introduction family Bunyaviridae and in susceptible animals is manifested clinically by high fever, and causes abortion in susceptible pregnant animals irrespective of the gestation period and high mortality in newborn animals [9]. In humans, RVF can be asymptomatic, but can also cause mild illness (associated with headache, fever, muscle and joint pains) or severe illness (associated with hemorrhagic fever, encephalitis or ocular disease) [10–12].The disease was first described in the early 1910s and the aetiological agent was isolated in the 1930s in Kenya [13]. The disease pattern in the Eastern Africa region and the horn of Africa is driven by climatic conditions linked to the El Nin˜o/Southern Oscillation (ENSO) phenomenon, which leads to unusual high rainfall and floods alternated by long dry spells [2]. In other parts of Africa, RVF emerged in relation to the construction of Rift Valley fever (RVF) is known to occur in outbreaks in cycles of 5–15 years in the Eastern Africa region and the Horn of Africa, following unusual high precipitations that lead to sustained flooding [1,2]. In recent years, evidence of RVF transmission during the inter-epidemic periods in some parts of the African continent has increasingly been reported [3–5]. The inter- epidemic transmissions generally pass undetected clinically, but can be revealed where active serological surveillance is regularly done in either livestock or human populations [4,6,7]. Rift Valley fever is a mosquito borne viral zoonosis that affects both livestock and wild ruminants [4,5,8]. It is caused by Rift Valley fever virus (RVFV) belonging to the genus Phlebovirus of the August 2013 \| Volume 7 \| Issue 8 \| e2356 1 PLOS Neglected Tropical Diseases \| www.plosntds.org Inter-epidemic Transmission of RVF in Livestock Inter-epidemic Transmission of RVF in Livestock Design, sampling and data collection During this outbreak, the disease occurred in 10 of the 21 regions of Tanzania mainland, including Morogoro region where the Kilombero river valley is located, with clinical cases in people and livestock [30–32].Given the ecology and climatic factors that support mosquito vectors in the valley we hypothesized a continued transmission of RVFV during the inter-epidemic periods. The goal of our study was therefore to investigate potential inter-epidemic period transmission of RVF in an area experiencing seasonal floods annually. Specifically, the study aimed 1) to establish baseline information on the exposure status to Rift valley fever in livestock as determined by presence of antibodies against RVFV in livestock blood samples during the inter-epidemic period and 2) to assess the relationship between RVF seropositivity, animal characteristics and environmental factors. Study area Rift Valley fever (RVF) is an arthropod-borne viral disease that affects people, livestock and wild animals. It occurs mostly in Africa, and epidemics have been reported in the Arabian Peninsula. RVF is transmitted to humans and animals by mosquitoes, but people can also get the infection through direct contact with blood or tissues of infected animals. The disease occurs in epidemic form in a cycle of 5–15 years, but some reports also indicate occurrences of the disease during non-epidemic periods. We report here inter-epidemic period transmission of RVF in livestock population, evidenced by demonstration of RVFV antibodies in animals that were born after the 2006/ 07 RVF outbreak in Tanzania and demonstration of immunoglobulin M (IgM), a short lived class of antibodies, following infection by RVF virus in 9 samples. We have also identified hotspots of transmission in the study area, with exposure being higher away from the main flood plain. There was a linear increase in percent seropositivity from 1 year olds to age 5 years, implying a possible annual challenge. This study was conducted in the Kilombero river valley, which spans Kilombero and Ulanga districts in Morogoro region. The Kilombero valley is a seasonally inundated flood plain up to 52 km wide, at high water, between the densely forested escarpment of the Udzungwa mountains, which rise to 2576 m above sea level (asl) on the north-western side and the grass covered Mahenge mountains, which rise to 1516 m asl on the south-eastern side (Figure 1). The valley receives an average annual rainfall of 1200 mm–1800 mm and temperatures range between 25uC and 32uC. The valley has a diverse ecology and demography with villages consisting largely of numerous distinct groups of houses located on the margins of the flood plain where rice cultivation is the predominant economic activity. Other land use types include hunting, fishing, forestry, pastoral livestock rearing and other crops cultivation. There has been intense malaria transmission in the valley with the main vectors being Anopheles gambiae complex and Anopheles funestus [33]. Other mosquito species inhabiting the valley, some of which are vectors of RVF virus, include Culex spp, Aedes spp and Mansonia spp [34]. Methods Author Summary PLOS Neglected Tropical Diseases \| www.plosntds.org Serological assays To determine the individual animals’ longstanding exposure status to previous Rift Valley fever virus (RVFV), a commercial, inhibition enzyme-linked immunosorbent assay (c-ELISA) for the PLOS Neglected Tropical Diseases \| www.plosntds.org August 2013 \| Volume 7 \| Issue 8 \| e2356 August 2013 \| Volume 7 \| Issue 8 \| e2356 2 Inter-epidemic Transmission of RVF in Livestock Figure 1. Study area depicting spatial distribution pattern of RVF seroconversion in livestock. The graduated colors indicate output of IDW based on actual number of seropositive animals to RVFV antibodies in all species. The round dots represent location of households from which livestock were sampled and clustering pattern given by Z-scores (red, blue and white colorations indicating hotspot, cold spot and random pattern respectively). The black stars indicate households where IgM positive animals were sampled. The insert is Map of Tanzania showing districts of Kilombero and Ulanga, and location of study site within. doi:10.1371/journal.pntd.0002356.g001 Figure 1. Study area depicting spatial distribution pattern of RVF seroconversion in livestock. The graduated colors indicate output of IDW based on actual number of seropositive animals to RVFV antibodies in all species. The round dots represent location of households from which livestock were sampled and clustering pattern given by Z-scores (red, blue and white colorations indicating hotspot, cold spot and random pattern respectively). The black stars indicate households where IgM positive animals were sampled. The insert is Map of Tanzania showing districts of Kilombero and Ulanga, and location of study site within. doi:10.1371/journal.pntd.0002356.g001 detection of antibodies against RVFV in humans, domestic and wildlife ruminants was used (Biological Diagnostic Supplies Limited, Dreghorn, United Kingdom) [38]. Recent infection was determined using a commercial IgM ELISA kit (Biological Diagnostic Supplies Limited, Dreghorn, United Kingdom) [39].The IgM ELISA test was employed for c-ELISA positive samples only since the c-ELISA detects both IgG and IgM antibodies against RVFV [38]. spatially. The IDW is a deterministic interpolation model that assigns values to locations where no measurements have been taken to produce a surface pattern, based on how far those locations are to the sentinel locations where measurements have been taken. Ethics statement The blood collection procedure from livestock was performed by a qualified veterinarian following proper physical restraint of animals that ensured both personnel and animal safety. Livestock owners were explained the study purpose and procedures and upon agreeing to participate they provided a written consent prior to study procedures and blood collection from their animals. Ethical approval for this study protocol was obtained from the Institutional Review Board of the Ifakara Health Institute and Medical Research Coordination Committee of the Tanzania’s National Institute for Medical Research (permit number NIMR/ HQ/R.8a/Vol. IX/1101). Data analyses The data was analysed in STATA version 12 [Stata Corp., College Station, Texas, USA]. To examine the determinants for RVF seropositivity, first a univariable analysis of individual factors was performed by fitting a logistic regression model with wards and villages as random effects to account for clustering. Variables with p-value ,0.25 were selected as potential covariables in the multivariable analysis, where a p-value #0.05 was considered statistically significant. Forward model-building was done with subsequent models evaluated against sparser models by means of the Akaike information criterion (AIC). Two-way interactions between variables included in the model were also tested. Lastly, all factors that were dropped in the process of model building were later tested for any confounding effect. Factors were considered a confounder if they led to a change of $25% in the coefficient estimates of other determinants. PLOS Neglected Tropical Diseases \| www.plosntds.org Results A total of 1680 livestock serum samples were tested by RVF c- ELISA, of which 1234 samples were from Kilombero district and 446 samples were from Ulanga district. Out of the samples tested 970, 455 and 255 were from cattle, goats and sheep respectively. Several potential animal-level risk factors were investigated; table 1 shows the univariable logistic regression model output of the risk factors. The proportion of seropositive animals by c-ELISA was 11.3%. A seroprevalence of 5.5% was recorded among animals that were born after the 2006/07 RVF outbreak (less than 4 years of age), compared to 22.7% in those that were born before and thus present during the outbreak. There was a linear increase in The spatial analysis of the seropositivity was performed using ArcGIS software version 10 (ESRI, Redlands, USA) using hot spot analysis and inverse distance weighted (IDW) tools. The Getis-Ord Gi* statistic for each feature (household) was computed with the resultant Z-score values indicating where households with either high (hot spot), median (random) or low (cold spot) values cluster August 2013 \| Volume 7 \| Issue 8 \| e2356 PLOS Neglected Tropical Diseases \| www.plosntds.org August 2013 \| Volume 7 \| Issue 8 \| e2356 3 Inter-epidemic Transmission of RVF in Livestock Table 1. Potential animal level risk factors associated with RVF sero-positivity for cattle, goats and sheep in the Kilombero Valley, Tanzania based on a univariable random effect logistic regression model. Variable Level %Positive (n) OR 95% CI Species Cattle 11.03 (970) 1 Goat 11.86 (455) 1.06 0.74–1.52 Sheep 11.37 (255) 1.07 0.68–1.69 Sex Female 14.16 (1144) 1 Male 5.22 (536) 0.30 0.19–0.46 Breed Exotic 9.54 (220) 1 Local 11.61 (1420) 1.23 0.70–2.15 Mixed 10.00 (40) 0.95 0.28–3.14 District Kilombero 10.69 (1234) 1 Ulanga 13.00 (446) 1.17 0.68–2.03 Present during RVF outbreak No 5.55 (1116) 1 Yes 22.69 (564) 5.29 3.79–7.38 Herd composition One species 10.27 (360) 1 Two species 10.30 (359) 1.08 0.64–1.83 Three species 12.07 (961) 1.24 0.80–1.92 Distance to the edge of floods (km) ,5 km 10.74 (1117) 1 5–10 km 14.01 (157) 1.27 0.72–2.21 10–15 km 7.94 (214) 0.75 0.41–1.37 .15 km 16.14 (192) 1.62 0.91–2.86 Feeding practices Mixed 4.76 (21) 1 Grazing 11.30 (1592) 2.29 0.29–18.18 Cut & carry 13.43 (67) 2.80 0.31–25.06 Altitude (m) Continuous n/a 1.007 0.99–1.01 OR Odd ti CI C fid i t l Table 1. Results Potential animal level risk factors associated with RVF sero-positivity for cattle, goats and sheep in the Kilombero Valley, Tanzania based on a univariable random effect logistic regression model. Table 1. Potential animal level risk factors associated with RVF sero-positivity for cattle, goats and sheep in the Kilombero Valley, Tanzania based on a univariable random effect logistic regression model. Figure 2. Seropositivity to Rift Valley fever virus in cattle, sheep and goats pooled by age. The error bars indicate 95% confidence intervals of the percentage. doi:10.1371/journal.pntd.0002356.g002 Table 1. Potential animal level risk factors associated with RVF sero-positivity for cattle, goats and sheep in the Kilombero Valley, Tanzania based on a univariable random effect logistic regression model. Variable Level %Positive (n) OR 95% CI Species Cattle 11.03 (970) 1 Goat 11.86 (455) 1.06 0.74–1.52 Sheep 11.37 (255) 1.07 0.68–1.69 Sex Female 14.16 (1144) 1 Male 5.22 (536) 0.30 0.19–0.46 Breed Exotic 9.54 (220) 1 Local 11.61 (1420) 1.23 0.70–2.15 Mixed 10.00 (40) 0.95 0.28–3.14 District Kilombero 10.69 (1234) 1 Ulanga 13.00 (446) 1.17 0.68–2.03 Present during RVF outbreak No 5.55 (1116) 1 Yes 22.69 (564) 5.29 3.79–7.38 Herd composition One species 10.27 (360) 1 Two species 10.30 (359) 1.08 0.64–1.83 Three species 12.07 (961) 1.24 0.80–1.92 Distance to the edge of floods (km) ,5 km 10.74 (1117) 1 5–10 km 14.01 (157) 1.27 0.72–2.21 10–15 km 7.94 (214) 0.75 0.41–1.37 .15 km 16.14 (192) 1.62 0.91–2.86 Feeding practices Mixed 4.76 (21) 1 Grazing 11.30 (1592) 2.29 0.29–18.18 Cut & carry 13.43 (67) 2.80 0.31–25.06 Altitude (m) Continuous n/a 1.007 0.99–1.01 OR = Odds ratio; CI = Confidence interval. doi:10.1371/journal.pntd.0002356.t001 PLOS Neglected Tropical Diseases \| www plosntds org 4 August 2013 \| Volume 7 \| Issue 8 \| e2356 Figure 2. Seropositivity to Rift Valley fever virus in cattle, sheep and goats pooled by age. The error bars indicate 95% confidenc intervals of the percentage. doi:10.1371/journal.pntd.0002356.g002 Tanzania based on a univariable random effect logistic regression model. Inter-epidemic Transmission of RVF in Livestock There was no significant difference in seropositivity between the different species. The sex difference in seroprevalence in animals born after the previous epidemic was not significant (6.1% vs. 4.6% for females and males respectively, p = 0.158) whereas there was a significant sex difference in seroprevalence in animals that were present during the outbreak (26.0% vs. 7.8% for females and males respectively, p,0.001) and overall (table S1). proportion sero-conversion up to age 5, Figure 2. The samples included less than one year olds in which the youngest positive individual was 7 months old at which age the maternally acquired immunity has waned out. There was no significant difference in seropositivity between the different species. The sex difference in seroprevalence in animals born after the previous epidemic was not significant (6.1% vs. 4.6% for females and males respectively, p = 0.158) whereas there was a significant sex difference in seroprevalence in animals that were present during the outbreak (26.0% vs. 7.8% for females and males respectively, p,0.001) and overall (table S1). The contact between infected vectors and naı¨ve mammalian hosts is the main determinant of arboviral disease transmission [55]. Mosquito vector population dynamics are driven by environment and ecology, which provide essential life resources. The trend observed in this study of increased seroprevalence away from the main flood plain and in high altitude is contrary to findings in other studies [6,46]. This could be an indication of other factors playing a role including localized floods unrelated to the main flood area, but also proximity to dense vegetation (forested environments) that could harbor a variety of mosquito vectors [6], as the main forested areas are further away from the flood plain. Another explanation could be movement of previously exposed animals from one locality to another within the area through animal trade among livestock keepers, dowry or establishment of new households. In such circumstances, naı¨ve animals can be transferred to an infected area or infected animals can be introducing the disease into a naı¨ve location. In this way vector populations which are abundant and of diverse species within the valley [34] are exposed and maintenance mechanisms established where conditions are favorable. The observed separate hotspots further point to fine scale factors playing major role in transmission dynamics. Inter-epidemic Transmission of RVF in Livestock On the other hand the only cold spot is located around Ifakara town, a semi urban environment in which livestock keeping is characterized by small herds, sedentary and mixed feeding practices as compared to relatively large herds with extensive system in the villages. The urban environment might be unfavorable to the main vector species but also due to limited daily livestock movements there is little interaction between neighbour- ing herds thus possibility of infection to limit itself to isolated herds in the event of disease outbreak. Nine out of c-ELISA positive samples were positive for RVFV IgM antibodies indicating a recent infection. The IgM positive samples originated from 7 villages out of the 44 sampled. The spatial distribution of sero-conversion in the study area exhibited no particular pattern but rather a few hotspots (Figure 1). Each district had an almost equal share of the disease, and this was true for the wards. Very few villages had zero seroprevalence. There was a significant relationship between proportion seropositivity and being .15 km from the flood plains as compared to ,5 km (Table 2). PLOS Neglected Tropical Diseases \| www.plosntds.org Inter-epidemic Transmission of RVF in Livestock Inter-epidemic Transmission of RVF in Livestock Table 2. Animal level risk factors associated with RVF sero- positivity for cattle, goats and sheep in the Kilombero valley in multivariable analysis. Variable Levels OR 95% CI Sex Female 1 Male 0.41 0.26–0.63 Present during RVF outbreak No 1 Yes 4.67 3.33–6.55 Distance to the edges of floods (km) ,5 km 1 5–10 km 1.28 0.72–2.29 10–15 km 0.89 0.48–1.65 .15 km 1.92 1.04–3.56 OR = Odds ratio; CI = Confidence interval. doi:10.1371/journal.pntd.0002356.t002 sero-surveys in Madagascar, Nigeria and Senegal [47,48,50]. One of the few other studies reporting sex differences in RVF prevalence in livestock, a study in a slaughter house in Chad also found higher prevalence in female animals [51]. In contrast to our observation, a serosurvey in Madagascar reported higher preva- lence in male animals [50]. Our observation might be as a result of female animals staying longer in a herd due to their role in reproduction and consequently most of sampled female animals were relatively older than their male counterparts. The different timing of our survey and the Madagascar survey, i.e. 4 years versus 3 months post-outbreak, might explain the different result on risk associated with sex in livestock populations. Despite the sero-conversions observed in animals born after the 2006/07 outbreak, there have been no reports of epidemic or clinical disease in the area during the study period. This might be as a result of high herd immunity following the RVF outbreak as demonstrated by high prevalence in animals that were present during the previous epidemic in the study area. In such scenario high proportion of offspring born to immune dams would acquire maternal antibodies thus protected during vulnerable young age whereas old animals of local breeds are naturally less susceptible to clinical disease [52,53]. Another explanation could be a circulation of non-virulent strain of RVFV [54] during inter-epidemic period in the area as it has been hypothesized in other reports of sero- conversion with no previous epidemic or clinical disease reports [46]. The sporadic cases of RVF could easily be confused with other livestock diseases which present similar clinical features of fever, including sporadic abortions and thus overlooked and under reported [53]. proportion sero-conversion up to age 5, Figure 2. The samples included less than one year olds in which the youngest positive individual was 7 months old at which age the maternally acquired immunity has waned out. Results Variable Level %Positive (n) OR 95% CI Species Cattle 11.03 (970) 1 Goat 11.86 (455) 1.06 0.74–1.52 Sheep 11.37 (255) 1.07 0.68–1.69 Sex Female 14.16 (1144) 1 Male 5.22 (536) 0.30 0.19–0.46 Breed Exotic 9.54 (220) 1 Local 11.61 (1420) 1.23 0.70–2.15 Mixed 10.00 (40) 0.95 0.28–3.14 District Kilombero 10.69 (1234) 1 Ulanga 13.00 (446) 1.17 0.68–2.03 Present during RVF outbreak No 5.55 (1116) 1 Yes 22.69 (564) 5.29 3.79–7.38 Herd composition One species 10.27 (360) 1 Two species 10.30 (359) 1.08 0.64–1.83 Three species 12.07 (961) 1.24 0.80–1.92 Distance to the edge of floods (km) ,5 km 10.74 (1117) 1 5–10 km 14.01 (157) 1.27 0.72–2.21 10–15 km 7.94 (214) 0.75 0.41–1.37 .15 km 16.14 (192) 1.62 0.91–2.86 Feeding practices Mixed 4.76 (21) 1 Grazing 11.30 (1592) 2.29 0.29–18.18 Cut & carry 13.43 (67) 2.80 0.31–25.06 Altitude (m) Continuous n/a 1.007 0.99–1.01 OR = Odds ratio; CI = Confidence interval. doi:10.1371/journal.pntd.0002356.t001 PLOS Neglected Tropical Diseases \| www plosntds org 4 August 2013 \| Volume 7 \| Issue 8 \| e235 Figure 2. Seropositivity to Rift Valley fever virus in cattle, sheep and goats pooled by age. The error bars indicate 95% confidenc intervals of the percentage. OR = Odds ratio; CI = Confidence interval. doi:10.1371/journal.pntd.0002356.t001 Figure 2. Seropositivity to Rift Valley fever virus in cattle, sheep and goats pooled by age. The error bars indicate 95% confidence intervals of the percentage. doi:10.1371/journal.pntd.0002356.g002 August 2013 \| Volume 7 \| Issue 8 \| e2356 PLOS Neglected Tropical Diseases \| www.plosntds.org PLOS Neglected Tropical Diseases \| www.plosntds.org 4 References The Journal of Immunology 62: 213–227. 7. Labeaud AD, Ochiai Y, Peters CJ, Muchiri EM, King CH (2007) Spectrum of rift valley fever virus transmission in Kenya: Insights from three distinct regions. Am J Trop Med Hyg 76: 795–800. gy 24. Morita C (1988) Prevalence of Rift Valley fever in Lusaka and Mazabuka - Zambia. J Vet Med B Infect Dis Vet Public Health 35: 157–160. 25. Wilson ML, Chapman LE, Hall DB, Dykstra EA, Ba K, et al. (1994) Rift Valley Fever in Rural Northern Senegal: Human Risk Factors and Potential Vectors. Am J Trop Med Hyg 50: 663–675. 8. Anderson EC, Rowe LW (1998) The prevalence of antibody to the viruses of bovine virus diarrhoea, bovine herpes virus 1, rift valley fever, ephemeral fever and bluetongue and to Leptospira sp in free-ranging wildlife in Zimbabwe. Epidemiol Infect 121: 441–449. 26. Woods CW, Karpati AM, Grein T, McCarthy N, Gaturuku P, et al. (2002) An outbreak of Rift Valley fever in northeastern Kenya, 1997–98. Emerg Infect Dis 8: 138. 9. Swanepoel R, Coetzer JAW (2004) Rift Valley Fever. In: Coetzer JAW, Tustin RC, editors. Infectious Diseases of Livestock in Southern Africa: Oxford University Press. pp. 1037–1059. 27. Morrill JC, Peters C (2011) Mucosal immunization of rhesus macaques with Rift Valley fever MP-12 vaccine. J Infect Dis 204: 617–625. 10. McIntosh B, Russell D, Dos Santos I, Gear J (1980) Rift Valley fever in humans in South Africa. S Afr Med J 58: 803–806. 28. Pepin M, Bouloy M, Bird BH, Kemp A, Paweska J (2010) Rift Valley fever virus (Bunyaviridae: Phlebovirus): an update on pathogenesis, molecular epidemiol- ogy, vectors, diagnostics and prevention. Veterinary research 41: 61. 11. Al-Hazmi M, Ayoola EA, Abdurahman M, Banzal S, Ashraf J, et al. (2003) Epidemic Rift Valley fever in Saudi Arabia: a clinical study of severe illness in humans. Clin Infect Dis 36: 245–252. 29. Abu-Elyazeed R, El-Sharkawy S, Olson J, Botros B, Soliman A, et al. (1996) Prevalence of anti-Rift-Valley-fever IgM antibody in abattoir workers in the Nile delta during the 1993 outbreak in Egypt. Bull World Health Organ 74: 155. 12. Madani TA, Al-Mazrou YY, Al-Jeffri MH, Mishkhas AA, Al-Rabeah AM, et al. (2003) Rift Valley fever epidemic in Saudi Arabia: epidemiological, clinical, and laboratory characteristics. Clin Infect Dis 37: 1084–1092. during the 1993 outbreak in Egypt. Bull World Health Organ 74: 1 30. References 1. Davies FG, Linthicum KJ, James AD (1985) Rainfall and epizootic Rift Valley fever. Bull World Health Organ 63: 941–943. 1. Davies FG, Linthicum KJ, James AD (1985) Rainfall and epizootic Rift Valley fever. Bull World Health Organ 63: 941–943. based survey method to overcome the lack of sampling frame. Vet Microbiol 121: 249–256. 18. Hay SI, Myers MF, Burke DS, Vaughn DW, Endy T, et al. (2000) Etiology of interepidemic periods of mosquito-borne disease. Proc Natl Acad Sci USA 97: 9335–9339. 2. Anyamba A, Chretien J-P, Small J, Tucker CJ, Formenty PB, et al. (2009) Prediction of a Rift Valley fever outbreak. Proc Natl Acad Sci USA 106: 955– 959. 2. Anyamba A, Chretien J-P, Small J, Tucker CJ, Formenty PB, et al. (2009) Prediction of a Rift Valley fever outbreak. Proc Natl Acad Sci USA 106: 955– 959. 3. LaBeaud AD, Muchiri EM, Ndzovu M, Mwanje MT, Muiruri S, et al. (2008) Interepidemic Rift Valley fever virus seropositivity, Northeastern Kenya. Emerg Infect Dis 14: 1240. 19. Gora D, Yaya T, Jocelyn T, Didier F, Maoulouth D, et al. (2000) The potential role of rodents in the enzootic cycle of Rift Valley fever virus in Senegal. Microbes Infect 2: 343–346. 4. LaBeaud AD, Cross PC, Getz WM, Glinka A, King CH (2011) Rift Valley fever virus infection in African buffalo (Syncerus caffer) herds in rural South Africa: evidence of interepidemic transmission. Am J Trop Med Hyg 84: 641–646. 20. WHO (2009) Rift Valley fever outbreaks forecasting models. Rome, Italy. WHO/HSE/GAR/BDP/2009.2 WHO/HSE/GAR/BDP/2009.2. 25 p. 21. Archer BN, Weyer J, Paweska J, Nkosi D, Leman P, et al. (2011) Outbreak of Rift Valley fever affecting veterinarians and farmers in South Africa, 2008. S Afr Med J 101: 263–266. 5. Evans A, Gakuya F, Paweska JT, Rostal M, Akoolo L, et al. (2008) Prevalence of antibodies against Rift Valley fever virus in Kenyan wildlife. Epidemiol Infect 136: 1261–1269. 22. Kitchen SF (1934) Laboratory infections with the virus of Rift Valley fever. Am J Trop Med Hyg 1: 547–564. 6. Heinrich N, Saathoff E, Weller N, Clowes P, Kroidl I, et al. (2012) High Seroprevalence of Rift Valley Fever and Evidence for Endemic Circulation in Mbeya Region, Tanzania, in a Cross-Sectional Study. PLoS Negl Trop Dis 6: e1557. 23. Smithburn KC, Mahaffy AF, Haddow AJ, Kitchen SF, Smith JF (1949) Rift Valley Fever: Accidental Infections Among Laboratory Workers. Acknowledgments We would like to extend our sincere gratitude to district authorities of Kilombero and Ulanga, agriculture/livestock extension officers and village leaders for their support in the course of conducting this study. We are also indebted to livestock keepers from respective villages who willingly agreed to participate in this study and also by providing assistance in many ways thus making the smooth running of activities. We also thank the anonymous reviewers for their important critiques on this manuscript. Discussion The findings from this serosurvey indicate post-epidemic and recent transmission of RVFV in livestock populations in the Kilombero valley. The demonstration of RVFV antibodies in animals as young as one year old, and the observed linear increase in the proportion of sero-converted animals from the age of 1 year to 5 years implies a constant exposure to infectious mosquito bites. The IgM antibodies detected in some animals illustrate presence of active foci of recent transmission preceding the serosurvey as the median duration of IgM antibodies to RVFV is two months [39– 41]. These observations add to the increasing body of serological [3,4,42,43] and virological [44,45] evidence pertaining to RVFV transmission during the inter-epidemic periods in parts of Africa. Inter-epidemic transmission can be detected where active disease surveillance is in place in the livestock and/or human populations, as most of the inter-epidemic infections either are subclinical or mistaken for other diseases in the absence of public awareness of RVF presence [6,46,47]. Given the possible active transmission observed in this study within Kilombero valley and the moving out of livestock from the valley as a result of environmental degradation of wetland due to overstocking and overgrazing, there are chances for incubating or sick animals to introduce the disease into new areas. This might be The high prevalence observed in animals that were present during the outbreak is not surprising as during epidemics there is high exposure to RVFV with resulting high herd immunity [48,49]. Such increased prevalence with age was also reported in August 2013 \| Volume 7 \| Issue 8 \| e2356 PLOS Neglected Tropical Diseases \| www.plosntds.org August 2013 \| Volume 7 \| Issue 8 \| e2356 5 Inter-epidemic Transmission of RVF in Livestock river valley. The linear increase in prevalence of RVFV antibodies in the post-epidemic annual cohorts implies a constant exposure and presence of active foci of recent transmission preceding the survey. enhanced by the quick means of transportation employed and possibilities for animals to harbour the RVFV for up to three weeks [52,56]. In view of that, follow up of these moved herds and their new environment through serological and vector population monitoring will help to inform various stakeholders of the currently unidentified consequences, as livestock movement have been implicated to spread RVF in previously free areas [16,57]. Supporting Information Table S1 Comparison of RVF prevalence across species, sex and presence during the 2006/07 RVF epidemic. (DOC) The interaction of livestock keepers with their animals is intense and includes milking, taking care of sick animals, grazing, using as draft animals, slaughtering and butchering and even children playing with animals. Future work should establish to what degree this inter-epidemic zoonotic circulation of RVFV leads to human infection as well. If considerable transmission to humans exists, health care providers within the Kilombero valley should consider RVF in their differential diagnosis in all fever cases presented in their facilities as RVF in humans may present with similar clinical signs to malaria, which is thought to be the main cause of fever in the valley [58]. We think this should be of priority in particular when dealing with patients from agro-pastoralist communities, especially when the malaria test is negative. Conclusion Conceived and designed the experiments: RDS EG DB. Performed the experiments: RDS EM. Analyzed the data: RDS EG DB. Wrote the paper: RDS EG DB. Conceived and designed the experiments: RDS EG DB. Performed the experiments: RDS EM. Analyzed the data: RDS EG DB. Wrote the paper: RDS EG DB. The findings from this study indicate post-epidemic and recent transmission of RVFV in livestock populations in the Kilombero Inter-epidemic Transmission of RVF in Livestock Inter-epidemic Transmission of RVF in Livestock Inter-epidemic Transmission of RVF in Livestock control of endophagic filariasis, arbovirus and malaria vectors. PLoS Negl Trop Dis 4: e773. 46. Pourrut X, Nkoghe D, Souris M, Paupy C, Paweska J, et al. (2010) Rift Valley fever virus seroprevalence in human rural populations of Gabon. PLoS Negl Trop Dis 4: e763. 35. Armstrong-Schellenberg J, Mukasa O, Abdulla S, Marchant T, Lengeler C, et al. (2002) Chapter 11. Ifakara DSS, Tanzania. 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Addis Ababa, Ethiopia (International Livestock Centre for Africa). 287pp p. p 37. ILCA (1990) Livestock systems research manual. Add (International Livestock Centre for Africa). 287pp p. 38. Paweska JT, Mortimer E, Leman PA, Swanepoel R (2005) An inhibition enzyme- linked immunosorbent assay for the detection of antibody to Rift Valley fever virus in humans, domestic and wild ruminants. J Virol Methods 127: 10–18. 50. Jeanmaire EM, Rabenarivahiny R, Biarmann M, Rabibisoa L, Ravaomanana F, et al. (2011) Prevalence of Rift Valley fever infection in ruminants in Madagascar after the 2008 outbreak. Vector Borne Zoonotic Dis 11: 395–402. 39. Paweska JT, Burt FJ, Anthony F, Smith SJ, Grobbelaar AA, et al. (2003) IgG- sandwich and IgM-capture enzyme-linked immunosorbent assay for the detection of antibody to Rift Valley fever virus in domestic ruminants. J Virol Methods 113: 103–112. 51. Ringot D, Durand JP, Tolou H, Boutin JP, Davoust B (2004) Rift Valley fever in Chad. Emerg Infect Dis 10: 945–947. 52. Swanepoel R, Paweska JT (2011) Rift valley fever. References Mohamed M, Mosha F, Mghamba J, Zaki SR, Shieh WJ, et al. (2010) Epidemiologic and clinical aspects of a Rift Valley fever outbreak in humans in Tanzania, 2007. Am J Trop Med Hyg 83: 22–27. 13. Daubney R, Hudson JR (1931) Enzootic hepatitis or Rift Valley fever: an undescribed virus disease of sheep, cattle and man from East Africa. Journal of Pathology 34: 545–579. 31. WHO (2007) Rift Valley Fever in Kenya, Somalia and the United Republic of Tanzania: Distribution Map. gy 14. Chevalier V, Thiongane Y, Lancelot R (2009) Endemic transmission of Rift Valley fever in Senegal. Transbound Emerg Dis 56: 372–374. 32. WHO (2007) Outbreaks of Rift Valley fever in Kenya, Somalia and United Republic of Tanzania, December 2006–April 2007. Wkly Epidemiol Rec 82: 169–178. 15. Zeller HG, Fontenille D, Traore-Lamizana M, Thiongane Y, Digoutte J-P (1997) Enzootic Activity of Rift Valley Fever Virus in Senegal. Am J Trop Med Hyg 56: 265–272. 33. Killeen GF, Tami A, Kihonda J, Okumu FO, Kotas ME, et al. (2007) Cost- sharing strategies combining targeted public subsidies with private-sector delivery achieve high bednet coverage and reduced malaria transmission in Kilombero Valley, southern Tanzania. BMC Infect Dis 7: 121. 16. Balkhy HH, Memish ZA (2003) Rift Valley fever: an uninvited zoonosis in the Arabian peninsula. Int J Antimicrob Agents 21: 153–157. Kilombero Valley, southern Tanzania. BMC Infect Dis 7: 121. Arabian peninsula. Int J Antimicrob Agents 21: 153–157. 34. Ogoma SB, Lweitoijera DW, Ngonyani H, Furer B, Russell TL, et al. (2010) Screening mosquito house entry points as a potential method for integrated 17. Soumare B, Tempia S, Cagnolati V, Mohamoud A, Van Huylenbroeck G, et al. (2007) Screening for Rift Valley fever infection in northern Somalia: A GIS PLOS Neglected Tropical Diseases \| www.plosntds.org August 2013 \| Volume 7 \| Issue 8 \| e2356 6 PLOS Neglected Tropical Diseases \| www.plosntds.org Inter-epidemic Transmission of RVF in Livestock Oxford Textbook of Zoonoses: Biology, Clinical Practice, and Public Health Control. pp. 423. 40. Geering WA, Davies FG, Martin V (2002) Preparation of Rift Valley fever contingency plans. FAO Animal Health Manual No. 15. 7–18 p. 53. Davies FG, Martin V (2006) Recognizing Rift Valley fever. Vet Ital 42: 31–53. 54. Muller R, Saluzzo J-F, Lopez N, Dreier T, Turell M, et al. (1995) Characterization of clone 13, a naturally attenuated avirulent isolate of Rift Valley fever virus, which is altered in the small segment. Am J Trop Med Hyg 53: 405. 41. Morvan J, Rollin PE, Laventure S, Roux J (1992) Duration of immunoglobulin M antibodies against Rift Valley fever virus in cattle after natural infection. 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Contrib Epidemiol Biostat 3: 83–91. 58. Hetzel M, Alba S, Fankhauser M, Mayumana I, Lengeler C, et al. (2008) Malaria risk and access to prevention and treatment in the paddies of the Kilombero Valley, Tanzania. Malar J 7: 7. 45. Linthicum KJ, Davies FG, Kairo A, Bailey CL (1985) Rift Valley fever virus (family Bunyaviridae, genus Phlebovirus). Isolations from Diptera collected during an inter-epizootic period in Kenya. J Hyg (Lond) 95: 197–209. August 2013 \| Volume 7 \| Issue 8 \| e2356 PLOS Neglected Tropical Diseases \| www.plosntds.org 7 7
https://openalex.org/W4304161337	https://econjournals.com/index.php/ijefi/article/download/13405/6962	English	null	Effectiveness of the Asset Price Channel as a Monetary Policy Transmission Mechanism in Malawi: Evidence from Time Series Data	International journal of economics and financial issues	2,022	cc-by	7,572	ABSTRACT This paper first, investigates the responsiveness of stock prices to changes in interest rates in Malawi, a low-income country in sub-Saharan Africa. Secondly, determines whether Gross Domestic Product of such economies respond significantly to changes in stock prices. Time series data from January 2001 to December 2019 are utilised. Unit root tests indicate that each variable is integrated of order one and the Johansen test for cointegration reveal that the variables are cointegrated, with one cointegrating equation. Therefore, a vector error correction model has been adopted to investigate the short and long-run dynamic relationship among the variables. Results of Granger causality tests and impulse response analysis indicate that stock prices do respond to changes in interest rates. Moreover, Gross Domestic Product also responds to changes in stock prices. Thus, contrary to previous studies, this paper finds that an asset price channel operating through stock prices exists in Malawi. More specifically, this study finds that variations in interest rates cause significant changes in stock prices which in turn cause changes in Gross Domestic Product. Among others, the study recommends that the monetary authorities should raise public awareness on investment opportunities available in stock markets. Keywords: Econometric Modelling, Informal Economies, Monetary Policy, Time Series Models JEL Classifications: C32, C50, E26, E52 Keywords: Econometric Modelling, Informal Economies, Monetary Policy, Time Series Models JEL Classifications: C32, C50, E26, E52 International Journal of Economics and Financial Issues, 2022, 12(5), 160-168. 1. INTRODUCTION This study assesses the effectiveness of the financial asset prices as a channel of monetary policy transmission in low-income countries. It uses data on stock prices, interest rates and Gross Domestic Product (GDP) per capita from Malawi, a low-income country in sub-Saharan Africa (SSA) with a GNI per capita of about US$360 (World Bank, 2018). This study assesses the effectiveness of the financial asset prices as a channel of monetary policy transmission in low-income countries. It uses data on stock prices, interest rates and Gross Domestic Product (GDP) per capita from Malawi, a low-income country in sub-Saharan Africa (SSA) with a GNI per capita of about US$360 (World Bank, 2018). Monetary policy is formulated to influence the trajectory of economies towards a more desirable state. For example, when economic output is declining or when unemployment is rising, authorities may lower interest rates or expand the money supply with an aim of encouraging consumption and investment (Blanchard, 2017). Although scholars have long been studying transmission mechanisms of monetary policy (Bernanke and Gertler, 1995; Ireland, 2010), the interest continues to grow even in recent times (Cesa-Bianchi et al., 2020) especially given the influence it has in changing the structure of the economy (European Central Bank, 2010). An understanding of monetary policy transmission mechanisms enables authorities to determine both the required level and the proper timing of policy interventions (European Central Bank, 2010). In itself, the determination of which channels operate within an economy can be a black box (Bernanke and Gertler, This Journal is licensed under a Creative Commons Attribution 4.0 International License 160 Betchani H. M. Tchereni1, Ahmed Makawa2, Fredrick Banda3 1Department of Management Studies, Faculty of Commerce, University of Malawi, the Polytechnic, Private Bag 303, Chichiri, Blantyre 3, Malawi, 2Department of Management Studies, University of Malawi-The Polytechnic, Private Bag 303, Chichiri, Blantyre 3, Malawi, 3Department of Business Administration, University of Malawi, the Polytechnic, Private Bag 303, Chichiri, Blantyre 3, Malawi. Email: btchereni@poly.ac.mw Accepted: 29 August 2022 DOI: https://doi.org/10.32479/ijefi.13405 Received: 16 June 2022 Received: 16 June 2022 International Journal of Economics and Financial Issues ISSN: 2146-4138 available at http: www.econjournals.com International Journal of Economics and Financial Issues, 2022, 12(5), 160-168. International Journal of Economics and Financial Issues ISSN: 2146-4138 available at http: www.econjournals.com International Journal of Economics and Financial Issues, 2022, 12(5), 160-168. International Journal of Economics and Financial Issues \| Vol 12 • Issue 5 • 2022 ess of the Asset Price Channel as a Monetary Policy Transmission Mechanism in Malawi: Evidence from Time Series Data Tchereni, et al.: Effectiveness of the Asset Price Channel as a Monetary Policy Transmission Mechanism in Malawi: Evide 1995). Low-income countries face additional challenges in their determination of channels of monetary policy transmission due to their underdeveloped institutional frameworks. As observed by Mishra et al. (2012), this results in weak or non-existent interest rate and asset price-based channels. stock prices rise. When stock prices rise, q increases because the market value of a firm is the product of average share price and the number of issued shares. Firms can therefore raise more funds by issuing a small number of additional shares. When faced with the need to decide whether to make new investment spending or use existing equipment by taking over existing firms, companies find it cheaper to issue stock and get a high price relative to the cost of the facilities and equipment they are buying. This means investment spending will rise as firms will be able to buy new equipment with an issue of a few stocks. This leads to an increase in national income. Studies have either explicitly dismissed the idea of an asset price channel, while others have not incorporated asset prices such as stock prices when investigating transmission mechanisms of monetary policy. For example, citing a study by Phiri (2002) who proposed that stock prices may act as a possible conduit of monetary policy transmission in Malawi, Ngalawa and Viegi (2011) observed that the stock exchange was immature to operate as a possible channel of monetary policy and therefore did not empirically test it using stock price data. A similar approach was echoed by Mwabutwa et al. (2013) whose study on the evolution of monetary policy transmission mechanisms in Malawi did not include stock prices. On the other hand, if share prices decline, the cost of buying replacement assets is high relative to the market value of firms. When faced with a need for additional assets, companies find it cheaper to just buy another firm and obtain its assets instead of purchasing new assets. As a result, no new assets are purchased by firms and investment falls. International Journal of Economics and Financial Issues \| Vol 12 • Issue 5 • 2022 It is easy to dismiss entirely the potential role that can be played by stock prices in the transmission of monetary policy in low-income countries on the basis of the immaturity of their stock markets. The motivation to take a closer look at stock prices in Malawi is due, in part, to the fact that there has been a dramatic rise in the volume of trade conducted at the Malawi Stock Exchange since its inception at the end of the 20th century. For example, during the first quarter of 2003, the market recorded a total of 22,307,347 shares (Malawi Stock Exchange, 2003). By the third quarter of 2019, the number of traded shares had risen to 209,366,267 (Malawi Stock Exchange, 2019) representing an average steady increase of 15% per annum. With such an increase in the popularity of trade at the Malawi Stock Exchange, it is imperative to know whether stock prices can now act as a viable channel of monetary policy transmission. This study is adding on to the existing literature on monetary policy transmission mechanisms of low-income economies. In addition to Tobin’s Q Theory, the Wealth Effect provides a theoretical explanation of the asset price channel of monetary policy transmission. It is based on the Life Cycle Hypothesis (Ando and Modigliani, 1963) which states that consumption spending is a function of the lifetime resources owned by individuals and not just their current income. Therefore, consumers are more likely to increase their consumption if their wealth increases, and reduce their consumption spending if their wealth declines. Thus, during expansionary monetary policy, stock prices increase. Individual investors will now value their wealth as being higher than before. According to Modigliani’s Life Cycle Hypothesis, the perceived increase in wealth leads to an increase in consumption. Ultimately, this leads to an increase in national income. In essence, this study utilises data collected from Malawi to check the applicability of Tobin’s Q Theory and the Wealth Effect in the context of a low-income economy. This study therefore aimed to assess the responsiveness of stocks to changes in interest rates or money supply and to determine whether GDP responds to changes in stock prices in Malawi. The rest of the paper is organized as follows. Section 2 is literature review followed by methodology in section 3Results and conclusions of the study are presented in Section 4 and 5 respectively. International Journal of Economics and Financial Issues \| Vol 12 • Issue 5 • 2022 Figure 1 highlights the relationship between stock prices and a monetary policy variable (interest rates) on one hand, and that of stock prices and real macroeconomic variable (y) on the other hand. 2.1. Theoretical and Conceptual Framework The investigation of monetary policy transmission mechanisms using the Vector Autoregressive model (VAR) or Vector Error Correction Model (VECM) techniques has intensified since the publication of the pioneering work by Sims (1980). Researchers throughout the world have attempted to analyse monetary policy transmission mechanisms by applying this method. The theoretical basis of the asset price channel operating through stock prices was laid down in Tobin’s Q Theory proposed by Kaldor (1966) as a ratio of the market value of a firm to the cost of replacement of its assets. = MarketValueof Firm q ReplacementCost of its Assets (1) = MarketValueof Firm q ReplacementCost of its Assets (1) (1) Although Mishkin (2017) identified the traditional transmission mechanisms of monetary policy such as the interest rate and exchange rate channels, novel transmission mechanisms continue to be explored (see for example the deposits channel by Drechsler et al. (2017) as well as the risk-taking channel by Delis et al. (2017)). Moreover, due to the importance attached to the Although Mishkin (2017) identified the traditional transmission mechanisms of monetary policy such as the interest rate and exchange rate channels, novel transmission mechanisms continue to be explored (see for example the deposits channel by Drechsler et al. (2017) as well as the risk-taking channel by Delis et al. (2017)). Moreover, due to the importance attached to the Essentially, this states that lowering interest rates (an act of expansionary monetary policy) make investors prefer stocks (equity) relative to bonds whose relative yields decline. As a result, Essentially, this states that lowering interest rates (an act of expansionary monetary policy) make investors prefer stocks (equity) relative to bonds whose relative yields decline. As a result, 161 International Journal of Economics and Financial Issues \| Vol 12 • Issue 5 • 2022 veness of the Asset Price Channel as a Monetary Policy Transmission Mechanism in Malawi: Evidence from Time Series Da Tchereni, et al.: Effectiveness of the Asset Price Channel as a Monetary Policy Transmission Mechanism in Malawi: Evide Figure 1: Theoretical relationship between interest rates, stock prices and income Figure 1: Theoretical relationship between interest rates, stock prices and income did not find evidence of the asset price channel but instead suggest the existence of an exchange rate channel. did not find evidence of the asset price channel but instead suggest the existence of an exchange rate channel. understanding of monetary policy transmission mechanisms by central bankers (European Central Bank, 2010), several studies have investigated transmission mechanisms of monetary policy in different low-income economies such as Sierra Leone (Lavally and Nyambe, 2019) and Tanzania (Alexianu, 2020), as well as in medium-income economies such as Vietnam (Nguyen et al., 2019) and Ethiopia (Nuru, 2019). In Malawi, the study by Ngalawa and Viegi (2011) and Mwabutwa et al. (2013) are among the major works on transmission mechanisms of monetary policy. The fact that many studies of transmission mechanisms of monetary policy do not specifically test for the existence of the asset price channel can be explained by the widely held view that low-income countries have a weak financial institutional framework (Bolnick, 1991; Mishra et al., 2012). Thus in Kenani et al. (2012), the immaturity of the Malawi Stock Exchange has been cited as an indication that the financial sector in Malawi is still underdeveloped. The study is aimed at investigating any short-run and long-run relationships between stock prices and exchange rates in Malawi for the period from 1999 to 2010. One conclusion from the study is that both internal and external macroeconomic shocks do not have immediate effect on the stock and foreign exchange markets. This implies that stock prices do not show a significant responsiveness to changes in monetary policy variables such as interest rates and money supply. Ngalawa and Viegi (2011) investigated how monetary policy decisions affect economic activity in Malawi. Using a Structural Vector Autoregression (S-VAR) approach, the study illustrates that bank lending, exchange rates and aggregate money supply are important transmission processes of monetary policy shocks in Malawi. International Journal of Economics and Financial Issues \| Vol 12 • Issue 5 • 2022 However, Ngalawa and Viegi (2011) highlighted that the Malawi Stock Exchange which was opened in 1996 (Malawi Stock Exchange, 2020) was still immature to act as a viable channel of monetary policy transmission. Consequently, despite their use of 7 macroeconomic variables, Ngalawa and Viegi (2011) did not include the stock price index in their S-VAR model. In this aspect, this study is unique in that it utilises the Malawi All Share Index (MASI) in the analysis of the transmission mechanism of monetary policy. However, in a study that analyses secondary data sets of thirteen local companies listed on the Malawi Stock Exchange for the period 2008 to 2014, Majanga (2015) employs correlation analysis and finds a strong positive relationship between a firm’s dividends and its stock price on the stock market despite the underdeveloped financial sector and immaturity of the stock markets in low- income countries suggested by Kenani et al. (2012) and Mishra et al. (2012). Mwabutwa et al. (2013) investigateed how the monetary transmission mechanism in Malawi evolved between 1981 and 2010 through the use of a Time Varying Parameter Vector Autoregressive (TVP-VAR) model. The study concludes that inflation, real output and exchange rate responses to monetary policy shocks varied significantly over the reviewed period. Although it adopts the TVP-VAR approach in order to avert the limitations of the traditional VAR model such as price puzzles and omitted variable bias, the study did not specifically incorporate the stock price index among the 5 variables it utilized. With such a mixed array of findings, and the increase in the volume of trade at the Malawi Stock Exchange as well as the clear absence of a study that specifically tests for the asset price channel in Malawi, this study examines the transmission process of monetary policy in Malawi. Next section presents the methodology. 3.1.Research Design hi d d i Lungu et al. (2012) attempted to derive a money demand function and explored its implications for monetary policy conduct in Malawi from 1985 to 2010. Through cointegration tests in a VECM approach, the study finds a long-run relationship between real money balances, prices, income and exchange rates. It however This study adopts a positivist philosophy (Saunders et al., 2019) in which transmission mechanisms of monetary policy in Malawi are regarded as an objective reality capable of being measured independent of the researcher. In contrast to this, interpretivism is a perspective adopted by researchers who acknowledge that 162 3.2. Variable Selection and Data Sources In this study, the primary focus is the determination of the effectiveness of the asset channel as a monetary policy transmission mechanism thereby establishing an empirical link between interest rate and Gross Domestic Product (GDP) through stock prices. It is standard practice in time-series analysis to begin by checking whether the series are stationary in levels or if they are integrated. A time-series variable is said to be stationary if it has a time invariant mean and variance, and if the correlation between any two observations only depend on the time between observations, not on the actual time that the observations are made (Hill et al., 2018). This is important since there is a possibility that the series may be non-stationary and cointegrated, in which case estimation of Equation 2 using Ordinary Least Squares (OLS) method would lead to spurious regression. This is undesirable, as regression results may indicate that the variables have significant relationships when in actual fact, they are unrelated (Gujarati, 2013). In their study of the dynamic effects of monetary policy in Malawi, Ngalawa and Viegi (2011) use seven variables in their S-VAR model. These variables were output, consumer price level, commercial bank loans, exchange rates, aggregate money supply, bank rate and reserve money. Although they admit that the number of variables in their study is higher than those in comparable studies, it is interesting to note that conspicuously missing from their long list is stock prices. Given that the authors had already overruled the possibility of the asset channel as a monetary policy transmission mechanism in Malawi, it is unsurprising to find that movement of stock prices is not captured in their study. It will therefore be inappropriate to adopt their choice of variables in this study. Thus, the study adopts a quantitative methodology following the Structural Vector Auto Regression (SVAR) approach developed by Sims (1980) which has been widely applied, even in contemporary studies such as the one for the Democratic Republic of Congo (Yemba et al., 2020), Vietnam (Nguyen et al., 2019) as well as the study for Pakistan (Anis et al., 2019). Thus, let Yt be an (n × 1) matrix representing a vector of autoregressive endogenous variables which can be expressed as a function of their own lags. International Journal of Economics and Financial Issues \| Vol 12 • Issue 5 • 2022 International Journal of Economics and Financial Issues \| Vol 12 • Issue 5 • 2022 International Journal of Economics and Financial Issues \| Vol 12 • Issue 5 • 2022 ess of the Asset Price Channel as a Monetary Policy Transmission Mechanism in Malawi: Evidence from Time Series Data Tchereni, et al.: Effectiveness of the Asset Price Channel as a Monetary Policy Transmission Mechanism in Malawi: Evide their subjective experiences cannot be separated from the study but aim at gaining an in-depth meaning and understanding of a social phenomenon (Cresswell, 2018). between changes in interest rates (INTR) and stock prices (LOGMASI), and if changes in stock prices affect per capita output (LOGGDP) in Malawi. Ideally, this can be investigated by estimating a linear regression model in which LOGGDP is the dependent variable while LOGMASI and INTR are independent variables as in equation 2: The research approach adopted is deductive, as an attempt to use data to prove the applicability of the asset price channel (Mishkin, 2017) in Malawi is made. Therefore, the methodology adopted in this study is quantitative, being an econometric investigation of the dynamic relationship between selected macroeconomic variables in Malawi within the study period using secondary data. (2) Apriori, the sign of β1 is expected to be negative since any reduction in interest rates is an instance of expansionary monetary policy which is expected to lead to higher output. However, β2 is expected to be positive since from Tobin’s Q Theory, increases in stock prices lead to increases in GDP. 3.2. Variable Selection and Data Sources Taking natural logarithms of the variables in Table 1 except for interest rates, we have: However, in a study of monetary policy transmission mechanisms in Vietnam, Canh and Vo (2014) use a rotating variable approach by combining three endogenous variables with one proxy variable for each transmission mechanism they investigated. The three endogenous variables were the Industrial Production Index, the Consumer Price Index (CPI) and the Vietnamese Interbank Offer Rate. Unlike that of Ngalawa and Viegi (2011), the study by Cahn and Vo (2014) investigated the effectiveness of the asset channel using the Vietnam Stock Market Index as the proxy variable for stock prices. → =       t t t LOGGDP Y t INTR LOGMASI (3) (3) This study therefore adopts the approach taken by Canh and Vo (2014) as it is the one which investigated the asset channel. However, this study does not incorporate the CPI because it only focusses on the asset price channel as outlined in Tobin’s Q theory (Mishkin, 2017). Instead, this study uses the following variables: Interest rates (INTR), the natural logarithm of the Malawi All Share Index (LOGMASI), and the natural logarithm of real Gross Domestic Product (LOGGDP) per capita for Malawi. The use of INTR is justified on the basis of the observation by Ngalawa and Viegi (2011) that it is a more effective operating target variable of monetary policy than reserve money. Moreover, LOGMASI represents the channel variable of interest while LOGGDP is a macroeconomic policy goal variable. In a VAR model, the variables are expressed as a function of their own previous lags, and the lags of all other variables (Asteriou and Hall, 2016). Thus, in order to test for the existence of the asset price channel through Tobin’s Q Theory, one must establish the link In a VAR model, the variables are expressed as a function of their own previous lags, and the lags of all other variables (Asteriou and Hall, 2016). Thus, in order to test for the existence of the asset price channel through Tobin’s Q Theory, one must establish the link Table 1: Variables and data sources VARIABLE DESCRIPTION SOURCE INTR Interest rate Reserve bank of malawi (RBM) https://www.rbm.mw/Statistics/ FinancialData/ MASI Malawi all share index Malawi stock exchange (MSE) https://mse.co.mw/index. php?route=market/market/report GDPC GDP per capita Federal reserve economic data (FRED) https://fred.stlouisfed.org/series/ NYGDPPCAPKDMWI VARIABLE DESCRIPTION SOURCE INTR Interest rate Reserve bank of malawi (RBM) https://www.rbm.mw/Statistics/ FinancialData/ MASI Malawi all share index Malawi stock exchange (MSE) https://mse.co.mw/index. php?route=market/market/report GDPC GDP per capita Federal reserve economic data (FRED) https://fred.stlouisfed.org/series/ NYGDPPCAPKDMWI International Journal of Economics and Financial Issues \| Vol 12 • Issue 5 • 2022 4. RESULTS AND DISCUSSION (5) 4.1. Summary Statistics This section presents the results of the study on the asset price channel of monetary policy transmission in Malawi. Figure 2 indicate that overall, interest rates declined during the study period whereas both stock prices and per capita income showed a positive trend. If the series are cointegrated, a vector error correction (VEC) model is estimated which enable the determination of both the short run and long run relationship of the variables (Engle and Granger, 1987). Stated in general for the 3 variable case as follows: It can be seen from Table 3 that the average value of interest rates in Malawi was 21.1% with a standard deviation of 11.7% indicating that there was great interest rate volatility during the study period. Moreover, the average value of the stock price index was 8433 while that of per capita GDP was about US$38 per month. Note that this amounts to an average per capita income of $1.27 per day. 1 1 1 0 1 1 1 1 α β γ λ φ ε − − − − − − − = = = ∆ = + ∆ + ∆ + ∆ + + ∑ ∑ ∑ p p p t i t i j t j j t k t t i j k Y Y X Z ECT (7) (7) Where ECT is the error correction term which represent the long- run relationship, p is the optimum lag length and φ is the speed of adjustment. Apriori, this is expected to be negative and represents the proportion of the error in the current period that is corrected towards the long-run relationship. The variables Y, X and Z are the endogenous variables (in this case they represent LOGGDP, LOGMASI and INTR) and ∆ is the difference operator. 3.3. Econometric Modelling and Data Analysis 3.3. Econometric Modelling and Data Analysis As a starting point in the modelling process, it must be emphasised that this study seeks to establish whether there is any relationship 163 International Journal of Economics and Financial Issues \| Vol 12 • Issue 5 • 2022 ess of the Asset Price Channel as a Monetary Policy Transmission Mechanism in Malawi: Evidence from Time Series Data Tchereni, et al.: Effectiveness of the Asset Price Channel as a Monetary Policy Transmission Mechanism in Malawi: Evide between interest rates and stock prices on one hand, and between stock prices and GDP on the other hand. Hence the following system of equations is estimated as a VAR, assuming for the time being that there is only one lag (the actual number of lags are obtained from lag length criteria test undertaken in the study, Table 2): In the analysis, the first stage tested for unit roots on each of the series using the widely adopted Augmented Dickey-Fuller (ADF) test (Cheung and Lai, 1995) and to ensure robustness of the results, the Phillips-Perron (PP) test is also used (Phillips and Perron, 1988). This is followed by the basic VAR which is used to carry out lag-length selection tests. The Johansen Test for cointegration (Johansen, 1991) is next and, depending on the results, either VAR in levels or a vector error correction model (VECM) will be estimated. Section 4 presents the results. LOGGDPt = α0 + α1LOGGDPt−1 + α2LOGMASIt−1 + α3INTRt−1 + εt (4) LOGMASIt = β0 + β1LOGGDPt−1 + β2LOGMASIt−1 + β3INTRt−1 + µt (5) INTRt = γ0 + γ1LOGGDPt−1 + γ2LOGMASIt−1 + γ3INTRt−1 + νt (6) 4.2. Results of Unit Root, Lag Order Selection and Cointegration Testsi The ADF tests and PP tests confirm that all series are integrated of order 1. For the basic VAR involving INTR, LOGMASI and LOGGDP, results of lag selection test (Table 2) show that the optimum lag length is either 6 or 5, being the lag lengths selected by the widely adopted Akaike Information Criteria (AIC) (Akaike, International Journal of Economics and Financial Issues \| Vol 12 • Issue 5 • 2022 164 Table 2: Lag length selection test Lag LogL LR FPE AIC SC HQ 0 –734.2695 NA 0.163461 6.702450 6.748726 6.721137 1 905.5555 3220.020 5.95e‑08 –8.123232 –7.938125 –8.048481 2 1304.759 773.0029 1.71e‑09 –11.67054 –11.34660 –11.53972 3 1317.493 24.30979 1.66e‑09 –11.70448 –11.24171 –11.51760 4 1395.162 146.1602 8.88e‑10 –12.32875 –11.72715 –12.08581 5 1436.147 76.00858 6.64e‑10 –12.61952 –11.87909* –12.32052* 6 1450.864 26.89125 6.31e‑10* –12.67149* –11.79223 –12.31642 7 1454.552 6.638872 6.63e‑10 –12.62320 –11.60511 –12.21207 8 1465.136 18.76248* 6.54e‑10 –12.63760 –11.48068 –12.17041 Figure 2: (a-c) Graphs of variables in the study b a c International Journal of Economics and Financial Issues \| Vol 12 • Issue 5 • 2022 164 Table 2: Lag length selection test Lag LogL LR FPE AIC SC HQ 0 –734.2695 NA 0.163461 6.702450 6.748726 6.721137 1 905.5555 3220.020 5.95e‑08 –8.123232 –7.938125 –8.048481 2 1304.759 773.0029 1.71e‑09 –11.67054 –11.34660 –11.53972 3 1317.493 24.30979 1.66e‑09 –11.70448 –11.24171 –11.51760 4 1395.162 146.1602 8.88e‑10 –12.32875 –11.72715 –12.08581 5 1436.147 76.00858 6.64e‑10 –12.61952 –11.87909* –12.32052* 6 1450.864 26.89125 6.31e‑10* –12.67149* –11.79223 –12.31642 7 1454.552 6.638872 6.63e‑10 –12.62320 –11.60511 –12.21207 8 1465.136 18.76248* 6.54e‑10 –12.63760 –11.48068 –12.17041 Figure 2: (a-c) Graphs of variables in the study b a c Table 2: Lag length selection test Table 2: Lag length selection test Figure 2: (a-c) Graphs of variables in the study Figure 2: (a-c) Graphs of variables in the study b a c b c a 164 International Journal of Economics and Financial Issues \| Vol 12 • Issue 5 • 2022 ess of the Asset Price Channel as a Monetary Policy Transmission Mechanism in Malawi: Evidence from Time Series Data Tchereni, et al.: Effectiveness of the Asset Price Channel as a Monetary Policy Transmission Mechanism in Malawi: Evide 1974), and the Bayesian Information Criterion (BIC) also known as the Schwarz Criterion (SC) (Schwarz et al., 1978) respectively. 4.4. Granger Causality Tests Granger causality tests reveal that changes in interest rates (INTR) cause significant changes in stock prices (LOGMASI) (Table 4). Having found our variables to be cointegrated with one cointegrating equation, we now estimate a vector error correction model (VECM). This is a restricted VAR estimated in first differences. It therefore has one lag less than the corresponding VAR, hence our VEC model has 5 lags. From the results of the Johansen test for cointegration, the cointegration equation in our VEC model (normalised on LOGGDP) is as follows: This confirms the first link in Tobin’s Q Theory and hence the existence of the asset price channel in Malawi. The null hypothesis that INTR does not Granger cause LOGMASI is rejected, even at the 0.01 significance level as the p-value of the test statistic is 0.0009, being much smaller than the standard 0.05 significance level. This means changes in interest rates do significantly cause changes in stock prices. ECTt−1 = LOGGDPt−1 − 0.065089LOGMASIt−1 + 0.002758INTRt−1 − 3.1499 (8) t−1 (8) We interpret this as follows. In the long run, stock prices (LOGMASI) in Malawi have a positive impact on per capita income (LOGGDP), ceteris paribus. This is consistent with the existence of the asset price channel through stock prices since Tobin’s Q Theory (see Section 2.1) indicates that when stock prices rise (Ps↑), there should be an increase in the value of q represented as q ↑ which should ultimately lead to an increase in income (Y ↑) as illustrated in Equation 2. Moreover, the value of coefficient φ of the error correction term (ECM) in Equation 7 is found (from the estimated VECM) to be -0.001854. This is negative as expected, confirming that the short run movements of the variables in the system do converge as expected to the long run relationship expressed in the ECT. This can be interpreted as follows. A unit short run change in the endogeneous variables in the current period is corrected by a factor of 0.19% towards the long run equilibrium. Moreover, Granger causality test results between LOGMASI and LOGGDP also indicate that changes in stock prices (LOGMASI) do have a significant effect on LOGGDP. This is illustrated in Table 5 where the null hypothesis that LOGMASI does not Granger cause LOGGDP is rejected at the 0.05 significance level. 4.4. Granger Causality Tests Thus, both tests confirm the existence of the asset price channel operating through stock prices, contrary to earlier findings by Ngalawa and Viegi (2011) and Mwabutwa et al. (2013). 4.2. Results of Unit Root, Lag Order Selection and Cointegration Testsi rates represent expansionary monetary policy and is expected to lead to increases in stock prices (according to Tobin’s Q Theory outlined in Section 2.1) which should lead to increases in wealth of equity investors (according to the Wealth Effect presented in Section 2.1 and illustrated in Equation 3). Results of the cointegration test (Johansen, 1991) with 6 lags show both the trace test and the max-eigenvalue test indicate 1 cointegrating equation at the 0.05 significance level. The Johansen test for cointegration is adopted in this study, as opposed to the Engel Granger approach because the Johansen test can be utilized for more than one cointegrating relationships and is more reliable in large samples (Hill et al., 2018). Therefore, our estimated VECM from data on stock prices obtained from the Malawi Stock Exchange and other variables reveal the existence of the asset price channel, contrary to earlier findings by Ngalawa and Viegi (2011) as well as Mwabutwa et al. (2013). International Journal of Economics and Financial Issues \| Vol 12 • Issue 5 • 2022 4.5. Impulse Response Analysis 30925.03 17500000000 5147.591 Observations 228 228 228 L N Table 3: Summary statistics Table 5: Granger causality test between LOGMASI and LOGGDP using 6 lags Table 5: Granger causality test between LOGMASI and LOGGDP using 6 lags Null hypothesis Obs F‑Statistic P‑value LOGGDP does not granger cause LOGMASI 222 2.23134 0.0415 LOGMASI does not granger cause LOGGDP 2.34076 0.0329 ess of the Asset Price Channel as a Monetary Policy Transmission Mechanism in Malawi: Evidence from Time Series Data Figure 3: Impulse response function Figure 3: Impulse response function Figure 3: Impulse response function recommendations. These recommendations are directed towards a wide range of audience, including the Reserve Bank of Malawi (RBM), the Malawi Stock Exchange (MSE), institutional and private investors.i change in the interest rates innovations by one standard deviation, stock prices (LOGMASI) decline, as expected in the presence of an asset price channel. This is because a unit increase in interest rates (INTR) is an act of contractionary monetary policy that is expected to lead to a decline in stock prices consistent with Tobin’s Q Theory. Moreover, the change of LOGGDP to Cholesky one standard deviation (SD) of innovations of LOGMASI also indicate an initial positive response. Again, this is consistent with economic theory and points towards the existence of the asset price channel because a positive standard deviation change in stock prices is expected to lead to an increase in income (LOGGDP) according to Tobin’s Q Theory and the Wealth Effect as explained in Section 2.1 i. The RBM should intensify its financial literacy and educational activities to the wider public. Among other topics, the RBM should aim at encouraging members of the general public to develop a culture of saving and investment. This should result in a well-informed public about investing opportunities available in the country including those at the MSE ii. ii. The MSE should initiate an intensive promotional campaign aimed at engaging both corporate and individual investors to discuss their concerns over the investment process and incorporate them in its policies and operations 5. CONCLUSION AND RECOMMENDATIONS iii. The MSE should fast track its computerization process to ensure that all share certificates and any other information related to investors are kept in a secure electronic form. The aim is to discourage duplication of work as well as minimizing the problem of forged shareholder certificates, both of which can discourage actual and potential investors and increase stock trading at the MSE 5.1. Conclusion This study examines the relationships between short term interest rates in Malawi with the stock price from the Malawi Stock Exchange. In addition, the study assesses the responsiveness of income (GDP) to changes in stock prices. Results of Granger causality tests and impulse response functions developed in the study confirm that interest rates significantly affect stock prices in Malawi for the period under study. Moreover, these results also indicate that stock prices do significantly affect GDP in Malawi. Thus, contrary to the findings in Ngalawa and Viegi (2011) as well as in Mwabutwa et al. (2013), the study finds statistical evidence based on the estimated VECM of the asset price channel operating through stock prices. iv. As the number and average size of limited companies remain small in Malawi, the MSE should create a viable alternative investment market (AIM). This should allow smaller companies to get listed without fulfilling the strict requirements of full public company listing v. There should be policies to minimize insider dealing activities in which directors make use of their knowledge of the current or likely financial position of their corporations to purchase or dispose shares. This discourages other investors and is likely to lead to reduced trading at the stock exchange. The MSE, in consultation with the RBM, should involve different stakeholders to find out exactly 4.5. Impulse Response Analysis The impulse response function (IRF) is a useful tool in the analysis of VAR results. It allows researchers to trace the impact on present and future values of an endogenous variable of one standard deviation shock of one of the innovations in the system (Lutkepohl, 2005). We present the IRF graphs of the three variables in our study in Figure 3. Our interest is the response of LOGMASI to Cholesky one standard deviation (SD) of innovations of INTR. This shows that initially, following a positive Similarly, our error correction term shown in Equation 8 indicate that there is negative relationship between interest rates (INT) and per capita income (LOGGDP). Again, this is consistent with the existence of the asset price channel since decreases in interest Table 4: Granger causality test results between INTR and LOGMASI using 6 Lags Table 4: Granger causality test results between INTR and LOGMASI using 6 Lags Null hypothesis Obs F‑Statistic P‑value LOGMASI does not granger cause INTR 222 1.73976 0.1132 INTR does not granger cause LOGMASI 3.98353 0.0009 Table 5: Granger causality test between LOGMASI and LOGGDP using 6 lags Null hypothesis Obs F‑Statistic P‑value LOGGDP does not granger cause LOGMASI 222 2.23134 0.0415 LOGMASI does not granger cause LOGGDP 2.34076 0.0329 Table 4: Granger causality test results between INTR and LOGMASI using 6 Lags Null hypothesis Obs F‑Statistic P‑value LOGMASI does not granger cause INTR 222 1.73976 0.1132 INTR does not granger cause LOGMASI 3.98353 0.0009 Table 4: Granger causality test results between INTR and LOGMASI using 6 Lags Table 3: Summary statistics INTR % MASI (2010 = 100) GDPC 2010 USD Mean 21.1 8433.0 38.0 Median 19.4 5068.5 40.25 Maximum 52.4 32373 44.2 Minimum 5.7 279 30.0 Std. Dev. 11.7 8792.6 4.76 Skewness 0.64 1.23 –0.445 Kurtosis 2.5 3.6 1.599 Jarque‑Bera 18.0 61.1 26.2 Probability 0.000126 0 0.000002 Sum 4822 1922713 8665.875 Sum Sq. Dev. International Journal of Economics and Financial Issues \| Vol 12 • Issue 5 • 2022 REFERENCES Kaldor, N. (1966), Marginal productivity and the macro-economic theories of distribution: Comment on Samuelson and Modigliani. The Review of Economic Studies, 33(4), 309-319. Akaike, H. (1974), A new look at the statistical model identification. IEEE Transactions on Automatic Control, 19(6), 716-723. Kenani, J.M., Maoni, F., Kaunda, S., Nyirenda, D. (2012), Short-run and long-run dynamics of stock prices and exchange rates in developing economies: Evidence from Malawi. European Journal of Business and Management, 4(18), 174-184.f Alexianu, M. (2020), Monetary Policy in Tanzania: Looking Back on 10 Years of Bank of Tanzania and International Growth Centre Contributions (tech rep). London: The International Growth Centre. 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The aim is to reduce its reliance on listed companies and minimize the charges paid by companies to the MSE when making an initial public offer (IPO) in order to encourage more firms to get listed. vi. The MSE should engage in various fundraising and alternative financing activities. This should result in a more commercially operated stock exchange with its own reliable sources of finance. The aim is to reduce its reliance on listed companies and minimize the charges paid by companies to the MSE when making an initial public offer (IPO) in order to encourage more firms to get listed. vi. The MSE should engage in various fundraising and alternative financing activities. This should result in a more commercially operated stock exchange with its own reliable sources of finance. The aim is to reduce its reliance on listed companies and minimize the charges paid by companies to the MSE when making an initial public offer (IPO) in order to encourage more firms to get listed. 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Econometrica, 59(6), 1551-1580. 5.2. Recommendations Folowing our finding of a viable asset price channel of monetary policy transmission in Malawi, we make the following 166 International Journal of Economics and Financial Issues \| Vol 12 • Issue 5 • 2022 Tchereni, et al.: Effectiveness of the Asset Price Channel as a Monetary Policy Transmission Mechanism in Malawi: E REFERENCES Canh, N., Vo, X.V. (2014), Monetary policy transmission in Vietnam: Evidence from a VAR approach. 1st Asia Conference on Business and Economic Studies (pp. 2-15). Ho Chi Minh City, Vietnam: University of Economics Ho Minh City. Mishkin, F. (2017), The Economics of Money Banking and Financial Markets. 12th ed. London: Pearson Education Limited. Mishra, P., Montiel, P.J., Spilimbergo, A. (2012), Monetary transmission in low-income countries: Effectiveness and policy implications. International Monetary Fund Economic Review, 60(1), 270-302. Cesa-Bianchi, A., Thwaites, G., Vicondoa, A. 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Applied Economics, 51(44), 4800-4819.f and output effects of monetary policy in Vietnam: Evidence from a VAR analysis. Applied Economics, 51(44), 4800-4819.f Nuru, N.Y. (2020), The dynamic effects of monetary policy innovations in Ethiopia. African Journal of Economic and Management Studies, 11(1), 169-180. World Bank. REFERENCES (2018), Gross National Income Per Capita, Atlas Method (Current US$)-Malawi. Washington DC: World Bank. Available from: https://www.data.worldbank.org/indicator/NY.GNP.PCA. CD?Locations=MW [Last accessed on 2020 Mar 12].i Phillips, P.C., Perron, P. (1988), Testing for a unit root in time series regression. Biometrika, 75(2), 335-346. World Bank. (2019), New Country Classifications by Income Level: 2019-2020. Washington: World Bank. Available from: https://www. blogs.worldbank.org/opendata/new-country-classificationsincome- level-2019-2020 [Last accessed on 2020 Mar 12]. Phiri, G. (2002), The Monetary Transmission Mechanism in Malawi. Lilongwe: Reserve Bank of Malawi Working Paper Series. Saunders, M., Lewis, P., Thornhill, A. (2019), Research Methods for Business Students. 8th ed. Essex: Pearson Education Limited. Yemba, B.P., Tang, B., Kitenge, E., Nsumbu, J. (2020), Divisia monetary aggregate and monetary transmission mechanism in the democratic republic of Congo (DRC). Applied Economics Letters, 27(4), 291-297. Schwarz, G. (1978), Estimating the dimension of a model. The Annals of Statistics, 6(2), 461-464. 168 International Journal of Economics and Financial Issues \| Vol 12 • Issue 5 • 2022
https://openalex.org/W3168857492	https://link.springer.com/content/pdf/10.1007/s00146-021-01224-x.pdf	English	null	Introduction: special issue—critical robotics research	AI & society	2,021	cc-by	5,323	1 Introduction does not build on humanist or social scientific research tra- ditions, or on design traditions from the arts to any great extent (Lupetti et al. 2021). In response to this, voices have been raised advocating for more human-centered and holistic approaches to question established assumptions of design in HRI research (Ljungblad et al. 2016; Lupetti et al. 2021; Šabanović 2010). In particular, the need to critically address underlying technology-driven values in our research prac- tices have been emphasized (Fernaeus et al. 2009; MacKen- zie and Wajcman 1999). Without a community that is open for alternative design approaches and critical perspectives, experiments actually conducted may go unreported, simply because there were no significant results in favor of the robot intervention (cf. Richardson 2018). In recent years, the design, use and study of robots in a vari- ety of social settings has increased, ranging from therapy and care for older adults and children, to education and domestic life. The typical discourse around these devices, in both the robotics and Human–robot interaction (HRI) fields, as well as in political debates and commercial develop- ments, is that the robotic permeation of society is somehow inevitable. Robots are predominantly viewed as engineering problems, but are usually designed to solve societal chal- lenges (Šabanović 2010). A resulting concern is that alter- native design perspectives and more critical perspectives of the consequences of using robots in social settings remain under-researched in HRI (Lupetti et al. 2021). When technologies are designed to address social chal- lenges (such as in the context of care or education), it is important to interrogate how the challenges in question are conceptualized. This is because the development and diffu- sion of new technologies are grounded in contextual under- standings and ideas about what their function and role might be. Critical analyses of technological innovations have been fruitful in other disciplines. For instance, Mansell (2012) interrogated the diffusion of the Internet, its expected role in the promotion of democracy, equality and the good life. Mansell's analysis highlights the need to address conflict- ing ideas about technologies, and how they are grounded in broader power relations and ideas about well-being. Critical approaches are, therefore, necessary to investigate techno- logical innovations and to identify the sometimes paradoxi- cal understandings that underpin their design and develop- ment (cf. Selwyn 2019). Robotic technologies raise fundamental existential ques- tions about what it means to be human. Introduction: special issue—critical robotics research Accepted: 12 April 2021 / Published online: 16 June 2021 © The Author(s) 2021, corrected publication 2021 Accepted: 12 April 2021 / Published online: 16 June 2021 © The Author(s) 2021, corrected publication 2021 AI & SOCIETY (2022) 37:417–423 https://doi.org/10.1007/s00146-021-01224-x AI & SOCIETY (2022) 37:417–423 https://doi.org/10.1007/s00146-021-01224-x 1 Introduction As Turkle (2011) points out, technologies that promise remedies to human vulnerabilities are very enticing. However, new technolo- gies also affect their environments, bringing with them both intended and unintended consequences (Stahl et al. 2013). These consequences are often complex and cannot be fully understood using a single perspective or approach. At the same time, the types of questions that researchers address are heavily affected by the research tradition and commu- nity that they act within. Current HRI research, for example, * Sofia Serholt sofia.serholt@ait.gu.se Sara Ljungblad sara.ljungblad@chalmers.se Niamh Ní Bhroin n.n.bhroin@media.uio.no 1 Department of Applied Information Technology, University of Gothenburg, Gothenburg, Sweden 2 Department of Computer Science and Engineering, University of Gothenburg, Chalmers University of Technology, Gothenburg, Sweden 3 Department of Media and Communication, University of Oslo, Oslo, Norway In this Special Issue, we seek to bring Critical Robotics Research to the fore by giving space to research that inves- tigates the introduction and use of robots in social contexts. This research identifies challenges and dilemmas that arise when using robots both in communication with, and in the immediate surroundings of, humans. It also introduces new approaches to understanding innovations in robotics and their potential social consequences. Philosophical discus- sions of potential futures, along with more contemporary 2 Department of Computer Science and Engineering, University of Gothenburg, Chalmers University of Technology, Gothenburg, Sweden 3 Department of Media and Communication, University of Oslo, Oslo, Norway (0121 3456789) 3 3456789) 3 AI & SOCIETY (2022) 37:417–423 418 critical design in HRI held at the International Conference on Human–Robot Interaction (Lee et al. 2019), and a propo- sition to consider more exploratory design approaches famil- iar to HCI in HRI (Luria et al. 2019)). issues in HRI are presented. These contributions interro- gate some of the fundamental ideas of HRI from a critical perspective. Our own work has also developed in these directions and includes interrogations of ethical issues surrounding educa- tional robots (Serholt et al. 2017; Toft Norgaard et al. 2018), scrutinizing problematic aspects of interactions between humans and robots (Serholt 2018; Serholt et al. 2020), focus- ing on understanding challenging experiences and practices of existing robot technology in society, such as professional use of drones (Ljungblad et al. 2021), and use of robotic toys in families (Fernaeus et al. 2010). 1 Introduction We have previously also done studies with artists to understand how artistic projects may question myths about robots (Jacobsson et al. 2013), and early work to address the need to ground ethical considerations in empirical studies and real practices rather than fictive use situations (Ljungblad et al. 2011; Nylander et al. 2012). Yet, the shaping and defining of the concept of Critical Robotics is an ongoing process that requires collec- tive effort from the research community. This Special Issue provides a conceptual interdisciplinary backdrop to further critical approaches in HRI and robotics research. 3 Key contributions This Special Issue consists of 12 articles by authors from a range of disciplines, each with a unique perspective to offer the Critical Robotics discussion. Some focus explicitly on the design of robots, while in others, implications for design can be understood as a by-product. Several focus on robots in care settings (Burema; Maibaum, Bischof, Herge- sell, and Lipp; van Wynsberghe), one focuses explicitly on children (Pashevich), and another focuses on professional service work (Dobrosovestnova, Hannibal, and Reinboth). Two contributions encompass several application settings (Fronemann, Pollman, and Loh; Weiss and Spiel), whereas another two utilize art installations to situate their research (Gemeinboeck and Saunders; Yolgormez and Thibodeau). Finally, three of the contributions consider robots in society (Balle; Hildebrand; Nørskov), although they also touch upon different contexts in their discussions. In the following, we synthesize and reflect upon these contributions, according to converging themes. In parallel with these disciplines, the robotics field is currently experiencing a similar shift as demonstrated for example by the Robophilosophy Conference Series2 that began in 2014, the establishment of the Foundation for Responsible Robotics,3 as well as notable research projects exploring issues of ethics, sustainability, and responsibility in social robotics, including, “Responsible Ethical Learn- ing With Robotics”4 and “Integrative Social Robotics—A New Framework for Culturally Sustainable Technology Solutions”.5 Emerging research related to Critical Robot- ics has also begun to take form (cf. a recent workshop on 2 History of critical robotics In 2018, we introduced the concept Critical Robotics in an attempt to foster what we observed as an emerging para- digm in HRI and related research. Through international and interdisciplinary collaboration, we organized the workshop Critical Robotics—Exploring a New Paradigm at the Nordic forum for human–computer interaction (NordiCHI) (Ljun- gblad et al. 2018). This brought together scholars interested in exploring critical human-centered perspectives in robot- ics research. We also discussed whether critical questions within the area of HRI could be considered an emerging paradigm, namely Critical Robotics. The workshop was ini- tiated by Applied Robotics,1 a group of researchers affiliated with the University of Gothenburg in Sweden. This group was formed due to shared concerns about robotics-centered research and, in particular, a perceived need to look beyond the social robot as a taken-for-granted solution to a range of societal challenges. Our view of critical robotics was inspired by paradigm shifts that have occurred in the field of Human–computer interaction (HCI) in recent years, where research moved away from the optimization of man–machine interaction, towards theories about the computer and the human mind, to finally focus on interaction as phenomenologically situated. In this, approaches to participation, values, philosophy and ethics began to play a more prominent role (Bødker 2006; Harrison et al. 2007; Koskinen et al. 2012).i 1 https://www.gu.se/en/research/applied-robotics-in-gothenburg. 2 https://conferences.au.dk/robo-philosophy/. 3 https://responsiblerobotics.org/. 4 https://reeler.eu/. 5 https://www.carlsbergfondet.dk/da/Forskningsaktiviteter/Forsk ningsprojekter/Semper-Ardens-forskningsprojekter/Johanna-Seibt_ Integrative-Social-Robotics. 3.1 Robotification of society These authors all identify an oversimplification of the role of humans in care and service work, or a reduction in the complexity of the tasks that they carry out. In her paper, Burema (2021) sheds light on representations of older adults in HRI, and how ageism may occur when older adults are depicted as dependent, fragile and vulnerable people. This renders them as “potentially burdensome care recipients'', and robot technologies are presented as an optimal solu- tion to this social problem. In a similar vein, Maibaum et al. (2021) problematize the understanding of caregivers and care-receivers: how care practices are deconstructed into tasks to give form to well-defined technical problems. This leads to the incremental mechanization of care, rather than to a more holistic understanding of it. As van Wynsberghe (2021) emphasizes, the practice of care constitutes instead a bi-directional relationship between caregiver and care- receiver, where reciprocity is of central importance. If a relationship based on mutual care would manifest between a care-receiver and a caregiving robot, van Wynsberghe argues that this would require a certain level of deception. This is because robots lack the ability to engage in true reciproc- ity. From a different vantage point, Dobrosovestnova et al. (2021) provide a sociological account of the dimensions of affective and emotional labor in professional service work. They discuss how the implementation of social robots in such contexts risks reducing the complexities of affective labor and exacerbating stereotypes of professional service workers. Both Nørskov and Hildebrand argue that an environment permeated by robots (‘robotification’) will shape our line of thought, promoting certain ways of being while discourag- ing others. This means that robots (social or otherwise) will influence relationships between humans, and may reduce human values to the level of services. Nørskov argues that the collective moral standards represented in robots will influence human conduct, and eventually vice versa.i l By conceptualizing the robotification of society as an ecological enterprise, Hildebrand and Nørskov both make compelling cases for its disruptive potential. The ethical imperative is to look beyond fragmented understandings of innovations in society, e.g., where a robot in one context is understood as distinct from other elements of culture, and to develop more holistic perspectives. While it is yet debatable whether social robots are able to carry out even simpler care and service tasks in any meaningful way, this also depends on how care is concep- tualized. 3.1 Robotification of society 1 https://www.gu.se/en/research/applied-robotics-in-gothenburg. 2 https://conferences.au.dk/robo-philosophy/. 3 https://responsiblerobotics.org/. 4 https://reeler.eu/. 5 https://www.carlsbergfondet.dk/da/Forskningsaktiviteter/Forsk ningsprojekter/Semper-Ardens-forskningsprojekter/Johanna-Seibt_ Integrative-Social-Robotics. The narrative of a robot society has permeated and enticed our cultural imagination for a long time. It has been lik- ened to the Industrial Revolution, argued to be the natu- ral next step in human evolution. Ideas of ‘the singularity’, perpetuated through works of science fiction (such as the 1 3 419 AI & SOCIETY (2022) 37:417–423 Terminator or I, Robot), suggest that robots and artificial intelligence (AI) will someday evolve to a stage where they will reproduce themselves. In the worst case scenario, robots will then no longer need humans. While this scenario is often contested and regarded as highly unrealistic, the idea that robots (or autonomous machines) and AI will pervade many, if not all aspects of everyday life is closer at hand. It, therefore, requires consideration and interrogation. living at home. With technical advancement the field keeps developing. The idea of robots providing services that we would otherwise expect from humans forces us to think about the aspects of these services that may, and may not, be replaceable. Several of the contributions to this Special Issue consider what constitutes care, and how research char- acterizes care and people in need of care (Burema; Maibaum et al.; van Wynsberghe). One additional contribution ana- lyzes complex interactions between service providers and receivers in the professional service sector (Dobrosovest- nova et al.).i In this Special Issue, Nørskov (2021) develops a phil- osophically informed, speculative outlook of what the robotification of society could mean, including its ethical implications. He defines robotification as ‘massive robotic integration’, where robots permeate practically all areas of life. By drawing on Heidegger, Nishitani, Bauman and oth- ers, Nørskov makes the case that robotification could lead to ethical cleansing, “a sanitation of culture by the calcu- lus of science and technology”. Specifically, he argues that robotics research has ushered in a technological mode of being, where everything can be measured objectively. Nør- skov posits that technology can never simply be added to an existing environment. It will always change its environ- ment. Building on a media ecology perspective, Hildebrand (2021) also argues that advancements in robotics are not simply additions to what already exists, but fundamentally change us and our understanding of the world, amputating and extending us as human beings. 6 http://www.parorobots.com/. 3.1 Robotification of society The authors writing in this Special Issue suggest that oversimplification, rooted in misconceptions about the provision of care, the process of ageing, affective labor in professional service work, etc., can influence the design and implementation of social robots. Fragmented and inaccurate understanding of work practices and human needs currently prevail. However, it should also be possible to address these questions in the design of robots. 3.4 Problematizing socio‑emotional relationships with robots Social robots influence human–robot and human–human relations. Aspects of social interaction and empathy are foundational for human relationships, and, therefore, gar- ner much attention from the HRI research community. A particular concern is the potential replacement of human care providers with robotic technologies. Even so, introduc- ing robots as complementary technologies in social settings raises important ethical questions. Several of the contributions to this Special Issue highlight and problematize how UCD processes are currently con- ducted in HRI research. Even rigorous attempts to involve stakeholders and target groups in design risk falling short of the ideals of UCD. For instance, Weiss and Spiel (2021) provide a much-needed exposé of what goes on behind the scenes of HRI-projects, and direct criticism (and self- reflection) towards the situations they have observed. Even in cases, where stakeholders are involved in phases of con- ceptualization, design, and later evaluations, it is not always clear to what extent stakeholders actually come to influence design outcomes. p q Drawing on the ethics of care, van Wynsberghe (2021) considers the possible effects of nudging in reciprocal relationships between humans and robots. She argues that “social robots designed for reciprocity use reciprocity as an instrumental value to enhance acceptability of the robot”, and that this is ethically questionable. In contrast, Balle's (2021) philosophical analysis, focuses on how humans develop empathic responses to robots. He proposes, based on the ethics of Danish theologian K. E. Løgstrup, that human empathy is inherently good, because it turns people away from their own self-focus (inturnedness), and argues that this applies also to relations with robots. In effect, he makes the case that claiming moral patienthood for robots is ethi- cally sound based on extrinsic grounds (i.e., that it benefits the person responding empathically to the robot). Even so, van Wynsberghe is not focused on immediate relational reactions to robots; instead, her focus is on what happens once the relationship develops. While she acknowledges that reciprocity is indeed a component of moral development, and is in no way harmful in itself, she argues that it is uncer- tain whether reciprocity fostered in HRI would transfer to human–human interaction, where it would provide the most benefit. Instead, she argues that in a world of finite resources, concerted efforts should rather be focused on fostering reci- procity among humans. 7 https://www.interaction-design.org/literature/article/5-stages-in-the- design-thinking-process. 3.2 Oversimplification of care and service work The care and service sectors have been heavily targeted with envisioned applications for social robots. Readers will be familiar with the use of zoomorphic robots, such as the robot seal ‘Paro’,6 which has for example been introduced to the therapy and care of dementia patients. State-of-the-art projects have developed robot companions for older adults 1 3 420 AI & SOCIETY (2022) 37:417–423 Technology Studies to problematize designer intentions and ideas about users (Burema 2021). 3.3 The (not so) straightforward path to robot design Furthermore, it is generally agreed that a rigorous design process should raise ethical questions and involve reflection on alternatives. Here, Fronemann et al. (2021) provide an example of reflective practice, discussing the tensions and tradeoffs between designing robots for a nice user experi- ence (UX) versus ethical design. They discuss the delicate balance that can arise between user acceptance, on the one hand, and autonomy and privacy on the other. They argue that the design and evaluation of a robot solution must, therefore, be understood holistically. In contemporary HRI-projects, typically ranging between 3 and 5 years, design processes constitute a substantial part of project time. Engaging in stakeholder practices and exper- tise is essential for a successful design outcome. However, depending on how user-centered design (UCD) or participa- tory design (PD) approaches are employed, such approaches can also lead to an oversimplification of practices, as well as to stereotyped views and problematic representations of the target group. This can result in suboptimal design. Without engaging in all the design steps, i.e., empathize, define, ideate, prototype, and test (as suggested by the Hasso Plattner Institute of Design7) there is a high risk that the overall ‘problem’ and the related opportunities for robotic solutions are misunderstood. 3.4 Problematizing socio‑emotional relationships with robots As Balle also reasons, one can only make a weak claim of moral patienthood for robots, i.e., it should be regarded as “permissible rather than obligatory and individual rather than universal” as far as it happens to occur in human–robot interactions. If stakeholders are not involved in design before a robotic solution exists, there is no space for them to reframe the problem or change the design idea. The analysis submit- ted by Dobrosovestnova et al. (2021) resonates with this. Open and critical reporting on existing practices, followed by ideation and other design activities could be useful for researchers working in the same design spaces. It could also prove informative for upcoming projects by ensuring sound starting points and avoiding the repetition of (early) mistakes and unsubstantiated assumptions of robotic ideas. Accordingly, it is important to report on aspects of the research process that did not work well, including the overall limitations of the user-centered approach employed. UCD and PD require flexibility in their implementation, with par- ticular regard to the approaches taken and the potential for alternative design methods. This in turn requires research- ers to have sufficient knowledge and skills that extend beyond the application of established research methods, and take inspiration from other fields, such as Science and 1 3 421 AI & SOCIETY (2022) 37:417–423 Related to this, Pashevich (2021) investigates how the use of social robots by pre-school children could influence the development of empathy. She finds that significant knowl- edge gaps exist, because the robots that are currently avail- able are not sophisticated enough. Children's actual experi- ences of interacting with social robots are limited, and these are usually not investigated over time. As such, the debate on unintended or undesirable consequences of empathic responses to robots requires continued attention. Moreover, as Balle notes, when research shows that humans tend to respond empathetically to robots, there is potential for mali- cious intent and exploitation in robot design and develop- ment. Taken together, these contributions show that ethical socio-emotional relationships with robots are not straightfor- ward and require further consideration and analyses. grounded in imaginaries of competition and control. They argue that these ideas influence our capacity to consider human–machine relations beyond frameworks of instru- mentalization and utility. 3.5 Challenging sociality in human–robot interactions In exploring dimensions of human–robot relations, the two final contributions to this Special Issue question the basic premise of what (social) robots are and should be. When it comes to designing and studying social robots within HRI, the robots that are utilized are typically purchased from robotics companies. These may be humanoid or zoomorphic in morphology and behavior. While some research projects build robots from scratch (cf. Weiss and Spiel in this issue, or the robots developed at Ishiguro Lab8), most projects pur- chase existing robots. Either way, robots tend to resemble the appearance, behavior and movement of biological entities we are familiar with. However, the following two contribu- tions question the focus on achieving similarity or human likeness as a goal by illustrating alternatives in designing robots for human interaction. Taken together, the contributions underline the impor- tance of questioning current research activities and perspec- tives, in particular with regard to the funding and design of robotic solutions for social problems. While doing so, they also offer alternatives to understanding and developing the role of robots in society. These are not easy topics to deal with, particularly, since it is difficult to foresee exactly how robotics will develop in the years to come, and what role our research efforts may play in shaping it. First, Gemeinboeck and Saunders (2021) present experi- ences from a research-based art installation. The authors propose another way of thinking about communication and relationships between humans and machines. As both van Wynsberghe (2021) and Balle (2021) also discuss, their core premise is that sociality is not something that can be a prop- erty of a machine, but is rather something that is enacted in an encounter, or an evolving relationship, between a human and a machine. Based on their theoretical and empirical observations, the authors argue for the importance of focus- ing on the enactment of social agency, rather than its repre- sentation, in the design of social robots.i f While our conceptualization of what it means to conduct Critical Robotics Research is still in its infancy and in no way exhaustive, we hope that this Special Issue serves to outline a set of ideas, concepts and approaches to assist other researchers in further developing this field. Acknowledgements We thank all the authors for contributing to this special issue. 4 The future of critical robotics research The contributions to this Special Issue clearly show how Critical Robotics Research can benefit and further mature HRI as a research field. Each article provides a unique per- spective on important aspects that are usually hidden or overlooked in robotics research. They articulate challenges to the robotification of society, highlight the oversimplifica- tion of care and service practices and of user centred design processes. They also untangle ethical dilemmas arising from socio-emotional relationships between humans and robots and challenge the need for human likeness in robots designed as social companions. 8 https://eng.irl.sys.es.osaka-u.ac.jp/. 3.4 Problematizing socio‑emotional relationships with robots By designing 'useless' machines, or machines that are not designed to serve human physical or social needs, and exhibiting these machines in public spaces, they explore different approaches to socializing with robots. They argue that sustained everyday interaction between human and non-purposeful machines requires a process of 'attunement', and that this should be considered as an alter- native approach to understanding human–robot relations. References Balle SN (2021) Empathic responses and moral status for social robots: an argument in favor of robot patienthood based on K. E. Løgstrup. Ai Soc. https://doi.org/10.1007/s00146-021-01211-2 (This issue) Ljungblad S, Serholt S, Milosevic T, Toft Nørgård R, Ni Bhroin N, Lindgren P, Ess C, Barendregt W, Obaid M (2018) Critical robotics: exploring a new paradigm. In: Proceedings of the 10th Nordic conference on human-computer interaction (NordiCHI '18). Association for Computing Machinery, New York, NY, USA, pp 972–975. https://doi.org/10.1145/3240167.3240267 Bødker S (2006) When second wave HCI meets third wave chal- lenges. In: Proceedings of the 4th Nordic conference on Human- computer interaction: changing roles (NordiCHI '06). Associa- tion for Computing Machinery, New York, NY, USA, pp 1–8. https://doi.org/10.1145/1182475.1182476 Ljungblad S, Man Y, Aydin Baytas MG, Obaid M, Fjeld M (2021) What matters in professional drone pilots’ practices? An inter- view study to understand the complexity of their work and inform human-drone-interaction research. In: Proceedings of the 2021 CHI conference on human factors in computing sys- tems (CHI '21). Association for Computing Machinery, New York, NY, USA, Article 159, pp 1–16. https://doi.org/10.1145/ 3411764.3445737 Burema D (2021) A critical analysis of the representations of older adults in the field of human-robot interaction. AI Soc. https:// doi.org/10.1007/s00146-021-01205-0 (This issue) Dobrosovestnova A, Hannibal G, Reinboth T (2021) Service robots for affective labor: a sociology of labor perspective. AI Soc. https://doi.org/10.1007/s00146-021-01208-x (This issue) Fernaeus Y, Jacobsson M, Ljungblad S, Holmquist LE (2009) Are we living in a robot cargo cult? In: Proceedings of the 4th ACM/ IEEE international conference on Human robot interaction (HRI '09). Association for Computing Machinery, New York, NY, USA, pp 279–280. https://doi.org/10.1145/1514095.1514175 Lupetti ML, Zaga C, Cila N (2021) Designerly ways of knowing in HRI: broadening the scope of design-oriented HRI through the concept of intermediate-level knowledge. In: Proceedings of the 2021 ACM/IEEE international conference on human-robot interaction (HRI '21). Association for Computing Machinery, New York, NY, USA, pp 389–398. https://doi.org/10.1145/ 3434073.3444668 Fernaeus Y, Håkansson M, Jacobsson M, Ljungblad S (2010) How do you play with a robotic toy animal? a long-term study of Pleo. In: Proceedings of the 9th International Conference on Interaction Design and Children (IDC '10). Association for Computing Machinery, New York, NY, USA, pp 39–48. https:// doi.org/10.1145/1810543.1810549 Luria M, Zimmerman J, Forlizzi J (2019) Championing research through design in HRI. arXiv preprint arXiv:1908.07572 MacKenzie D, Wajcman J (1999) Introductory essay: the social shaping of technology. 3.5 Challenging sociality in human–robot interactions We would also like to thank our colleagues who collabo- rated with us on the development of Critical Robotics: Tijana Milo- sevic, Rikke Toft Nørgård, Pamela Lindgren, Charles Ess, Wolmet Barendregt, and Mohammad Obaid. Furthermore, this special issue would not have been possible without the excellent work of all the peer reviewers. Finally, thanks to our fellow researchers within Applied Robotics for their continued support, and the Division of Learning, Communication and IT at the Department of Applied IT, University of Gothenburg for investing in open access fees for this editorial. Focusing on a specific relational aspect, and based on a research-creation project entitled 'Machine Ménagerie', Yolgormez and Thibodeau (2021) emphasize how con- temporary understandings of human–robot relations are 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 1 3 422 AI & SOCIETY (2022) 37:417–423 In: Proceedings of the 14th ACM/IEEE international conference on human-robot interaction (HRI), 2019, pp 681–682. https:// doi.org/10.1109/HRI.2019.8673130. as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Ljungblad S, Nylander S, Nørgaard M (2011) Beyond speculative ethics in HRI? Ethical considerations and the relation to empiri- cal data. In: Proceedings of the 6th international conference on Human-robot interaction (HRI '11). Association for Computing Machinery, New York, NY, USA, pp 191–192. https://doi.org/ 10.1145/1957656.1957726 Ljungblad S, Serholt S, Barendregt W, Lindgren P, Obaid M (2016) Are we really adressing the human in human-robot interaction? Adopting the phenomenologically-situated paradigm. In: Seibt J, Nørskov M, Schack Andersen S (eds) What social robots can and should do: proceedings of robophilosophy 2016/TRAN- SOR 2016. 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https://openalex.org/W4283792069	https://philpapers.org/archive/BLAMFA-3.pdf	English	null	Marquard Freher and the presumption of goodness in legal humanism	History of European ideas	2,022	cc-by	13,819	History of European Ideas ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/rhei20 Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=rhei20 Marquard Freher and the presumption of goodness in legal humanism Andreas Blank ABSTRACT ABSTRACT One of the most detailed early modern discussions of the morality of esteem can be found in the work of the reformed jurist and historian Marquard Freher (1565–1614). Since the question of how much esteem others deserve is fraught with a high degree of uncertainty, Freher relied on the work of other legal humanists, who discussed questions of esteem from the perspective of arguments from the presumption of goodness. The humanist approach to the presumption of goodness integrated considerations about presumed non-delinquency with considerations concerning natural needs, natural rights and natural duties grounded in natural rational capacities common to all (grown-up, healthy) human beings. It advised us to fulﬁll natural duties that derive from the natural need of being held in good esteem by others, and to develop socially valuable attitudes such as trust and positive emotions toward others. At the same time, this approach was responsive to considerations of prudence, both in grounding the presumption of goodness in considerations concerning causal powers and in balancing the presumption of goodness against a variety of presumptions of badness. Freher applied this understanding of the presumption of goodness to political practice in his role as an advisor to the Palatinate. © 2022 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. CONTACT Andreas Blank andreasblank@hotmail.com Philosophy, Alpen Adria Universitat Klagenfurt, Universitätsstr. 65 67, Klagenfurt, 9020 Austria Andreas Blank To cite this article: Andreas Blank (2023) Marquard Freher and the presumption of goodness in legal humanism, History of European Ideas, 49:3, 491-505, DOI: 10.1080/01916599.2022.2095524 To link to this article: https://doi.org/10.1080/01916599.2022.2095524 © 2022 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group Published online: 04 Jul 2022. Submit your article to this journal Article views: 290 View related articles View Crossmark data © 2022 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group Published online: 04 Jul 2022. Submit your article to this journal Article views: 290 View related articles View Crossmark data Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=rhei20 HISTORY OF EUROPEAN IDEAS 2023, VOL. 49, NO. 3, 491–505 https://doi.org/10.1080/01916599.2022.2095524 HISTORY OF EUROPEAN IDEAS 2023, VOL. 49, NO. 3, 491–505 Marquard Freher and the presumption of goodness in legal humanism Andreas Blank Philosophy, Alpen-Adria-Universitat Klagenfurt, Klagenfurt, Austria KEYWORDS Esteem; reputation; glory © 2022 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.o which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Blank andreasblank@hotmail.com Philosophy, Alpen-Adria-Universitat Klagenfurt, Universitätsstr. 65- 0 Austria 1. Introduction The desire for esteem expresses one of the basic human needs, and probably there is no period in intellectual history where there was no theoretical reﬂection about the role of this desire in human life. Early modern natural law theories are no exception to this. Typically, however, the issue is dis- cussed in texts primarily concerned with other issues, and often discussion of the morality of the desire for esteem takes the form of shorter passages and scattered remarks.1 An exception to this is the work of the Reformed jurist and historian Marquard Freher (1565–1614). Freher graduated under the great legal humanist Jacques Cujas (1522–1590) in Bourges. After a few years as professor of law in Heidelberg, he became a judge, counsellor and diplomat in the service of the Palatinate and one of the pioneers in the historiography of medieval German law and literature.2 Freher devoted two lengthy books to the legal aspects of esteem, reputation, glory and infamy: Tractatus de fama publica (1588; second edition 1591) and Tractatus de existimatione acquirenda, conservanda et amittenda (1591). Like many works of the legal humanists, these books give advice to legal prac- titioners (for instance, by giving procedural rules for assessing the reputation of witnesses) but also oﬀer extremely detailed discussions of legal history and theoretical reﬂections concerning CONTACT Andreas Blank 67, Klagenfurt, 9020 Austria 492 A. BLANK the natural-law foundations of aspects of legal practice. And as the dedications to the Duke of Bavaria, Johannes Casimir, and to Elector Friedrich IV show, Freher also had the hope that his work would help princes to develop the right attitude toward fulﬁlling duties of esteem in their per- sonal and political relations. While Freher’s work as a historian has found some scholarly attention,3 his work on esteem and reputation has not yet been studied in any detail. Three reasons come to my mind why Freher’s books deserve to be studied. First, when we try to assess the personal qualities of others, we are usually faced with a high degree of uncertainty. In early modern legal thought, the Roman-law con- cept of presumption fulﬁlled an indispensable function in dealing rationally with situations of decisions under uncertainty. 1. Introduction In addition to presumptions that were laid down in law (the praesump- tio iuris et de iure and the praesumptio iuris), the Roman-law tradition operated with presumptions that are formed by individuals and that concern the actions and qualities of individuals (the so- called praesumptiones hominis). While presumptions of the ﬁrst type derive their validity from the law and were taken to be immune to contrary evidence, presumptions of the latter type were understood to be assumptions that are taken to be true until and unless contrary evidence makes it inevitable to revise them.4 Freher relied on the work of other sixteenth-century legal humanists such as Jacopo Menochio (1532–1607), André Tiraqueau (1488–1558), Andrea Alciato (1492– 1550), Aimone Cravetta (1504–1569), and Nicolaus Everardus (1462–1532), who discussed ques- tions of esteem from the perspective of arguments from praesumptiones hominis, in general, and of arguments from the presumption of goodness (praesumptio bonitatis), in particular. Freher’s work can function as an insider’s guide to sixteenth-century debates about how to form presump- tions about the personal qualities of others, in general, and about how to argue from the presump- tion of goodness, in particular. Another reason for studying Freher’s views on these matters is that he discussed the ethical implications of the presumption of goodness much more explicitly than any other legal humanist. Although the group of sixteenth-century jurists just mentioned ﬁgures prominently in recent research concerning early modern historical scholarship5 and theories of juridical interpretation,6 and although early modern arguments from presumption have been studied in some detail,7 the question of how the relation between legal and ethical aspects of the presumption of goodness was understood by the legal humanists has, as far as I can determine, not been addressed in recent scholarly work. The neglect into which this facet of legal humanism has fallen is regrettable because the versions of natural law theory found in the legal humanists integrate aspects of legal theory with considerations concerning natural needs, natural rights and natural duties that we would now clas- sify as belonging to ethics. Taking Freher’s work as a point of departure makes clear that the pre- sumption of goodness found in legal humanism did not reduce to the presumption of innocence; rather, it included the duty to presume that others possess ethically good qualities, until and unless contrary considerations force us to revise this presumption. 2. Teleology and the presumption of goodness Freher’s treatment of the duties of esteem is a useful starting point because it makes clear that the conception of goodness implied by Roman law included legal and ethical aspects. Freher referred to the deﬁnition given by the Roman jurist Callistratus according to which esteem is to be understood as ‘the standing of undamaged dignity, approved by laws and customs, which by our delict is dimin- ished or exhausted on the basis of the authority of laws.’8 As Freher explained, what matters here is ‘standing with respect to dignity, according to which some are held to be honorable, suitable, legal, worthy of trust, above all suspicious and (as it is called) authentic; others are held to be notorious, suspect, infamous, shameful, and excluded from testimony: and the verdict about reputation is introduced partly through laws and the edicts of the magistrate, partly it depends on the habits and good customs of the individual localities.’9 Evidently, according to this deﬁnition laws and other legal documents play a role in determining dignity, but so do also other social conventions. g p y g g y Accordingly, dignity does not reduce to non-delinquency. Rather, Freher understood dignity as ‘the quality of a person, in virtue of which she deserves to be counted among the good and honor- able citizens; in virtue of which she is thought to be capable of receiving oﬃces and honors and suitable to carry out all actions of civil society, and on the contrary is thought to be unworthy of any resentment, as long as he cares about a moderate performance in oﬃce and keeps clear of all contagion with crime and shame, through which the purity and sincerity of his reputation can be hurt immediately.’10 Crime is thus one of the elements that destroys reputation, but so is behavior that, according to the conventions of a locality, give rise to shame. 1. Introduction This conception allowed to fulﬁl natu- ral duties that derive from the natural need that we have for others’ thinking well of our character traits, and to develop socially valuable attitudes such as trust and positive emotions toward others. A third reason for studying Freher’s work is that he shared with the legal humanists an acute awareness of the problem that the advantages that they presumption of ethical goodness may have for social relations may be cancelled out by prudential concerns. If we fulﬁl the natural need of others to be held in good esteem and if we trust them and like them without good reasons, we may have to pay a high price for our self-deception. The legal humanists answered these pru- dential concerns by grounding the presumption of goodness in presumptions concerning natural rational and emotional capacities common to all (grown-up, healthy) human beings, and also by counterbalancing the presumption of goodness with evidence-based presumptions of badness. Such prudential considerations were taken to be important not only in legal but also in political contexts. As we will see, in his role as counsellor, Freher himself used this strategy of balancing the presumption of goodness with a variety of presumptions of badness to support the political interests of the Palatinate. 493 HISTORY OF EUROPEAN IDEAS I will proceed as follows: First, I will establish that Freher, like other legal humanists, invoked teleological considerations to distinguished the presumption of goodness from the presumption of innocence. Subsequently, I will argue that Freher, like other legal humanists, appealed to con- siderations concerning causal powers to answer prudential concerns raised by including ethical aspects into the juridical presumption of goodness. Finally, I will analyse how Freher, like other legal humanists, limited the presumption of goodness—both by restricting it to ordinary, non- excellent virtue and by balancing it with a variety of presumptions of ethical badness that are equally based on considerations concerning causal powers. 2. Teleology and the presumption of goodness Freher oﬀers some teleological considerations: ‘I do not how nature itself brought it about in the minds of everyone that they give great weight to the opinions of others about them- selves and that they direct a great part of their lives toward this opinion.’18 In this sense, it is a brute fact concerning human nature that social esteem is a source of happiness, more so than is self- esteem. But for the very reason that being held in good esteem is a natural need, Freher maintained, it is one of the needs that have to be taken into consideration in natural law: traits. The question, of course, is why one should presume that individuals exemplify all three var- ieties of goodness. Freher oﬀers some teleological considerations: ‘I do not how nature itself brought it about in the minds of everyone that they give great weight to the opinions of others about them- selves and that they direct a great part of their lives toward this opinion.’18 In this sense, it is a brute fact concerning human nature that social esteem is a source of happiness, more so than is self- esteem. But for the very reason that being held in good esteem is a natural need, Freher maintained, it is one of the needs that have to be taken into consideration in natural law: Since most people think that it is of highest importance, what others, who are endowed with the same reason, who use the same laws and customs, and who live in the same community (with whom hence one necessarily has to have a lot of interaction and communication), think about them; the opinion has arisen among them that without any doubt good reputation and esteem has to be sought by everyone. This is why Cicero in book 1 of De legibus, where he discusses natural law, says that the mind of all humans have in common that that all desire pleasure and all ﬂee death and pain; likewise, that, due to the similarity between what is honorable and glory, they judge those to be happy who are honored and miserable those who lack glory.19 Freher added that there is also instrumental value to social esteem. 2. Teleology and the presumption of goodness One dimension concerns trust: ‘The one is not said to be overly credulous who believes a person worthy of trust,’ a formulation that alludes to Baldus’s view that ‘to believe a good person is not acting against natural law.’20 Another instrumental dimension of social esteem concerns positive emotions—a dimension that is brought out by Freher’s quotation from Cicero’s De oﬃciis: ‘Because what it decent and honorable pleases by itself, moves the mind of humans by its nature and kind … , we are forced by nature herself to like those in whom we believe these virtues to reside.’21 If ethical aspects are included in the juridical conception of goodness, then the presumption of goodness may be capable of fulﬁlling natural needs that the presumption of innocence could not fulﬁl. Still, saying that the more inclusive way of thinking about the presumption of goodness would fulﬁl some natural needs does not answer prudential concerns. Perhaps one could achieve these nice eﬀects only at an exceedingly high price. If one is too optimistic about the virtuous char- acter traits of others, one will often be disappointed—very much to one’s own detriment. Would it not be good advice to defer trusting and liking others until they have given some conﬁrmation that they deserve to be trusted and liked? The answer that the legal humanists could give to these con- cerns is that their conception of the presumption of goodness was not only motivated by teleological considerations but also by assessments of causal powers. In this sense, the presumption of goodness is a special case of how presumptions, more generally, can be supported by causal considerations. The speciﬁc diﬀerence is that, while other presumptions are based on evidence concerning the qual- ities of particular individuals, the presumption of goodness was understood to be based on con- siderations concerning human nature. 2. Teleology and the presumption of goodness This is why non-delin- quency is only one of the factors that grounds social esteem: ‘This esteem of being a good person is acquired through integrity of customs, striving for virtue and innocence of living … ’11 Freher also observed that the dimension of custom is built into how the Roman jurist Aemilius Papinianus spelled out the content of the presumption of goodness: ‘Those acts that hurt our piety, esteem, and shame and that (if I may speak in general) happen against good customs, of these, too, it should not be believed that we are able to do so.’12 In addition to conformity with law and customs, virtue is a further factor constitutive of being held in good esteem. This can be seen in Freher’s treatment of glory, which he took to be a form of esteem that is particularly widespread but has the same foundation as esteem.13 ‘Since glory is to be sought only on the basis of true virtue, and this in such an order … that we strive for virtue not out of the desire for praise but in order to deserve praise for virtue … ’14 With a view to princes, Freher held that striving for glory is essential for upholding power because it is essential for them to be perceived as virtuous persons; while ‘through contempt for reputation, virtue is despised’ (con- temptu famae contemni virtutes).15 More generally, he noted that ‘the aﬀect relating to praise and glory … was always believed to oﬀer a perspicuous indication of character traits and minds that are able to strive for virtue … ’16 And, as he commented, ‘since the love of glory contributes so much to triggering in mortals the cultivation and the study of virtue, the wise have consequently judged that glorious reputation and celebration must be the reward for excellent and accomplished virtue.’17 The presumption of goodness in the Roman-law tradition thus comprised three elements: (1) belief in non-delinquency, (2) belief in conformity to customs, and (3) belief in virtuous character A. BLANK A. BLANK 494 traits. The question, of course, is why one should presume that individuals exemplify all three var- ieties of goodness. 3. Causal powers and the presumption of goodness It will be useful to begin with an analysis of how evidence-based presumptions were treated by the legal humanists, on whose work Freher drew extensively. The general idea was brought into a suc- cinct formula by Menochio: ‘Presumption and conjecture are derived from causal powers.’22 For instance, ‘we say that it is to be believed that the one died earlier who was weaker and more vul- nerable … Likewise, we say that the one is presumed to be the aggressor in a brawl who is bodily stronger than the other … ’23 Menochio also mentioned virtues, vices, studies, education, arts and inertia as mental qualities the give rise to presumptions.24 Evidently, these mental qualities can be inferred on the basis of evidence concerning particular individuals, and not every individual will exemplify these qualities in the same way. Freher, too, used evidence-based presumptions of goodness side by side with the view that the presumption of goodness could be based on observations concerning human nature. As to the for- mer pattern of argument, Freher quoted a passage from the Roman jurist Atrius Menander con- cerning the legal standing of a soldier returning from enemy territory: 495 HISTORY OF EUROPEAN IDEAS If he had been esteemed to be a good soldier before, his assertion should be believed that he had been captured by the barbarians and that he did not desert: But if he had been one who exceeds his furlough, or negligent of those close to him, or sluggish, or acting outside his companionship, he is not to be believed when it happens that he returns after a long time.25 Freher commented: ‘[O]n the ground of the previous life, we can estimate what we should judge about the subsequent conversation and how the deeds and words of humans are to be under- stood.’26 To explain what allows forming presumptions based on evidence from previous times, he referred to André Tiraqueau’s De poenis. There, Tiraqueau developed the point by giving atten- tion to Virgil’s portrayal of the relation between Dido and Aeneas: In these matters [Dido] was turned from a temperate person (which is the disposition of virtue itself) into a self-controlled person, but not yet into a dissolute one. Sexual desire is presumed to diminish in old age … And generally, those qualities are presumed that are pre- sent according to the diﬀerent ages: from whence an old person is presumed to die easily … You could say that 3. Causal powers and the presumption of goodness For Dido was at the beginning moved to love Aeneas both through virtue … and through those factors that in human aﬀairs are esteemed not only to be good but to be the highest goods. For who would not count the nobility of descent, the dignity and excellence of ﬁgure, and outstanding eloquence among the highest goods? Finally, when to so many austere enemies the persuasion of the ﬂattering sister Anna were added, a more powerful appetite emerged and overwhelmed unwilling and resisting reason; and she became not only an intemperate but dissolute woman … 27 As Tiraqueau explained, ‘this story teaches us how many steps there are from the disposition toward virtues to the disposition toward vices; and how reluctantly and with how great diﬃculty we are diverted from the former to the latter … ’28 Or to put it diﬀerently: ‘Change of life of humans is not easy but takes place with labor of the soul.’29 This is a thought that Freher took up: What has become a habit is rarely corrected … The only reason for this presumption is what Cicero says in Pro Sulla: ‘ … and no-one of us can rapidly pretend or suddenly change his way of living or turn his nature upside down, no-one becomes suddenly the best, no-one becomes suddenly the worst.’30 This consideration, of course, can be illustrated by particular examples; but applying it to new cases does not presuppose any new evidence concerning a particular individual. In this sense, even those presumptions of goodness or badness that invoke previous experiences with the personal qualities of particular individuals need to make use of a presumption against change that is based on an assumption that concerns human nature in general. In fact, such general background assumptions play a crucial role in how presumptions are formed in the absence of evidence concerning the character traits of particular individuals. Meno- chio put it as follows: A presumption is derived from that nature of a person, of an action or of a thing. 3. Causal powers and the presumption of goodness BLANK old age properly speaking is not a disease; but once old age is proven, there results a praesumptio juris that this person is weakened … Likewise, an old person is not as easily presumed to ﬂee as a young person … 38 old age properly speaking is not a disease; but once old age is proven, there results a praesumptio juris that this person is weakened … Likewise, an old person is not as easily presumed to ﬂee as a young person … 38 Presumptions of this kind are grounded in the distinction between essential and accidental qual- ities. Alciato noted that, as to the thematic realm to which such revisable legal presumptions could be applied, two seemingly contradictory rules were often quoted. The ﬁrst rule says that ‘a presumption relates only to the realm of the factual since only there, conjectures have their place.’39 The second rule says that ‘regularly, facts are not presumed but have to be proven.’40 As Alciato explained, the second rule holds especially for accidental facts—those that do not derive from the essence of things—because ‘extrinsic accidents are not presumed.’41 There is no contra- diction between the two rules because the second rule is not applicable to cases ‘in which facts are presumed on the basis of a previously existing cause.’ In such cases, ‘always once something certain is presupposed, the presumption follows from thence. But we call the presuppositions signs; for as arguments have their seats or places from where they arise … , so also do presumptions arise from certain places … ’42 p But not all presumptions require evidence concerning individual cases. Alciato’s treatment of the rule that tells us that ‘change is not to be presumed’ illustrates this point.43 As Alciato clariﬁed, this rule is based on an ontological analysis of the nature of change: ‘[T]he rationale of this rule is that changing means that something happens accidentally; and accidents are not presumed … ’44 What he had in mind is the distinction between intrinsic accidents (which can be presumed because they arise from the essence of a thing) and extrinsic accidents (which cannot be presumed because they arise from an event that is external to the essence of the thing). 3. Causal powers and the presumption of goodness For instance, he contrasted the insight that ‘extrinsic accidents are not presumed’45 with the insight that someone who was poor in the past is not to be presumed poor now because everyone is presumed to strive to become weal- thier.46 Here, the striving for material well-being seems to be understood as one of the natural qual- ities that are presumed to be present in every person. What about the possibility of forming presumptions concerning personal qualities without evi- dence concerning particular individuals? Freher described this possibility as follows: ‘Unless esteem is contaminated or deleted through some delict, it always persists and always inheres; in such a way that it is always presumed in favor of good reputation, probity and innocence; because this is con- venient for, friendly to and in agreement with nature.’47 One of the arguments developed by Freher concerns the professional duties of merchants. He ascribed to Baldus the view that ‘[o]ne has to rely on the books of merchants more than on those of others, for the very reason that merchants are persons of more proven faith and reputation and more committed to the law than others.’48 As Fre- her commented, ‘this reason seems to be founded in what should happen than in what happens.’49 In fact, this seems to correspond closely to Baldus’s treatment of the question of when one should rely on the balance books of merchants. Consider cases where these books contain contradictory entries, for example when a merchant in one entry writes that he has received a sum connected with a certain transaction and in a subsequent entry that he expects to receive the same sum. Baldus gave the following assessment: In this case, the task and equity of the one who judges demand circumspection. For when the merchant is noble and trustworthy, and the quantity small, one should believe his record, otherwise the business relation would be fraudulent and the duty to keep a balance book (to which he is obliged due to the necessity to give an account) would be most dangerous for him. 3. Causal powers and the presumption of goodness For … it has to be presumed that each person and each thing and each action is according to its nature and as nature requires … A pre- sumption is derived from nature itself, as when each person is presumed to be good, and hence not ungrateful unless it is argued for and proved … Contracts are presumed to be made according to their nature, that is, by someone who is mentally healthy. And hence the one who asserts that someone is furious and mentally impaired must prove it, for, according to nature, everyone is presumed to be mentally healthy … 31 A presumption is derived from that nature of a person, of an action or of a thing. For … it has to be presumed that each person and each thing and each action is according to its nature and as nature requires … A pre- sumption is derived from nature itself, as when each person is presumed to be good, and hence not ungrateful unless it is argued for and proved … Contracts are presumed to be made according to their nature, that is, by someone who is mentally healthy. And hence the one who asserts that someone is furious and mentally impaired must prove it, for, according to nature, everyone is presumed to be mentally healthy … 31 Presumptions based on what belongs to human nature were ubiquitous in sixteenth-century legal discourse. One rule discussed by Alciato says that ‘the quality that naturally is in human beings is presumed to be present always.’32 This rule is based on an understanding of what the natural qualities of humans are—for instance, an understanding of emotional states—as well as an understanding of qualities that are essential to human beings—such as sensation and natural reason.33 To mention some of the examples that Alciato gave: Fathers are presumed to love their sons, and vice versa;34 fathers are presumed to have more fear about their sons than about themselves;35 brothers are not presumed to hate each other but ‘rather it has verisimilitude that brothers like each other;’36 and ‘sense and natural reason are presumed in each human being unless the contrary is proven.’37 A similar pattern can be found in Alciato’s example concerning the nature of aging persons: 496 A. 3. Causal powers and the presumption of goodness And it does not have anything in common with them, if humans wanted to use it rightly.’52 As he explained the normative side of his argument, ‘the houses of merchants must be houses of truth and equity, especially because one has commonly recourse to trusting them.’53 Hence, because trade can be carried on only if the customers trust in merchants, and because usually customers show such trust, merchants have a duty to be trustworthy and equitable. Some- thing analogous holds for the role of trust and equity in the relations between merchants: Contracts are ‘formed between merchants, among whom, for the most part, there is the custom of judging according to what is good and equitable, and not according to the rigor of the law.’54 This is why good faith and a commitment to equity is required from those who engage in trade. Going one step further, Freher argued that something analogous holds for the assumption that (healthy, grown-up) person will understand the demands of natural law. As one of the sources of the idea that the presumption in favour of good reputation agrees with nature, Freher mentioned Aimone de Cravetta (1504-1569).55 Take Cravetta’s discussion of a case where the captain of an old vessel refuses to transport some heavy goods in stormy weather. The discussion began with a pre- sumption based on an assumption concerning a natural quality: ‘The desire for money, without which we cannot live, is natural to all humans; therefore, absence of bad faith should be presumed in someone whose act does not result in any pleasurable result for the agent.’56 This is why it should be presumed that the captain’s refusal has been motivated by the intention to avoid shipwreck. Cra- vetta used the concept of verisimilitude to express this thought: ‘Because this cause possesses ver- isimilitude, it should be presumed … When something possesses verisimilitude, it is said to be in accordance with natural law, because verisimilitude is said to be cognate to nature.’57 Here, one encounters a characterization of ‘being cognate to nature’ as what is in accordance with natural law. Moreover, accordance with natural law is understood as a criterion for the rationality of a pre- sumption. 3. Causal powers and the presumption of goodness Also, because he wrote this book as if out of a tacit mandate and out of the necessity of his duty, in particular when he himself has given an oath that he will practice his art faithfully … 50 Baldus’s claim seems to be that one should presume that the second entry in the balance book should be understood to be a correction of the ﬁrst entry, otherwise one would ascribe a fraudulent intention to the merchant; and ascribing such an intention would be contrary to the duties that come naturally with his chosen profession. At the same time, Baldus limited the weight of such HISTORY OF EUROPEAN IDEAS 497 a presumption: As soon as the sum is big enough to constitute a substantial damage to the other party, arguing from presumptions is not enough but requires evidence-based arguments. The presumption in favour of the faithfulness of a merchant thus arises from the assumption that merchant will understand the ethical demands arising from the nature of his chosen profession. In this context, Freher also referred to Tiraqueau, who made the reasoning relating to merchants more explicit. As the French humanist expressed it, ‘in merchants … most extensive trust is required. But it is not as if trust abounds in them; rather, it is much smaller and, if I may say, more sterile than in others, because they are dedicated to ruses, frauds, lies, perjuries more than all others … ’51 In this sense, Tiraqueau did not regard trust in the personal qualities of merchants to derive from a gen- eralization based on what happens most frequently. Rather, he distinguished between the personal qualities of merchants and the ethical demands that derive from the nature of trade relations. As to the vices of merchants, Tiraqueau held that ‘they belong not to the art but to persons … For even if in most cases those exert the art make an admixture of ruses, lies, frauds and perjuries: The art itself is far away from them. 4. Balancing the presumption of goodness against presumptions of badness Forming the presumption of goodness, for the legal humanists, thus was not only guided by their respect for the natural needs of others but also by their view that the rationality of presumptions derives from their verisimilitude. But the answer that the legal humanists found to prudential con- cerns had further aspects, and these aspects, like the assessment of verisimilitude, derive from con- siderations concerning causal powers. One line of thought limited the presumption of goodness by restricting the degree of ethical goodness that should be presumed. Immediately after the passages mentioned by Freher, Tiraqueau cautioned that the presumption in favour of the good qualities of members of particular professions does not involve the ascription of anything like ethical perfection. Rather, he believed that, while considerations concerning human nature speak in favour of that normal goodness required to fulﬁl professional roles, such considerations also speak against presuming excellent ethical qualities in others. The reason he gave derives from an assumption concerning the role of self-love: It seems to be rather absurd and in no way safe for someone to be a judge in his own case, no matter how decent and good a person he may be; for in almost everyone, self-love is so ingrained … that you will not ﬁnd anyone so decent, so modest, so attentive, so sharp-eyed that he would not, in estimating and inspecting his own cause, become blind, dizzy, half-awake, wavering, dull, and delirious by the propensity of his mind.64 Here, everyday observations about the eﬀects of self-love provide the basis for forming the pre- sumption that no-one is a good judge in his own aﬀairs. This presumption is not based on obser- vations concerning the character traits of a particular individual; rather, it is a presumption based on an assumption concerning human nature in general. Similarly, Freher emphasized that ‘no-one is presumed to be good in the most excellent degree.’65 Also, he held that when someone ‘alleges his goodness or good reputation as the foundation of his intention, then it must necessarily be proven … ’66 Likewise, he argued that when the law requires witnesses to have a good reputation, it has to be proven whether these requirements are fulﬁlled, otherwise the law would be superﬂuous.67 In the view of the legal humanists, the presumption of goodness thus did not amount to a presumption of ethical excellence. 3. Causal powers and the presumption of goodness Freher’s reference to Cravetta is informative because it indicates how the presumption of goodness can be understood as an instance of arguing from verisimilitude: Persons should be pre- sumed to recognize what is naturally good for them. This sense of verisimilitude understood as what is in agreement with the demands of natural law58 seems also to be at stake when Freher wrote: ‘The verisimilitude of the thing usually recommends good reputation if the reputation concerns those things that are in agreement with nature, such as the probity and innocence of someone, in favor of which one should form a presumption … ’59 Alciato, too, held that claims concerning what we should presume because it corresponds to the nature of a thing are not restricted to matters of fact but also can reﬂect demands of natural law. Among the presumptions concerning the presence of a natural quality, Alciato mentioned the pre- sumption that a member of an older generation wants to leave his estate to his descendants, because a legitimate part of the estate is owed to descendants by natural law.60 Thereby, Alciato connected an aspect of inheritance law that is highly relevant for cases where someone passes away without having made a last will with a level of natural law that the Roman jurists regarded as being common to humans and other animals—the demands of caring for oﬀspring without which the survival and A. BLANK A. BLANK 498 well-being of a biological species cannot be secured.61 Hence, these are duties that are understood as arising from the natural constitution of biological beings of our kind. Placing this presumption in the context of the rule that recommends grounding presumptions on natural qualities thus is a con- sequence of the idea that the demands of natural law are understood as being grounded in natural qualities. From this perspective, it is not surprising to see that Alciato understood the presumption of goodness to be grounded in assumptions concerning natural duties. With respect to the one who proﬁts from the presumption of goodness, he held that ‘because he has to be good, he deserves to be presumed to be such … .’62 And since presuming the goodness of another is itself a natural duty, there is a justiﬁed presumption that this duty will be fulﬁlled: ‘everyone is not only presumed to be good but also presumed to be held in good esteem and reputation.’63 HISTORY OF EUROPEAN IDEAS Everyone is presumed to care about his own aﬀairs before caring about the aﬀairs of others … This is why it is presumed that someone who dares to treat himself badly will commit worse things against others. And note this against those who accept administrators or overseers who have consumed their own goods: Because they will consume the good of others more easily … And this is conﬁrmed by the consideration that he who failed to provide advice in his own aﬀairs is even more presumed to fail to provide advice in the aﬀairs of others.69 As in the cases of evidence-based presumptions of goodness, such presumptions are based on a combination of observations concerning particular individuals and assumptions concerning human nature. This gives the legal humanists’ conception of how both presumptions of goodness and presumptions of badness are formed a high degree of internal coherence and explains why the same argumentative foundations that lead to evidence-based presumptions of goodness also allow revision of these presumptions. Both evidence-based presumptions of goodness and presumptions of badness thus involve the presumption against change. p p g g But, as Menochio pointed out, the presumption against change does not apply indiscriminately: [I]t does not apply when things are variable through the passage of time, out of either the necessity of nature, or verisimilitude, or quality of acting. As when we say that the one who was once a child is not presumed to be one now … Likewise, we say out of verisimilitude that the one who did not know letters in childhood is not presumed not to know them now, since it has verisimilitude that humans can learn daily … Likewise, out of the quality of acting we say that it is not presumed that the one who stabbed someone in the past will also stab someone today … 70 In this way, assumptions concerning human nature can also ground presumptions in favour of change. And this example makes it clear that even the presumption of badness to which a former crime gives rise can be overturned by the passage of time in cases where assumptions about human nature tell us that emotions that have prompted the misdemeanour are by their nature transitory. HISTORY OF EUROPEAN IDEAS The interplay between the presumption of goodness that could be overturned by more speciﬁc presumptions of badness, which again could be overturned by equally speciﬁc presumptions of goodness, is also highly relevant for Freher’s political activity as a counselour for the Palatinate. The constitutional situation of the Palatinate at the end of the sixteenth century was controversial because large parts of the territory (Oberpfalz and Rheinpfalz) had fallen under the dominion of the dukes of Bavaria. While the Bavarian side argued that electoral rights should be regarded as terri- torial rights, Freher and his colleague at the University of Heidelberg, Denis Godefroy (1549–1622), argued that electoral rights should be regarded as personal rights unaﬀected by territorial losses.71 One element of their case for the personal nature of electoral rights was their justiﬁcation of the validity of testamentary provisions about tutelage for the ﬁrst-born heirs who have not yet reached adulthood. Both the Bulla aurea of Emperor Charles IV (1356) the Bulla aurea Sigismundi (1434), two of the fundamental laws of the German Empire, unambiguously conceded to the Electors the right to confer in their last wills tutelage for their underage heirs upon some of their agnates (that is, their male relatives who descend from a common male ancestor).72 As Freher explained, this right was understood to be crucial for securing the electoral dignity for their ﬁrst-born son (in the con- crete case, Friedrich IV, who was under the tutelage of his uncle Johann Casimir until 1592).73 By including legal questions arising from tutelage, arguments from presumption became highly pertinent for the controversy about the nature of electoral rights. Alciato discussed a bundle of pre- sumptions relevant for tutelage as one of the applications of the rule that advises us to presume natural qualities to be present. 4. Balancing the presumption of goodness against presumptions of badness Rather, it amounted to the presumption that individuals will respect law and cus- tom, and that they will develop a moderate degree of ethical goodness. In this sense, the presump- tion of goodness did not involve some imprudently high expectations. g p y g p Placing the presumption of goodness in the context of a variety of presumptions of badness was a further strategy to fulﬁl the demands of prudence. The presumption of goodness was not only understood to be defeasible by something as strong as contrary evidence but also by something as weak as contrary presumptions. And as in the case of other presumptions, these presumptions were understood to be motivated by considerations concerning causal powers. Recall the case of the soldier returning from the barbarians, where the presumption against the change of character traits allows forming a presumption against someone who has been held to be an unreliable soldier in the past.68 Alciato discussed a similar case, where the inability to take good care of oneself can give rise to a presumption against the ability to take good care of others: HISTORY OF EUROPEAN IDEAS 499 HISTORY OF EUROPEAN IDEAS Most importantly, he took this rule to imply that, due to vulnerability of minors, there cannot be a presumption of goodness in favour of the tutor: ‘In a minor ignorance is presumed … And for this reason, a minor is presumed to be deceived easily.’74 This is why the burden of proof lies on the side of the tutor: ‘From this it is inferred that a minor who takes out a loan from a lender is presumed to have been injured, unless it is proven that he has become wealthier … Also, a sale that injures a pupil is presumed to have been made on the basis of a fabricated lie.’75 Likewise, Menochio observed that a tutor who has neglected the duty of having the goods belonging to the pupil registered in an inventory cannot invoke any presumption in his own favour.76 There was even a praesumptio iuris et de iure against the tutor that goods 500 A. BLANK included in the inventory cannot be goods belonging to the tutor. As Menochio argued, ‘it does not correspond to natural reason, nor does it have in any way verisimilitude, that tutors … accept that in making the inventory of the goods of a pupil … their own goods be described among the goods of the pupil … ’77 These are example of how the presumption of goodness can be overruled by presumptions of badness based on considerations concerning causal propensities. The case of tutelage, however, is interesting because it also provides examples of how presumptions of badness can be overruled by speciﬁc presumptions of goodness. HISTORY OF EUROPEAN IDEAS For instance, Menochio noted that ‘the opinion of interpreters is commonly accepted and true that it is permitted to a tutor to contract with his pupil publicly, but not secretly … because by contracting publicly the suspicion of fraud ceases … ’78 As he explained, ‘publicly’ can mean something like ‘in the presence of a co-tutor’, or ‘in the presence of a well-informed judge’, or ‘in the presence of friends and relatives.’79 Alciato, too, held that ‘it is presumed that blood relatives and family members will not fail to defend their own relative … From which it follows that someone is not presumed to be devoid of defense when he gives rise to some verisimilitude that at least his relatives or friends will defend him … ’80 For this reason, ‘someone who contracted through the intervention of family members and blood relatives is not presumed to have contracted out of fear … ’81 And for the same reason, Alciato argued, relatives should be chosen with priority as tutors.82 Analogous considerations shaped the defense of the personal nature of electoral rights. As to the question of whether the electors’ right of making testamentary provisions concerning tutelage has been used in a legitimate way, Godefroy argued that the deceased Elector (Ludwig VI) had sought the advice of friends, legal experts and German and foreign rulers. In Godefroy’s view, this process of consultation created a presumption in favour of the person nominated as tutor: ‘Suspect tutors and curators are to be rejected through the advice or intervention of such persons; hence, once it has been proven that one has decided and acted on the basis of their advice, it has to be presumed that decision has been reached in good faith and without deception.’83 One of the reasons for why the suspicion of fraud can be overcome through a process of consultation invoked emotional factors relevant not only the question at hand: ‘In other cases, due to friendship, princes are never pre- sumed to transgress rights because they can assist the one for whom they have an aﬀection in a diﬀerent way, which allows them to gain both securer gratitude and truer glory.’84 Thus, assump- tion about emotions toward friends and assumptions about expectations concerning gratitude and social esteem belong to the causal propensities that make presumptions in favour of those giving advice rational. HISTORY OF EUROPEAN IDEAS Freher justiﬁed the constitutional rule given by Emperor Sigismund in a similar way. According to Freher, Sigismund preferred tutelage by close relatives over tutelage by foreigners or more remote relatives for a good reason: This is so either because of the presumed faithfulness and aﬀection of agnates, or also because this is what is owed to the honor, connection and necessity of blood relations. And in their family, the members of the Pala- tinate have fulﬁlled this demand of the situation since ancient times, and never admitted foreigners or even relatives of a more remote degree at the cost of the envy of and contempt for the closer relatives … 85 The presumption in favour of the goodness of tutors chosen in accordance with the Bulla aurea Sigismundi thus is grounded on assumptions concerning the emotions that tend to be present in close relatives and on the assumption that they will have desire to act as tutors because this fulﬁls a widely shared norm of what honourable action consist in. Notes Ibid., 10: “eam hominis qualitatem, qua in numero bonorum virorum & honestorum civium censeri meretur; qua capax munerum & honorum, & ad omnes actus civilis societatis gerendos idoneus, rursumque iniuria & quavis obtrectatione indignus reputatur, quandiu oﬃcii mediocritatem tuetur, omnique sceleris & ﬂagitii con- tagio vacat, quo famae suae puritas & synceritas statim laedi posset.” 10. Ibid., 10: “eam hominis qualitatem, qua in numero bonorum virorum & honestorum civium censeri meretur; qua capax munerum & honorum, & ad omnes actus civilis societatis gerendos idoneus, rursumque iniuria & quavis obtrectatione indignus reputatur, quandiu oﬃcii mediocritatem tuetur, omnique sceleris & ﬂagitii con- tagio vacat, quo famae suae puritas & synceritas statim laedi posset.” g q p y p 11. Ibid.: “Haec autem existimatio viri boni ut morum integritate, virtutis studio, vitaeque innocentia adquiritur … ” On the concept of being a good person, see Welti, “‘Vir bonus’/ ‘homo bonus’/ ‘preudhomme.’” 11. Ibid.: “Haec autem existimatio viri boni ut morum integritate, virtutis studio, vitaeque innocentia adquiritur … ” On the concept of being a good person, see Welti, “‘Vir bonus’/ ‘homo bonus’/ ‘preudhomme.’” 12. Freher, Existimatio, 84, citing Papinianus, Dig. 28.7.15: “Quae facta laedunt pietatem, existimationem, vere- dundiam nostram, & (ut generaliter dixerim) contra bonos mores ﬁunt, ea nec facere nos posse credendum est.” 12. Freher, Existimatio, 84, citing Papinianus, Dig. 28.7.15: “Quae facta laedunt pietatem, existimationem, vere- dundiam nostram, & (ut generaliter dixerim) contra bonos mores ﬁunt, ea nec facere nos posse credendum est.” 13. Freher, Existimatio, 18. , , 14. Ibid., 69: “Cum enim gloria non nisi ex vera virtute petenda sit, idque eo ordine … ut non laudis studio vir- tutem aﬀectemus, sed ex virtute laudem mereamur … ” b d 14. Ibid., 69: “Cum enim gloria non nisi ex vera virtute petenda sit, idque eo ordine … ut non laudis studio vir- tutem aﬀectemus, sed ex virtute laudem mereamur … ” 16. Ibid., 38: “Sane hunc laudis & gloriae aﬀectum, ut a natura animis insitus est; ita praeclarae indolis animique ad virtutis studium apti signiﬁcationem non obscuram praebere, semper creditum est … ” 16. Ibid., 38: “Sane hunc laudis & gloriae aﬀectum, ut a natura animis insitus est; ita praeclarae indolis animique ad virtutis studium apti signiﬁcationem non obscuram praebere, semper creditum est … ” 17. 5. Conclusion By now it should be clear that the conception of the presumption of goodness that Freher derived from the legal humanists oﬀered an answer to how we could develop social emotions that fulﬁl the natural need for being esteemed (not only for non-delinquency), and also an answer to prudential concerns by grounding the presumption of goodness in considerations concerning causal powers. 501 HISTORY OF EUROPEAN IDEAS What the rules speciﬁed by the legal humanists have in common is that they rely on considerations concerning the nature of persons and their acts. Forming the presumption of goodness is an instance of forming presumptions on the basis of what happens most easily, given our background assumptions concerning human nature. This is why the presumption of goodness can be counter- balanced by presumptions of badness that likewise make use of assumptions concerning the causal powers of mental states. And presumptions of badness, in turn, can be counter-balanced by pre- sumptions of goodness that arise from considerations concerning the causal powers of other mental states. Including ethical aspects into the juridical conception of the presumption of goodness thus did not lead to a naïve and potentially dangerous form of trust; rather, it opened up the possibility of dealing rationally with situations of uncertainty about the personal qualities of others. And this is why the strategy of balancing presumptions of goodness and presumptions of badness turned out to be a powerful tool not only for juridical but also for political argumentation. 1. On the desire for esteem in early modern natural law theories, see Blank, “Besold on Confederation Rights and Duties of Esteem;” Blank, “Pufendorf and Leibniz on Duties of Esteem.” Notes 1. On the desire for esteem in early modern natural law theories, see Blank, “Besold on Confederation Rights and Duties of Esteem;” Blank, “Pufendorf and Leibniz on Duties of Esteem.” 2. On Freher’s biography, see Fuchs, “Freher, Marquard;” Adam, Vitae Germanorum Jureconsultorum et Politi- corum, 473-479; Brucker, Ehren-tempel der Deutschen Gelehrsamkeit, 106-110; Kornexl, Studien zu Marquard Freher. 3. On Freher’s editions of medieval sources, see Kühlmann, Die deutschen Humanisten. 4. On the types of presumption, see Alciato, De praesumptionibus, 15-33. 5. See Kelley, Foundations of Historical Scholarship; Plessis, Reassessing Legal Humanism. 6. See Sbriccoli, L’interpretazione dello statuto; Massironi, Nell’oﬃcina del’interprete; Maclean, Meaning and Interpretation in the Renaissance; Monheit, “Guillaume Budé, Andrea Alciato, Pierre de l’Étoile,” 27-31; Kriechbaum, “Verba und mens in den Interpretationslehren des Humanismus,” 47-56; Méniel, “La séman- tique d’un juriste.” 6. See Sbriccoli, L’interpretazione dello statuto; Massironi, Nell’oﬃcina del’interprete; Maclean, Meaning and Interpretation in the Renaissance; Monheit, “Guillaume Budé, Andrea Alciato, Pierre de l’Étoile,” 27-31; Kriechbaum, “Verba und mens in den Interpretationslehren des Humanismus,” 47-56; Méniel, “La séman- tique d’un juriste.” q j 7. See Gaskins, Burdens of Proof in Modern Discourse; Guiliani, “Civilian Treatises on Presumption;” Blank, Arguing from Presumptions. 7. See Gaskins, Burdens of Proof in Modern Discourse; Guiliani, “Civilian Treatises on Presumption;” Blank, Arguing from Presumptions. 8. Freher, Existimatio, 9: “Deﬁnitur … a Callistrato existimatio, dignitatis illaesae status, legibus ac moribus com- probatus, qui ex delicto nostro authoritate legum minuitur aut consumitur” (Dig. 50.13.5). 8. Freher, Existimatio, 9: “Deﬁnitur … a Callistrato existimatio, dignitatis illaesae status, legibus ac moribus com- probatus, qui ex delicto nostro authoritate legum minuitur aut consumitur” (Dig. 50.13.5). 9. Freher, Existimatio, 9-10: “[Status] dignitatis, quo alii honesti, idonei, legales, ﬁde digni, exceptione maiores, & (ut vocant) authentici; alii notati, suspecti, infames, turpesque & intestabiles habentur: quae famae censura partim a legibus & edictis magistratuum introducta est, partim a consuetudine & bonis moribus singulorum locorum dependet.” 9. Freher, Existimatio, 9-10: “[Status] dignitatis, quo alii honesti, idonei, legales, ﬁde digni, exceptione maiores, & (ut vocant) authentici; alii notati, suspecti, infames, turpesque & intestabiles habentur: quae famae censura partim a legibus & edictis magistratuum introducta est, partim a consuetudine & bonis moribus singulorum locorum dependet.” p 10. Notes Ibid., 44-45: “Cum itaque ad virtutis culturam & studium apud mortales excitandum tantum gloriae amor possit, consequenter est a sapientibus iudicatum, excellentis & consummatae virtutis praemium, gloriosam famam & celebrationem esse debere.” 17. Ibid., 44-45: “Cum itaque ad virtutis culturam & studium apud mortales excitandum tantum gloriae amor possit, consequenter est a sapientibus iudicatum, excellentis & consummatae virtutis praemium, gloriosam famam & celebrationem esse debere.” 18. Ibid., 14: “[N]escio quo modo in omnium animis ab ipsa natura comparatum est, ut aliorum de se opinionem magni pendant, & ad eam se magna ex vitae parte component.” 18. Ibid., 14: “[N]escio quo modo in omnium animis ab ipsa natura comparatum est, ut aliorum de se opinionem magni pendant, & ad eam se magna ex vitae parte component.” 19. Ibid., 14-15: “Cum itaque plurimum referre plerique homines putarent, quid de ipsis alii homines eadem ratione praediti, iisdemque legibus & moribus utentes, & in eadem civitate constituti (quibuscum ideo neces- sario plurimum consuetudinis & commercii habendum esset) sentirent; nata est inter illos haec opinio, ut non 19. Ibid., 14-15: “Cum itaque plurimum referre plerique homines putarent, quid de ipsis alii homines eadem ratione praediti, iisdemque legibus & moribus utentes, & in eadem civitate constituti (quibuscum ideo neces- sario plurimum consuetudinis & commercii habendum esset) sentirent; nata est inter illos haec opinio, ut non A. BLANK 502 dubitarent, bonam famam & existimationem esse cuilibet expetendam. Unde Tullius lib. 1. De legibus ubi de iure naturae disserit, in hoc omnium hominum ingeniis convenire ait, quod & voluptate capiantur omnes, & mortem doloremque fugiant; itemque quod propter honestatis & gloriae similitudinem beatos, qui honorati sunt, iudicent; miseros autem, qui inglorii.” See Cicero, De legibus, 1.32. 20. Ibid., 116: “Non dicitur nimium credulus, qui credit homini ﬁde digno … credere bono viro non est facere contra ius naturale” (see Baldus, Consiliorum … volumen primum, 144). 20. Ibid., 116: “Non dicitur nimium credulus, qui credit homini ﬁde digno … credere bono viro non est facere contra ius naturale” (see Baldus, Consiliorum … volumen primum, 144). 20. Ibid., 116: “Non dicitur nimium credulus, qui credit homini ﬁde digno … credere bono viro non est facere contra ius naturale” (see Baldus, Consiliorum … volumen primum, 144). p 21. Notes Freher, Existimatio, 114, citing Cicero, De oﬃciis, 2.9.32: “Etenim illud ipsum quod decorum honestumque est, quia per se nobis placet, animosque hominum natura & specie sua commovet … : idcirco eos in quibus illas virtutes esse remur, a natura ipsa diligere cogimur.” p 21. Freher, Existimatio, 114, citing Cicero, De oﬃciis, 2.9.32: “Etenim illud ipsum quod decorum honestumque est, quia per se nobis placet, animosque hominum natura & specie sua commovet … : idcirco eos in quibus illas virtutes esse remur, a natura ipsa diligere cogimur.” 21. Freher, Existimatio, 114, citing Cicero, De oﬃciis, 2.9.32: “Etenim illud ipsum quod decorum honestumque est, quia per se nobis placet, animosque hominum natura & specie sua commovet … : idcirco eos in quibus illas virtutes esse remur, a natura ipsa diligere cogimur.” p g g 22. Menochio, De praesumptionibus, 1: 20: “A viribus praesumptio atque coniectura desumitur.” p g g . Menochio, De praesumptionibus, 1: 20: “A viribus praesumptio atque coniectura desumitur.” 23. Ibid.: “dicimus eum prius decessisse credi, qui debilior imbecilliorque erat … Ita dicimus quod is p aggressor in rixa qui altero corpore fortior est … ” gg q p 24. Ibid.: “Bona animi, a quibus persaepe etiam coniectura ducitur, haec sunt, virtutes, vitia, studia, educatio, artes, inertia.” 24. Ibid.: “Bona animi, a quibus persaepe etiam coniectura ducitur, haec sunt, virtutes, vitia, studia, educatio, artes, inertia.” 24. Ibid.: “Bona animi, a quibus persaepe etiam coniectura ducitur, haec sunt, virtutes, vitia, studia, educatio, artes, inertia.” 25. Freher, Existimatio, 156, citing Dig. 49.16.5.6: “Si bonus miles ante existimatus fuit, credendum aﬃrmationi eius, quod se captum a barbaris fuisse dicat; non transfugisse: Si vero emansor fuerit, aut negligens suorum, aut segnis, aut extra contubernium agens, non esse credendum, si forte post multum temporis revertatur.” 25. Freher, Existimatio, 156, citing Dig. 49.16.5.6: “Si bonus miles ante existimatus fuit, credendum aﬃrmationi eius, quod se captum a barbaris fuisse dicat; non transfugisse: Si vero emansor fuerit, aut negligens suorum, aut segnis, aut extra contubernium agens, non esse credendum, si forte post multum temporis revertatur.” 25. Freher, Existimatio, 156, citing Dig. 49.16.5.6: “Si bonus miles ante existimatus fuit, credendum aﬃrmationi eius, quod se captum a barbaris fuisse dicat; non transfugisse: Si vero emansor fuerit, aut negligens suorum, aut segnis, aut extra contubernium agens, non esse credendum, si forte post multum temporis revertatur.” 26. Notes Ibid., 263: “Non facilis est vitae hominum mutatio, sed cum animae ﬁt labore.” . Ibid., 263: “Non facilis est vitae hominum mutatio, sed cum animae ﬁt labore.” 30. Freher, Existimatio, 160: “[R]aro corrigitur, quod venit in consuetudinem … Cuius praesumptionis haec sola est ratio quod Tullius Pro Sulla ait: ‘ … neque enim potest quisquam nostrorum subito ﬁngi, neque cuiusquam vita repente mutari aut natura converti, nemo repente ﬁt summus, nemo repente turpissimus.’” See Cicero, Pro Sulla, 25.69. 30. Freher, Existimatio, 160: “[R]aro corrigitur, quod venit in consuetudinem … Cuius praesumptionis haec sola est ratio quod Tullius Pro Sulla ait: ‘ … neque enim potest quisquam nostrorum subito ﬁngi, neque cuiusquam vita repente mutari aut natura converti, nemo repente ﬁt summus, nemo repente turpissimus.’” See Cicero, Pro Sulla, 25.69. 31. Menochio, De praesumptionibus, 1: 23: “sumitur praesumptio ex natura personae, actus vel rei. Nam … prae- sumendum est, quod quaelibet persona & quaelibet res, & quilibet actus sit secundum suam naturam sicut eius requirit natura … Sumitur praesumptio ex ipsa natura, ut quilibet bonus esse praesumatur, atque ita non ingratus nisi arguatur & probetur … Codicillos praesumi factos secundam suam naturam, idest ab eo qui sanae mentis est. Et ideo qui asserit aliquem essse furiosum & mentecaptum probare id debet, cum secundum naturam quilibet praesumatur sanae mentis … ” 31. Menochio, De praesumptionibus, 1: 23: “sumitur praesumptio ex natura personae, actus vel rei. Nam … prae- sumendum est, quod quaelibet persona & quaelibet res, & quilibet actus sit secundum suam naturam sicut eius requirit natura … Sumitur praesumptio ex ipsa natura, ut quilibet bonus esse praesumatur, atque ita non ingratus nisi arguatur & probetur … Codicillos praesumi factos secundam suam naturam, idest ab eo qui sanae mentis est. Et ideo qui asserit aliquem essse furiosum & mentecaptum probare id debet, cum secundum naturam quilibet praesumatur sanae mentis … ” 33. Ibid., 108: “Sensus & ratio naturalis praesumuntur in quolibet homine, nisi probetur contrarium … Probatur autem quis fuiosus per signa extrinseca, puta quia loquitur verba more furiosorum.” 33. Ibid., 108: “Sensus & ratio naturalis praesumuntur in quolibet homine, nisi probetur contrarium … Probatur autem quis fuiosus per signa extrinseca, puta quia loquitur verba more furiosorum.” 35. Ibid. d., 69: “imo est verisimile quod alter alterum diligat 36. Ibid., 69: “imo est verisimile quod alter alterum diligat.” q g 37. Notes Ibid.: “Ex quo textu fundata est nobilis illa praesumptio, quod qui ante fuit bonus, & nunc sit bonus. Quia ex transactae vitae ratione aestimari potest, quid de sequenti conversatione iudicemus, & quomodo facta dicta- que hominum sint accipienda.” 26. Ibid.: “Ex quo textu fundata est nobilis illa praesumptio, quod qui ante fuit bonus, & nunc sit bonus. Quia ex transactae vitae ratione aestimari potest, quid de sequenti conversatione iudicemus, & quomodo facta dicta- que hominum sint accipienda.” q p 27. Tiraqueau, De poenis, 215: “In quibus quidem ex temperata (qui est ipsius virtutis habitus) continens duntaxat eﬀecta est, nondum autem incontinens. Mota enim fuit … a principio Dido, ut Aeneam amet, tum virtute, quam unam in vita contemplatione dedita intuemur, tum & his quae humanis coetibus non solum bona, sed & summa bona existimabantur. Quis enim generis nobilitatem, quis formae dignitatem, atque excellen- tiam, quis denique multo ornatu insignem orationem inter summa non adnumeret? Tandem vero cum ad tot acerrimos hostes, blandae sororis Annae persuasiones accessissent, superior evasit appetitus, & rationem quanvis invitam & reluctantem traxit: factaque est demum mulier non prorsus intemperans, sed incontinens … ” q p 27. Tiraqueau, De poenis, 215: “In quibus quidem ex temperata (qui est ipsius virtutis habitus) continens duntaxat eﬀecta est, nondum autem incontinens. Mota enim fuit … a principio Dido, ut Aeneam amet, tum virtute, quam unam in vita contemplatione dedita intuemur, tum & his quae humanis coetibus non solum bona, sed & summa bona existimabantur. Quis enim generis nobilitatem, quis formae dignitatem, atque excellen- tiam, quis denique multo ornatu insignem orationem inter summa non adnumeret? Tandem vero cum ad tot acerrimos hostes, blandae sororis Annae persuasiones accessissent, superior evasit appetitus, & rationem quanvis invitam & reluctantem traxit: factaque est demum mulier non prorsus intemperans, sed incontinens … ” 28. Ibid., 215: “Quibus quidem monemur, quam multi sint a virtutum habitu ad habitum vitiorum gradus: quam- que aegre, & cum magna diﬃcultate ab illis ad ista divertimus … ” 28. Ibid., 215: “Quibus quidem monemur, quam multi sint a virtutum habitu ad habitum vitiorum gradus: quam- que aegre, & cum magna diﬃcultate ab illis ad ista divertimus … ” b d “ f l h d ﬁl b ” 28. Ibid., 215: “Quibus quidem monemur, quam multi sint a virtutum habitu ad habitum vitiorum gradus: quam- que aegre, & cum magna diﬃcultate ab illis ad ista divertimus … ” 29. Notes Menochio, De praesumptionibus, 1: 28: “Declaratur hic casus ut non procedat, quando res sunt variables ipso temporis cursu, vel ex ipsius naturae necessitate, vel verisimilitudine, vel agendi qualitate. Sicuti dicimus, quod ille, qui olim fuit infans non praesumitur etiam hodie infans … Ita quoque ex verisimilitudine dicimus, quod ille qui a pueritia nescivit litteras, non praesumitur hodie nescire, cum verisimile sit hominem quotidie discere posse … Ita ex qualitate agendi dicimus, non praesumi, quod ille, qui olim percussit, etiam hodie percutiat … ” 70. Menochio, De praesumptionibus, 1: 28: “Declaratur hic casus ut non procedat, quando res sunt variables ipso temporis cursu, vel ex ipsius naturae necessitate, vel verisimilitudine, vel agendi qualitate. Sicuti dicimus, quod ille, qui olim fuit infans non praesumitur etiam hodie infans … Ita quoque ex verisimilitudine dicimus, quod ille qui a pueritia nescivit litteras, non praesumitur hodie nescire, cum verisimile sit hominem quotidie discere posse … Ita ex qualitate agendi dicimus, non praesumi, quod ille, qui olim percussit, etiam hodie percutiat … ” 71. For detailed analysis of the legal and political situation of the Palatinate around 1600, see Messinger, Die Über tragung der pfälzischen Kurwürde auf das Herzogtum Bayern. 71. For detailed analysis of the legal and political situation of the Palatinate around 1600, see Messinger, Die Üb tragung der pfälzischen Kurwürde auf das Herzogtum Bayern. 72. The relevant passage from the Bulla aurea is reproduced in Freher, De legitima tutela, sig. ***recto-verso; the full text of the Bulla aurea Sigismundi is reproduced in Freher, Assertio, sig. I3verso-K2verso. 72. The relevant passage from the Bulla aurea is reproduced in Freher, De legitima tutela, sig. *recto-verso; the full text of the Bulla aurea Sigismundi is reproduced in Freher, Assertio, sig. I3verso-K2verso. 73 Freher Assertio 63 72. The relevant passage from the Bulla aurea is reproduced in Freher, De legitima tutela, sig. ***recto-verso; the full text of the Bulla aurea Sigismundi is reproduced in Freher, Assertio, sig. I3verso-K2verso. 73. Freher, Assertio, 63. 74. Alciato, De praesumptionibus, 112: “In minore praesumitur ignorantia … Et propterea talis praesumitur de facili circumscribi.” 74. Alciato, De praesumptionibus, 112: “In minore praesumitur ignorantia … Et propterea talis praesumitur de facili circumscribi.” 75. Ibid.: “inde infertur, quod minor contrahens mutuum cum foeneratore, praesumitur laesus, nisi probetur fac- tus locupletior … [E]tiam venditio quae pupillum laedit, praesumitur commento mendacio facta.” 75. Notes Freher, Existimatio, 144: “Quae quidem ratio fundata videtur magis in eo quod ﬁeri debebat, quam quod ﬁt.” , , Q q g q , q q 50. Baldus, In VII. VIII. IX X. & XI. partem Codicis commentaria, fol. 86 recto:“[T]unc oﬃcium, & aequitas iudi- cantis debet esse circumspecta. Nam quando mercator est nobilis, & ﬁde digna persona, & quantitas parva, credendum est suae scripturae, alioquin societas esset captiosa, & oﬃcium scibendi codicem rationum (ad quod de necessitate reddendarum rationum tenetur) esset ei periculosissimum. Item quia hanc scripturam facit quasi ex tacito mandato, & de necessitate oﬃcii sui, praesertim cum ipse iuraverit artem suam ﬁdeliter exercere … ” Q q g q q q 50. Baldus, In VII. VIII. IX X. & XI. partem Codicis commentaria, fol. 86 recto:“[T]unc oﬃcium, & aequitas iudi- cantis debet esse circumspecta. Nam quando mercator est nobilis, & ﬁde digna persona, & quantitas parva, credendum est suae scripturae, alioquin societas esset captiosa, & oﬃcium scibendi codicem rationum (ad quod de necessitate reddendarum rationum tenetur) esset ei periculosissimum. Item quia hanc scripturam facit quasi ex tacito mandato, & de necessitate oﬃcii sui, praesertim cum ipse iuraverit artem suam ﬁdeliter exercere … ” 50. Baldus, In VII. VIII. IX X. & XI. partem Codicis commentaria, fol. 86 recto:“[T]unc oﬃcium, & aequitas iudi- cantis debet esse circumspecta. Nam quando mercator est nobilis, & ﬁde digna persona, & quantitas parva, credendum est suae scripturae, alioquin societas esset captiosa, & oﬃcium scibendi codicem rationum (ad quod de necessitate reddendarum rationum tenetur) esset ei periculosissimum. Item quia hanc scripturam facit quasi ex tacito mandato, & de necessitate oﬃcii sui, praesertim cum ipse iuraverit artem suam ﬁdeliter exercere … ” 51. Tiraqueau, De poenis, 259: “In mercatoribus … ﬁdes exuberantissima requiritur. Sed tantum abest, ut is eis ﬁdes exuberet, ut in eis sit multo minor, & ut sic loquar, sterilior, quam in caeteris: quippe cum dolis, frau- dibus, mendaciis, periuriis sint ante omnes dediti … ” 51. Tiraqueau, De poenis, 259: “In mercatoribus … ﬁdes exuberantissima requiritur. Sed tantum abest, ut is eis ﬁdes exuberet, ut in eis sit multo minor, & ut sic loquar, sterilior, quam in caeteris: quippe cum dolis, frau- dibus, mendaciis, periuriis sint ante omnes dediti … ” 51. Tiraqueau, De poenis, 259: “In mercatoribus … ﬁdes exuberantissima requiritur. Notes Freher, Fama, 115: “ipsa verisimilitudo rei famam magnopere commendare solet, si fama sit de his, quae su naturae consentanea, veluti de probitate & innocentia alicuius, pro qua praesumendum est … ” 60. Alciato, De praesumptionibus, 45. 59. Freher, Fama, 115: “ipsa verisimilitudo rei famam magnopere commendare solet, si fama sit de his, quae sunt naturae consentanea, veluti de probitate & innocentia alicuius, pro qua praesumendum est … ” 60. Alciato, De praesumptionibus, 45. 61. See Scattola, Das Naturrecht vor dem Naturrecht, 58-59. 62. Alciato, De praesumptionibus, 245: “unde cum debeat esse bonus, merito debet etiam praesumi … ” 63 Ibid 247 “ ilib t l it b d ti it b i ti ti i & f 62. Alciato, De praesumptionibus, 245: “unde cum debeat esse bonus, merito debet etiam praesum 63. Ibid., 247: “quilibet non solum praesumitur bonus, sed etiam praesumitur bonae existimation 64. Ibid., 236: “Satis absurdum videri & minime tutum, quempiam quantumlibet probum ac bonum virum, iudi- cem esse in sua, aut suorum causa: cum omnibus usque adeo insita sit illa philautia … , ut neminem tam pro- bum, tam modestum, tam attentum, tam oculatum invenias, qui in sua causa aestimanda, at introspicienda quadam animi propensione corruptus caecutiat, calliget, lippiat, ﬂuctuet, hebetescat, deliret.” 65. Freher, Fama, 135. 64. Ibid., 236: “Satis absurdum videri & minime tutum, quempiam quantumlibet probum ac bonum virum, iudi- cem esse in sua, aut suorum causa: cum omnibus usque adeo insita sit illa philautia … , ut neminem tam pro- bum, tam modestum, tam attentum, tam oculatum invenias, qui in sua causa aestimanda, at introspicienda quadam animi propensione corruptus caecutiat, calliget, lippiat, ﬂuctuet, hebetescat, deliret.” 65. Freher, Fama, 135. 66. Ibid., 201: “Quando tamen bonitatem seu bonam famam pro suae intentionis fundamento allegat, tunc illa ei necessario probanda est … ” 67. Ibid., 135. b 66. Ibid., 201: “Quando tamen bonitatem seu bonam famam pro suae intentionis fundamento allegat, tunc illa ei necessario probanda est … ” 67. Ibid., 135. 67. Ibid., 135. 68. See above, note 25. 69. Alciato, De praesumptionibus, 140-141: “Quilibet praesumitur prius res curare proprias, quam alienas … Ubi praesumitur peius in alium committere, qui in seipsum malum audet. Et ista nota contra eos, qui accipiunt factores seu institores qui consumpserunt propria bona: quia facilius consument aliena … Et pro hoc etiam facit, quia qui suis in rebus consilio indiget, multo magis praesumitur in alienis rebus indigere.” 70. Notes Ibid., 108: “Sensus & ratio naturalis praesumuntur in quolibet homine, nisi probetur contrarium.” 37. Ibid., 108: “Sensus & ratio naturalis praesumuntur in quolibet homine, nisi probetur contrarium.” 108: “Sensus & ratio naturalis praesumuntur in qu 38. Ibid, 142: “praesumitur libido diminuere in senectute … Et generaliter, secundum aetates praesumuntur ea quae naturaliter insunt: unde senex praesumitur de facili moriturus … Potes dicere, quod senectus proprie loquendo non sit morbus: sed probata senectute, resultat praesumtio iuris, quod sit aeger … Item senex non ita de facili praesumitur fugiturus sicut iuvenis … ” 38. Ibid, 142: “praesumitur libido diminuere in senectute … Et generaliter, secundum aetates praesumuntur ea quae naturaliter insunt: unde senex praesumitur de facili moriturus … Potes dicere, quod senectus proprie loquendo non sit morbus: sed probata senectute, resultat praesumtio iuris, quod sit aeger … Item senex non ita de facili praesumitur fugiturus sicut iuvenis … ” 38. Ibid, 142: “praesumitur libido diminuere in senectute … Et generaliter, secundum aetates praesumuntur ea quae naturaliter insunt: unde senex praesumitur de facili moriturus … Potes dicere, quod senectus proprie loquendo non sit morbus: sed probata senectute, resultat praesumtio iuris, quod sit aeger … Item senex non ita de facili praesumitur fugiturus sicut iuvenis … ” 42. Ibid., 33. HISTORY OF EUROPEAN IDEAS 503 47. Freher, Existimatio, 11: “nisi aliquo delicto existimatio contaminetur vel deleatur, semper durat, semper inest; adeo ut pro bona fama, probitate, & innocentia usque praesumatur; quia naturae conveniens, amica, & con- sentanea est … ” 47. Freher, Existimatio, 11: “nisi aliquo delicto existimatio contaminetur vel deleatur, semper durat, semper inest; adeo ut pro bona fama, probitate, & innocentia usque praesumatur; quia naturae conveniens, amica, & con- sentanea est … ” 48. Ibid., 144: “propter hoc ipsum stari codicibus mercatorum potius quam aliorum, quod mercatores sint homines probatioris ﬁdei & famae & legaliores quam caeteri.” 48. Ibid., 144: “propter hoc ipsum stari codicibus mercatorum potius quam aliorum, quod mercatores sint homines probatioris ﬁdei & famae & legaliores quam caeteri.” 48. Ibid., 144: “propter hoc ipsum stari codicibus mercatorum potius quam aliorum, quod mercatores sint homines probatioris ﬁdei & famae & legaliores quam caeteri.” g 49. Freher, Existimatio, 144: “Quae quidem ratio fundata videtur magis in eo quod ﬁeri debebat, quam quod ﬁt.” g 49. Notes Sed tantum abest, ut is eis ﬁdes exuberet, ut in eis sit multo minor, & ut sic loquar, sterilior, quam in caeteris: quippe cum dolis, frau- dibus, mendaciis, periuriis sint ante omnes dediti … ” 52. Tiraqueau, De nobilitate, fol. 212v: “Nec mercaturae laudibus aﬀert praeiudicium quod de mercatorum vitiis dictum sit. Non enim artis sunt ista, sed hominum … Nam etsi plerique eam artem exercentes, dolos, men- dacia, fradues, periuria admiscent: Ars tamen ipsa longe ab iis distat. Neque quicquam cum iis habet com- mune, si ea recte uti velint homines.” 52. Tiraqueau, De nobilitate, fol. 212v: “Nec mercaturae laudibus aﬀert praeiudicium quod de mercatorum vitiis dictum sit. Non enim artis sunt ista, sed hominum … Nam etsi plerique eam artem exercentes, dolos, men- dacia, fradues, periuria admiscent: Ars tamen ipsa longe ab iis distat. Neque quicquam cum iis habet com- mune, si ea recte uti velint homines.” 52. Tiraqueau, De nobilitate, fol. 212v: “Nec mercaturae laudibus aﬀert praeiudicium quod de mercatorum vitiis dictum sit. Non enim artis sunt ista, sed hominum … Nam etsi plerique eam artem exercentes, dolos, men- dacia, fradues, periuria admiscent: Ars tamen ipsa longe ab iis distat. Neque quicquam cum iis habet com- mune, si ea recte uti velint homines.” 53. Tiraqueau, De poenis, 258: “domus mercatorum debet esse domus veritatis, & aequitatis, ob id possimum, quod ad ﬁdem eorum communiter confugitur.” 53. Tiraqueau, De poenis, 258: “domus mercatorum debet esse domus veritatis, & aequitatis, ob id possimum, quod ad ﬁdem eorum communiter confugitur.” 53. Tiraqueau, De poenis, 258: “domus mercatorum debet esse domus veritatis, & aequitatis, ob id possimum, quod ad ﬁdem eorum communiter confugitur.” g 54. Ibid.: “cum sunt inter mercatores initas inter quos ut plurimum iudicari solet ex bono & aequo, & non de rigore.” 54. Ibid.: “cum sunt inter mercatores initas inter quos ut plurimum iudicari solet ex bono & aequo, & non de rigore.” 54. Ibid.: “cum sunt inter mercatores initas inter quos ut plurimum iudicari solet ex bono & aequo, & non de rigore.” 55. Freher, Fama, 206. 56. Cravetta, Consilia, 1: 64. 57. Ibid. 57. Ibid. 58. On this sense of verisimilitude in sixteenth-century legal theory, see Blank, “Common Usage.” 58. On this sense of verisimilitude in sixteenth-century legal theory, see Blank, “Common Usage.” 59. Disclosure statement Disclosure statement No potential conﬂict of interest was reported by the author(s). No potential conﬂict of interest was reported by the author(s). Funding This work was supported by Austrian Science Fund (FWF): [grant number M-2097]. Bibliography Adam, Melchior, Vitae Germanorum Jureconsultorum et Politicorum (Heidelberg: Rosa, 1620). Adam, Melchior, Vitae Germanorum Jureconsultorum et Politic Alciato, Andrea, De Praesumptionibus (Lyon: Iunta, 1551). Adam, Melchior, Vitae Germanorum Jureconsultorum et Politicorum (Heidelberg: Rosa, 1620) Alciato, Andrea, De Praesumptionibus (Lyon: Iunta, 1551). Baldus, de Ubaldis, In VII. VIII. IX X. & XI. partem Codicis commentaria (Venice: Iunta, 1515). Baldus, de Ubaldis, Consiliorum sive responsionum … volumen primum (Venice: Nicolinus, 1580). Blank, Andreas, “Common Usage, Presumption and Verisimilitude in Sixteenth-Century Theor Interpretation.” History of European Ideas 43 (2017): 401–415. p y f p Blank, Andreas, Arguing from Presumptions. Essays on Early Modern Ethics and Politics (Munich: Philosophia, 2019). s, Arguing from Presumptions. Essays on Early Mode Blank, Andreas. “Christoph Besold on Confederation Rights and Duties of Esteem in Diplomatic Relations.” Intellectual History Review 32 (2022): 51–70. y Blank, Andreas. “Pufendorf and Leibniz on Duties of Esteem in Diplomatic Relations.” Journal o Political Theory 18 (2022): 186–204. y Brucker, Jacob, Ehren-tempel der Deutschen Gelehrsamkeit (Augsburg: Haid, 1747). Cicero, Marcus Tullius, De legibus, ed. Karl Büchner (Turin: Mondadori, 1973). g Cicero, Marcus Tullius, De oﬃciis, ed. Karl Atzert (Leipzig: Teubner, 1963). g Cicero, Marcus Tullius, De oﬃciis, ed. Karl Atzert (Leipzig: Teubner, 1963). arcus Tullius, De oﬃciis, ed. 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De testamentaria tutela curaque electorali libera et integra (Heidelberg: Voegelin, 16 Fuchs, Peter, “Freher, Marquard.” Neue Deutsche Biographie 5 (1961): 392-393. Gaskins, Richard H., Burdens of Proof in Modern Discourse (New Haven: Yale University Press, 1992). Gaskins, Richard H., Burdens of Proof in Modern Discourse (New Haven: Yale U Giuliani, Adolfo, “Civilian Treatises on Presumption, 1580–1620.” In The Law of Presumpti Giuliani, Adolfo, “Civilian Treatises on Presumption, 1580–1620.” In The Law of Presumptions. Notes Ibid.: “inde infertur, quod minor contrahens mutuum cum foeneratore, praesumitur laesus, nisi probetur fac- tus locupletior … [E]tiam venditio quae pupillum laedit, praesumitur commento mendacio facta.” 76. Menochio, De praesumptionibus, 2: 314 504 A. BLANK 504 A. BLANK 504 77. Ibid., 2: 858: “nec rationi conveniat naturali, nec aliquo modo verisimile sit, ut tutores … in confectione inuen- tarii bonorum pupilli … describi patiantur propria bona inter illa pupilli … ” 77. Ibid., 2: 858: “nec rationi conveniat naturali, nec aliquo modo verisimile sit, ut tutores … in confectione inuen- tarii bonorum pupilli … describi patiantur propria bona inter illa pupilli … ” 78. Ibid., 1: 415-416: “Recepta & vera est interpretum sententia, tutori permissum quidem esse, palam suo cum pupillo contrahere, clam vero non esse … quia contrahendo palam cessat fraudis suspicio … ” 79 Ibid 1 416 78. Ibid., 1: 415-416: “Recepta & vera est interpretum sententia, tutori permissum quidem esse, palam suo cum pupillo contrahere, clam vero non esse … quia contrahendo palam cessat fraudis suspicio … ” 79. Ibid., 1: 416. 80. Alciato, De praesumptionibus, 62: praesumitur consanguinei & aﬃnes non omissuri defensionem consangui- nei sui … Ex quo sequitur, quod non praesumatur indefensus, ex quo verisimile est, quod saltem aﬃnes vel amici eum defendent … 81. Ibid., 65: non praesumitur per metum contraxisse, qui contraxit intervenientibus aﬃnibus & con … 82. Ibid., 63: “Et ex hac ratione prius etiam admitarentur ad tutelam.” 83. Godefroy, De tutelis, 22: “ut huiusmodi personarum consiliis vel interventu Tutores & Curatores suspecti removendi sunt: ita quod eorum consilio factum, gestum esse probabitur, bona ﬁde & sine calumnia factum fuisse praesumendum fuerit.” p 84. Ibid., 22-23: “alioqui Principes ob amicitiam jura transgredi nunquam praesumuntur; quia ei quem diligunt, aliter prodesse possunt, unde & certiorem gratiam & veriorem gloriam adsequantur.” 85. Freher, De testamentaria tutela, 77-78: “Idque sive ob praesumptam agnatorum ﬁdem & aﬀectionem, sive etiam quod hoc quidquid est honoris, coniunctioni & necessitudini sanguinis deberetur. Et facile servarunt hoc prepon jam ab omni antiquitate ipsi Palatini in familia sua, neque extraneos aut etiam gradu remotiores in invidiam & contemptum propinquorum unquam admiserunt … ” On the notion of to prepon in ancient rhetoric, see Poulakos, “Toward a Sophistic Deﬁnition of Rhetoric.” Bibliography Essays in Comparative Legal History, ed. Richard H. Helmholz (Berlin: Duncker & Humblot, 2009), 21–72. Godefroy Denis De tutelis electoralibus testamentariis ([Heidelberg]: Voegelin 1611) Giuliani, Adolfo, Civilian Treatises on Presumption, 1580 1620. In The Law of Presumptions. Essays in Comparative Legal History, ed. Richard H. Helmholz (Berlin: Duncker & Humblot, 2009), 21–72. Godefroy, Denis. De tutelis electoralibus testamentariis ([Heidelberg]: Voegelin, 1611). Comparative Legal History, ed. Richard H. Helmholz (Berlin: Duncker & Humblot, 2009), 21–72 Godefroy, Denis. De tutelis electoralibus testamentariis ([Heidelberg]: Voegelin, 1611). HISTORY OF EUROPEAN IDEAS 505 Kelley, Donald, Foundations of Historical Scholarship: Language, Law and History in the French Renaissance (Princeton: Princeton University Press, 1970). y Kornexl, Dietrich. Studien zu Marquard Freher (1565-1614): Leben, Werke und gelehrtengeschichtliche Bedeutung (Dissertation, University of Freiburg, 1966). Kriechbaum, Maximiliane. “Verba und mens in den Interpretationslehren des Humanismus,” in Theorie der Interpretation vom Humanismus bis zur Romantik. Rechtswissenschaft, Philosophie, Theologie, edited by Jan Schröder (Stuttgart: Steiner, 2001), 47-74. g Kühlmann, Wilhelm, Die deutschen Humanisten: Dokumente zur Überlieferung der antiken und mittelalterlichen Literatur in der Frühen Neuzeit. Vol. 1,1: Marquard Freher (Turnhout: Brepols, 2005). Maclean, Ian, Interpretation and Meaning in the Renaissance. 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https://openalex.org/W3094903907	https://run.unl.pt/bitstream/10362/115251/1/energies_13_05811.pdf	English	null	Implementation Framework for Energy Flexibility Technologies in Alkmaar and Évora	Energies	2,020	cc-by	10,762	Implementation Framework for Energy Flexibility Technologies in Alkmaar and Évora Nienke Maas 1,* , Vasiliki Georgiadou 1 , Stephanie Roelofs 1 , Rui Amaral Lopes 2 , Anabela Pronto 2 and João Martins 2 1 TNO—Netherlands Organization of Applied Scientiﬁc Research, NL-2595 DA, 96800 The Hague, The Netherlands; vasiliki.geogiadou@tno.nl (V.G.); stephanie.roelofs@tno.nl (S.R.) 2 UNINOVA—Instituto Desenvolvimento de Novas Tecnologias, Faculdade De Ciências E Tecnologia, 2829-517 Caparica, Portugal; rm.lopes@fct.unl.pt (R.A.L.); amg1@fct.unl.pt (A.P.); jf.martins@fct.unl.pt (J.M.) * Correspondence: Nienke.maas@tno.nl; Tel.: +31-646847246 1 TNO—Netherlands Organization of Applied Scientiﬁc Research, NL-2595 DA, 96800 The Hague, The Netherlands; vasiliki.geogiadou@tno.nl (V.G.); stephanie.roelofs@tno.nl (S.R.) 2 UNINOVA—Instituto Desenvolvimento de Novas Tecnologias, Faculdade De Ciências E Tecnologia, 2829-517 Caparica, Portugal; rm.lopes@fct.unl.pt (R.A.L.); amg1@fct.unl.pt (A.P.); jf.martins@fct.unl.pt (J.M.) * Correspondence: Nienke.maas@tno.nl; Tel.: +31-646847246 Received: 30 September 2020; Accepted: 4 November 2020; Published: 6 November 2020 Received: 30 September 2020; Accepted: 4 November 2020; Published: 6 November 2020 Abstract: As energy generation based on renewable resources does not always match energy consumption proﬁles, Positive Energy Districts (PEDs) should embody energy ﬂexibility technologies to decrease possible negative impacts on existing grids due to, e.g., reverse power ﬂows. As part of the EU H2020 Smart Cities and Communities project POCITYF, the cities Alkmaar (NL) and Évora (PT) aim to support the deployment and market uptake of such districts and in doing so demonstrate innovative and integrated technologies to enable ﬂexibility in the energy system. This paper addresses implementation conditions for energy ﬂexibility technologies that help cities to engender the expected impact and ensure replication of these technologies to other sites. It aims to guide both urban planners and technology solution providers through pitfalls and opportunities that can appear during the design and implementation of PEDs. Taking this into consideration, the RUGGEDISED innovation and implementation framework for smart city technology was taken as a starting point to describe and analyze the experiences in Alkmaar and Évora. Keywords: governance; energy flexibility; positive energy districts; sustainable energy; smart city deployment energies energies 1. Introduction The need for cities to become more sustainable is high and several deﬁnitions of smart cities refer to this need. A deﬁnition of a smart city is “a sustainable and eﬃcient City with high Quality of life that aims to address Urban challenges (improve mobility, optimize use of resources, improve Health and safety, improve social development, support economic growth and participatory governance) by application of ICT in its infrastructure and services, collaboration between its key stakeholders (Citizens, Universities, Government, Industry), integration of its main domains (environment, mobility, governance, community, industry, and services), and investment in Social capital” [1]. However, retroﬁtting existing environments remains challenging [2]. Positive Energy Districts (PEDs) play an important role in more liveable and sustainable future cities. PEDs are districts producing energy from local and distributed renewable energy sources, presenting generation surplus over a speciﬁc balance period (typically one year) that may be transferred to areas outside a PED’s boundaries [3]. One deﬁnition of PEDs by JPI Urban Europe (2019) refers explicitly to the active management of energy ﬂows: “PEDs are energy-eﬃcient and energy-ﬂexible urban areas or groups of connected buildings which produce net zero greenhouse gas emissions and actively manage an annual local or regional surplus production of renewable energy” [4]. Energies 2020, 13, 5811; doi:10.3390/en13215811 www.mdpi.com/journal/energies www.mdpi.com/journal/energies Energies 2020, 13, 5811 2 of 16 The large deployment of distributed generation based on renewable energy (RE) can increase the complexity of grid management and operation due to several factors (see, for instance, the impact of reverse power ﬂows on distribution transformer aging [5]. Increased energy ﬂexibility in the existing energy systems is therefore a crucial mechanism to delay costly and overdesigned adaptation of the grid infrastructure itself [6]. In this context, technology can be of great help in linking resource eﬃciency and ﬂexibility in energy supply and demand with innovative, inclusive and more cost-eﬀective services for citizens and businesses. Such technologies can integrate infrastructures like smart grids that have been piloted in several cities in FP7 and H2020 projects [7]. In Alkmaar (Netherlands) and Évora (Portugal), innovative and integrated technical solutions are being implemented in order to support the deployment and market uptake of PEDs. This research and the pilots in Alkmaar and Évora are being conducted within the EU H2020 Smart Cities and Communities project POCITYF [8]. 1. Introduction The project started in October 2019 and includes the demonstration of solutions of a high technology readiness level (TRL ≥6) [9] for achieving ﬂexible and eﬃcient use of electricity in contexts with diﬀerent climatic conditions and regional characteristics (technical, ﬁnancial, social and legal). Alkmaar and Évora are proving grounds for innovative and integrated technical solutions for buildings and districts in which energy management systems are implemented to increase ﬂexibility. Starting points are lessons learnt from pre-pilots at other sites, i.e., locations and buildings where individual technologies have been implemented before. The second step, which is dedicated to demonstration activities, combines technologies toward integrated systems at the building, block and district levels in the areas of Alkmaar and Évora. The third step refers to replication of these integrated systems in other selected areas of Alkmaar and Évora and in the six fellow cities: Granada (Spain), Bari (Italy), Celje (Slovenia), Újpest in Budapest (Hungary), Ioannina (Greece) and Hvidovre (Denmark). Starting from the RUGGEDISED innovation and implementation framework developed in the EU H2020 RUGGEDISED project [10], this paper addresses implementation conditions for energy ﬂexibility technologies in Alkmaar and Évora in order to support the achievement of expected impacts and ensure replication of these technologies within and beyond POCITYF. The technologies under consideration are ReFlex [11] for Alkmaar and ﬂexibility control algorithms for Évora. Both aim at exploiting the energy ﬂexibility provided by the available controllable devices (e.g., batteries or electric water heaters) at the building, block and district levels in order to achieve speciﬁc objectives (e.g., improve matching between renewable generation and energy demand or decrease peak loads). The RUGGEDISED innovation and implementation framework is an analytical tool that helps city planners to assess important success factors in the implementation process of smart technologies well in advance. This framework focusses on smart city technologies in a broader sense without concentrating necessarily on energy ﬂexibility technologies alone. Therefore, this paper aims to guide both urban planners and technology solution providers through pitfalls and opportunities that can appear during the design and implementation of a PED. It describes practical examples of implementation conditions seen or experienced in Alkmaar and Évora (or that were missing). This can be used to describe valuable lessons for the implementation of energy ﬂexibility in future PEDs. 1. Introduction This framework identiﬁes both the implementation conditions that were of relevance and inﬂuence in either case of the two cities, and those conditions that did not play a role. In doing so, it provides insights in improving and adjusting projects and shifts the focus on conditions that matter. This paper discusses the application of the framework by presenting how it is being used in practice for the analysis of the implementation of PEDs in Alkmaar and Évora. 2. Materials and Methods The RUGGEDISED framework [10] can be used to assess the implementation process of smart city projects and therefore “advises” what should be in place for successful implementation. In POCITYF, records of progress on pre-pilots and foreseen demonstration activities in Alkmaar and Évora have been taken, using questionnaires. These questionnaires focused on key technical components 3 of 16 Energies 2020, 13, 5811 and speciﬁcations of the innovative solutions, on the demonstration sites (considering speciﬁc challenges related to the context), on problems and restrictions experienced and on lessons learnt (e.g., technical improvements, energy savings or socioeconomic beneﬁts). The project partners involved with each innovative solution took care of the respective questionnaires. These project partners work for diﬀerent organizations, e.g., research institutes, governments, utilities and technology providers. Despite common challenges, the cities of Alkmaar and Évora face city- and district-speciﬁc challenges due to divergent geography, geology, demography, climate and socio-economic and cultural characteristics. These characteristics mean that urban energy transition challenges are embarked upon from diﬀerent starting points and perspectives, thus enhancing the complementarity of the POCITYF solutions. Évora represents South European cities, which generally show lower investments on reducing the footprint of their households and business sector but can enjoy an abundant solar potential. Alkmaar represents West European cities that are strongly dependent on gas for electricity and heating. g In more detail, the RUGGEDISED framework provides a useful base for analyzing success factors and hindering factors for the implementation of smart technologies. In POCITYF, innovative smart city solutions are implemented in order to achieve PEDs. This framework (see Table 1) was consolidated in the EU H2020 RUGGEDISED project and allows the analysis of suppressing and enhancing factors in implementation processes of smart solutions. Such an analysis is beneﬁcial in designing successful implementation processes, in assessing the potential project impact and in selecting aspects that need further consideration for successful implementation. Table 1. RUGGEDISED innovation and implementation framework: enhancing and suppressing factors [10]. 2. Materials and Methods Energies 2020, 13, 5811 4 of 16 The implementation factors inﬂuencing the development of smart solutions are described per impact level. A division is made between hardware, software and orgware implementation factors. Hardware focuses on physical infrastructure topics such as energy storage, conversion and savings. Software refers to ICT, data-related factors and applications. Hardware and software must be supplemented with orgware, which refers to organizational and governance aspects such as stakeholder management, institutional and organizational arrangements and innovation platforms. Dividing impact levels and implementation factors in three categories allows for structuring the diﬀerent factors that inﬂuence projects in diﬀerent stages. Some factors help or hinder the realization and output of smart solutions, whilst others speciﬁcally impact the working together of multiple solutions to realize embedded outcomes. The framework shows that the importance of orgware implementation factors increases as the impact level rises. These orgware implementation factors, in turn, aﬀect the upscaling and replication of smart solutions. 2. Materials and Methods Hardware Software Orgware Level of impact 1: Realization and output of smart solutions Pre-deployment assessment Software Business models Technology assessment Privacy Data and data ownership Impact on energy grid Security Smart Grid ICT User Interfaces Level of impact 2: Embedded outcomes of multiple smart solutions Communicating infrastructure Interoperability Dashboards Integrated vision on the smart city Robustness of the system Smart governance Windows of opportunity Stakeholder management Ownership Business models and split incentives Level of impact 3: Upscaling and replication Integrated planning Innovation platforms Conditions for upscaling: ﬁnance, regulation (including standardization), access to information and social aspects Table 1. RUGGEDISED innovation and implementation framework: enhancing and suppressing factors [10]. The framework distinguishes three levels of impact that are needed in order to think beyond implementation of single solutions and consider the real impact of implementation, namely: • Impact Level 1 (Realization and output of smart solutions): the ﬁrst level of impact considers a single smart solution that is successfully implemented and delivers its output (it is an isolated realization of a smart solution). • Impact Level 1 (Realization and output of smart solutions): the ﬁrst level of impact considers a single smart solution that is successfully implemented and delivers its output (it is an isolated realization of a smart solution). • Impact Level 2 (Embedded outcomes of multiple smart solutions): A smart solution produces real output if it is well-embedded in the existing context. At the second level of impact, multiple smart solutions interact and produce outcomes because they are well-connected and efficiently work together. • Impact Level 2 (Embedded outcomes of multiple smart solutions): A smart solution produces real output if it is well-embedded in the existing context. At the second level of impact, multiple smart solutions interact and produce outcomes because they are well-connected and efficiently work together. • Impact Level 3 (Upscaling and replication): The third impact level is city-level outcomes. This occurs when smart solutions are taken beyond single projects and are successfully scaled-up to create smart urban structures. Real impact is made when smart solutions are replicated to other areas and projects with diﬀerent contexts. • Impact Level 3 (Upscaling and replication): The third impact level is city-level outcomes. This occurs when smart solutions are taken beyond single projects and are successfully scaled-up to create smart urban structures. Real impact is made when smart solutions are replicated to other areas and projects with diﬀerent contexts. 3. Results One of the objectives of the POCITYF project is to deploy and validate smart energy management and storage solutions to optimize energy ﬂows with the goal of maximizing self-consumption, reducing grid stress and valorizing ﬂexibility services. To that end, innovative solutions to be demonstrated and replicated include several individual elements: e.g., low-temperature waste heat, innovative short- and long-term storage solutions, such as hydrogen fuel cells or electrical vehicles (EVs) coupled with stationary batteries, smart ICT solutions to interconnect the energy management system at the household, building and district levels, the virtual power plant (VPP) concept, thermal grid controllers or market-oriented building ﬂexibility services. Figure 1 illustrates how the individual elements corresponding to the various layers, from concrete, device-speciﬁc to abstract, domain-speciﬁc, are stacked on top of each other and interact to bring about the full implementation and impact of an integrated innovative solution. On the left side, the data and control ﬂow is bidirectional for the full stacked solution to perform as per design, while on the right side, there is only sharing of information to gain insight into the system, evaluate diﬀerent options and decide for further optimization and improvement steps. The loop is closed by feeding these insights back to the operational system in place. Energies 2020, 13, x FOR PEER REVIEW 5 of 15 Figure 1. From the concrete, device-specific layer to the abstract, domain-specific one. Figure 1. From the concrete, device-speciﬁc layer to the abstract, domain-speciﬁc one. Figure 1. From the concrete, device-specific layer to the abstract, domain-specific one. Figure 1. From the concrete, device-speciﬁc layer to the abstract, domain-speciﬁc one. 5 of 16 Energies 2020, 13, 5811 Energies 2020, 13, 5811 Taking this into consideration, Tables 2 and 3 map the information associated with the technologies under analysis (i.e., ReFlex for Alkmaar and ﬂexibility control algorithms for Évora) based on the impact levels and implementation factors that comprise the RUGGEDISED framework described in Section 2. Table 2. Mapping Alkmaar. Implementation Factor Explanation Level 1 realization and output Hardware Pre-deployment assessment • Availability of multiple assets that may be a source of ﬂexibility. • Consideration of the objective to apply solutions that expose and valorize ﬂexibility and potential alternatives. • Feasibility study of physical implementation (all components) as modeled in silico. 3. Results Technology assessment • The battery systems placed in residences are by default connected to a diﬀerent phase of the 3-phase grid than both the solar panels and household energy usage. This originates from standards used in the installation sector. This means that the battery system cannot be used eﬀectively to balance energy within the residence. • Technical readiness level of components must be improved in order to use small-scale storage solutions (through VPP or vehicle-to-grid (V2G)) in a reliable way to control vital infrastructure, especially in brown ﬁeld situations. Improvements in the European and international standards can ensure compatibility of the components in all situations. Impact on energy grid • Potential network constraints due to excessive ﬂexibility operation within the system have been identiﬁed. Analysis of the implemented scenarios was carried out in which each ﬂexibility source was individually considered; it became clear that they can be used for network congestion management up to a certain limitation. It was concluded that operation of ﬂexibility and the diﬀerent forecasting systems can also cause a congestion in the network which changes the network operation. Next to this, aggregator strategies further impact the network operation. • Considering each ﬂexibility source in relation to network limitations, especially taking into account balancing concerns and guarantee of supply. • Good understanding of the layout of the low-voltage (LV) grid is a prerequisite for implementing smartiﬁcation eﬃciently. Without these accurate data, one cannot determine where to place the measurement equipment in the grid. State estimation is needed in order to be able to draw conclusions about the current situation of the grid. • VPP in trade mode at times might have a negative eﬀect on grid balance given the current pricing mechanism. There are instances where energy is sold, and thus delivered to the grid, during moments of local energy generation. This can increase the peak load on the low-voltage grid in the districts. • By using the VPP for energy trading, based on forecasts of energy consumption, PV generation, the energy prices on the trading market and the availability of the battery, overall energy costs can be decreased. A low density of VPP contributors in the LV grid (less than 5% of residences were equipped with battery systems) showed little impact on the grid. 3. Results The density of the VPP contributors should be increased to notice the eﬀect on the grid and to have accurate predictability of average household behavior. • The distribution system operator (DSO) can alleviate peak loads on the grid by using small storage (battery) systems. These batteries need to be operated in an inherently grid-supporting mode, and peak load shaving requires fast and reliable control mechanisms. From a business perspective, adequate ﬁnancial stimuli are needed. The DSO should have a mandate to overrule the settings of the VPP to ensure grid stability and prevent a power outage. Table 2. Mapping Alkmaar. • The battery systems placed in residences are by default connected to a diﬀerent phase of the 3-phase grid than both the solar panels and household energy usage. This originates from standards used in the installation sector. This means that the battery system cannot be used eﬀectively to balance energy within the residence. • The battery systems placed in residences are by default connected to a diﬀerent phase of the 3-phase grid than both the solar panels and household energy usage. This originates from standards used in the installation sector. This means that the battery system cannot be used eﬀectively to balance energy within the residence. • Technical readiness level of components must be improved in order to use small-scale storage solutions (through VPP or vehicle-to-grid (V2G)) in a reliable way to control vital infrastructure, especially in brown ﬁeld situations. Improvements in the European and international standards can ensure compatibility of the components in all situations. • Potential network constraints due to excessive ﬂexibility operation within the system have been identiﬁed. Analysis of the implemented scenarios was carried out in which each ﬂexibility source was individually considered; it became clear that they can be used for network congestion management up to a certain limitation. It was concluded that operation of ﬂexibility and the diﬀerent forecasting systems can also cause a congestion in the network which changes the network operation. Next to this, aggregator strategies further impact the network operation. • Potential network constraints due to excessive ﬂexibility operation within the system have been identiﬁed. Analysis of the implemented scenarios was carried out in which each ﬂexibility source was individually considered; it became clear that they can be used for network congestion management up to a certain limitation. 3. Results It was concluded that operation of ﬂexibility and the diﬀerent forecasting systems can also cause a congestion in the network which changes the network operation. Next to this, aggregator strategies further impact the network operation. p • Considering each ﬂexibility source in relation to network limitations, especially taking into account balancing concerns and guarantee of supply. • Good understanding of the layout of the low-voltage (LV) grid is a prerequisite for implementing smartiﬁcation eﬃciently. Without these accurate data, one cannot determine where to place the measurement equipment in the grid. State estimation is needed in order to be able to draw conclusions about the current situation of the grid. • VPP in trade mode at times might have a negative eﬀect on grid balance given the current pricing mechanism. There are instances where energy is sold, and thus delivered to the grid, during moments of local energy generation. This can increase the peak load on the low-voltage grid in the districts. • By using the VPP for energy trading, based on forecasts of energy consumption, PV generation, the energy prices on the trading market and the availability of the battery, overall energy costs can be decreased. A low density of VPP contributors in the LV grid (less than 5% of residences were equipped with battery systems) showed little impact on the grid. The density of the VPP contributors should be increased to notice the eﬀect on the grid and to have accurate predictability of average household behavior. • The distribution system operator (DSO) can alleviate peak loads on the grid by using small storage (battery) systems. These batteries need to be operated in an inherently grid-supporting mode, and peak load shaving requires fast and reliable control mechanisms. From a business perspective, adequate ﬁnancial stimuli are needed. The DSO should have a mandate to overrule the settings of the VPP to ensure grid stability and prevent a power outage. 6 of 16 Energies 2020, 13, 5811 Table 2. Cont. Table 2. Cont. Implementation Factor Explanation Level 1 realization and output Software Privacy • Including privacy by design in order to meet both national and European standards and adhere to GDPR guidelines. In doing so, privacy concerns will not pose obstacles to implementation. Security • Taking cyber security issues into account as they may arise. 3. Results Including privacy and security by design so that they will not become an obstacle for implementation and the implementation will meet the required national and European regulations and standards. It can be helpful to follow the eight principles of privacy by design [12]. Smart grid ICT • Internet of things software from ICT.eu links all electricity consumers per platform. The data are made accessible via the cloud, giving the energy supplier remote control. Via this platform, everything can be connected per household that uses or generates energy. User interfaces • Using future-proof standards (even with low maturity) to implement new use cases and future business models. Orgware Business models • Cost reduction of grid operations as a result of unlocking and exploiting ﬂexibility. • New revenue streams potential from valorizing ﬂexibility. • Analysis of pricing mechanisms and market liquidity has been carried out as well as a usability analysis. • Bundling multiple smaller devices to increase accessibility to the trade market. Data and data ownership • Handled according to the principle “as open as possible, as closed as necessary”. Level 2 embedded outcomes of multiple smart solutions Hardware Communicating infrastructure • Continuous internet connection in order to communicate in real time between assets, the network and the energy market. Robustness of the system • Fast response to disconnection. • Backup facility. • Risk management: what goes wrong if the system is down or unavailable? Taking measures appropriate to the risks (system overload or just some missing data). • The placement of bidirectional charging stations is currently not a standard job. They are still far more expensive than standard charging stations, they need a reliable internet connection to function and during the City-zen project, problems with the hardware were encountered frequently. • The reliability of OT/IT connections through 4G and Wi-Fi communication networks used for VPP and V2G applications is lower than that of the grid itself. The reliability needs to increase to match the reliability of the grid before a VPP or V2G can be used as part of the vital infrastructure balancing loads on the LV grid. Table 2. Cont. Implementation Factor Explanation Level 1 realization and output Software Privacy • Including privacy by design in order to meet both national and European standards and adhere to GDPR guidelines. In doing so, privacy concerns will not pose obstacles to implementation. 3. Results Security • Taking cyber security issues into account as they may arise. Including privacy and security by design so that they will not become an obstacle for implementation and the implementation will meet the required national and European regulations and standards. It can be helpful to follow the eight principles of privacy by design [12]. Smart grid ICT • Internet of things software from ICT.eu links all electricity consumers per platform. The data are made accessible via the cloud, giving the energy supplier remote control. Via this platform, everything can be connected per household that uses or generates energy. User interfaces • Using future-proof standards (even with low maturity) to implement new use cases and future business models. Orgware Business models • Cost reduction of grid operations as a result of unlocking and exploiting ﬂexibility. • New revenue streams potential from valorizing ﬂexibility. • Analysis of pricing mechanisms and market liquidity has been carried out as well as a usability analysis. • Bundling multiple smaller devices to increase accessibility to the trade market. Data and data ownership • Handled according to the principle “as open as possible, as closed as necessary”. Level 2 embedded outcomes of multiple smart solutions Hardware Communicating infrastructure • Continuous internet connection in order to communicate in real time between assets, the network and the energy market. Robustness of the system • Fast response to disconnection. • Backup facility. • Risk management: what goes wrong if the system is down or unavailable? Taking measures appropriate to the risks (system overload or just some missing data). • The placement of bidirectional charging stations is currently not a standard job. They are still far more expensive than standard charging stations, they need a reliable internet connection to function and during the City-zen project, problems with the hardware were encountered frequently. • The reliability of OT/IT connections through 4G and Wi-Fi communication networks used for VPP and V2G applications is lower than that of the grid itself. The reliability needs to increase to match the reliability of the grid before a VPP or V2G can be used as part of the vital infrastructure balancing loads on the LV grid. 7 of 16 Energies 2020, 13, 5811 Table 2. Cont. Table 2. Cont. 3. Results Implementation Factor Explanation Software Interoperability • ReFlex is compatible with several open smart grid standards such as EFI/S2, USEF and OCPI and is easy to implement in an aggregator’s existing architecture. Still, bidirectional translation of asset protocols to EFI is a necessary conﬁguration step to enable reliable communication. • Reliance on standards instead of proprietary protocols to allow interoperability is paramount. • The maturity of standards for interoperability is low; as such, in every single project, protocols and interfaces need to be (re)determined between diﬀerent suppliers of technology. Dashboards • Management and administration dashboards are included in ReFlex. Orgware Integrated vision on the smart city • A local needs assessment showed that good accessibility with a balanced mobility system and a healthy, clean, safe, economic future-resilient, green and climate-adaptive city where everyone can happily live, work and recreate were important. • Flexibility solutions can help the DSO in operating the network and prevent investments in grid infrastructure. Smart governance • Insights gained regarding both the ﬂexibility potential and its value within the selected pilot areas can further support decision making and planning. Windows of opportunity • Implementation of ReFlex instead of investments in grid reinforcement: awareness of DSO that avoiding grid reinforcements is in their own interests and that ReFlex is valuable for network operation so that diﬀerent problems and interests of stakeholders are aligned. • The higher the number of ﬂexible assets, including EVs, the more value can be extracted through ReFlex deployment. • Local communities can become sustainable by being as self-proﬁcient as possible while monitoring energy use at the individual and district levels and using this information for behavior change. The business district of Boekelemeer could initiate a Citizen Energy Community (CEC) or a Renewable Energy Community (REC). According to the EU directive [13], this could improve their position in the local energy market. They can use this institutional framework to represent interest of the local community in the park management. Stakeholder management • A guiding role for the municipality in building the relationship between stakeholders. • Involvement of and willingness to engage from residents is crucial in relation to user acceptance. Support of homeowners is needed but they lack information and knowledge on energy ﬂexibility as well as on practical consequences and potential beneﬁts. • Diﬀerent reward mechanisms for VPP participants should be tested to determine the ﬁnancial and general economic value of these systems. 3. Results Ownership • Multiple aggregators in order to guarantee freedom of choice for citizens (selling energy ﬂexibility) and the DSO (congestion management). • Citizen (local) communities, municipality and housing corporations can have ownership of the applications and should be determined per case. Business models and split incentives • Analysis of market liquidity and pricing mechanisms. • Management and administration dashboards are included in ReFlex. • A local needs assessment showed that good accessibility with a balanced mobility system and a healthy, clean, safe, economic future-resilient, green and climate-adaptive city where everyone can happily live, work and recreate were important. • A local needs assessment showed that good accessibility with a balanced mobility system and a healthy, clean, safe, economic future-resilient, green and climate-adaptive city where everyone can happily live, work and recreate were important. p • Flexibility solutions can help the DSO in operating the network and prevent investments in grid infrastructure. p • Flexibility solutions can help the DSO in operating the network and prevent investments in grid infrastructure. • Insights gained regarding both the ﬂexibility potential and its value within the selected pilot areas can further support decision making and planning. • Implementation of ReFlex instead of investments in grid reinforcement: awareness of DSO that avoiding grid reinforcements is in their own interests and that ReFlex is valuable for network operation so that diﬀerent problems and interests of stakeholders are aligned. • The higher the number of ﬂexible assets, including EVs, the more value can be extracted through ReFlex deployment. • Local communities can become sustainable by being as self-proﬁcient as possible while monitoring energy use at the individual and district levels and using this information for behavior change. The business district of Boekelemeer could initiate a Citizen Energy Community (CEC) or a Renewable Energy Community (REC). According to the EU directive [13], this could improve their position in the local energy market. They can use this institutional framework to represent interest of the local community in the park management. • Involvement of and willingness to engage from residents is crucial in relation to user acceptance. Support of homeowners is needed but they lack information and knowledge on energy ﬂexibility as well as on practical consequences and potential beneﬁts. p • Diﬀerent reward mechanisms for VPP participants should be tested to determine the ﬁnancial and general economic value of these systems. 3. Results Due to current regulations and sector standards, certain capabilities of both the VPP and V2G system cannot be utilized. Examples are the use of the V2G system for emergency power and the self-consumption of energy using the VPP by connecting the battery and solar panels of a household to the same phase. • Implementation of a variable tariﬀsystem in the NL in which energy taxes are coupled to the energy price and network tariﬀ. Changing the Dutch taxation tariﬀsystem can pave the way for using (local) ﬂexibility and prevent more grid reinvestments than necessary. This can be accomplished with, e.g., variable network tariﬀs. Coupling the energy tax not to the energy amount but the price of energy and network tariﬀs can have positive eﬀects. Further research is needed in order to determine which tariﬀsystem is best applicable. Social aspects: • Finding and retaining participants for VPP and V2G projects is a time-consuming task. Active participation and a substantial time investment are required which is not always possible or desired. In addition, participants sometimes move or retract from the project due to life events. The promoted deﬁnitions of CEC and REC from the EU Green Energy Package can provide a much-needed boost on this area. • Aggregator strategies can potentially greatly impact upon the energy system since their objective of pursuing economic maximization of their assets may not always align with optimization of operations for said assets; for example, stationery batteries or EV operation may be masked by the aggregator whose portfolio includes such assets by revealing only the worst case of the day instead of their real-time state. Nonetheless, it also opens the door for other aggregators to dive in with their ﬂexibilities. This might result in a game theory problem where in reality the aggregators would self-regulate themselves. Since there is no ﬂexibility market, the single aggregator in one network can act as a monopolist. p • Certain capabilities of both the VPP and V2G system cannot be utilized due to regulatory barriers, sector standards or both. See also below. • Certain capabilities of both the VPP and V2G system cannot be utilized due to regulatory barriers, sector standards or both. See also below. Regulation and standardization: Regulation and standardization: • Similarly, regulation must be adapted to enable better use of the current and future electricity system possibilities such as use of ﬂexibility and congestion management. 3. Results • Multiple aggregators in order to guarantee freedom of choice for citizens (selling energy ﬂexibility) and the DSO (congestion management). • Citizen (local) communities, municipality and housing corporations can have ownership of the applications and should be determined per case. • Multiple aggregators in order to guarantee freedom of choice for citizens (selling energy ﬂexibility) and the DSO (congestion management). • Citizen (local) communities, municipality and housing corporations can have ownership of the applications and should be determined per case. 8 of 16 Energies 2020, 13, 5811 Table 2. Cont. Table 2. Cont. Implementation Factor Explanation Level 3 upscaling and replication Orgware Integrated planning • It is important that processes continue to be managed and it is essential that the involved key ﬁgures remain available at crucial positions. • There was no technical supplier as a consortium partner or as oﬃcial project partner. It is recommended to seek active collaboration with a supplier of technical components. Innovation platforms Important element to create a successful local innovation ecosystem is a problem-solving environment providing structure and practices of innovation management, training and tools as well as ﬁnancial resources for innovation. In Alkmaar, this has been initiated but from an external perspective not well anchored in the city organization. Conditions for upscaling: ﬁnance, regulation (including standardization), access to information and social aspects Market development: • Aggregator strategies can potentially greatly impact upon the energy system since their objective of pursuing economic maximization of their assets may not always align with optimization of operations for said assets; for example, stationery batteries or EV operation may be masked by the aggregator whose portfolio includes such assets by revealing only the worst case of the day instead of their real-time state. Nonetheless, it also opens the door for other aggregators to dive in with their ﬂexibilities. This might result in a game theory problem where in reality the aggregators would self-regulate themselves. Since there is no ﬂexibility market, the single aggregator in one network can act as a monopolist. • Certain capabilities of both the VPP and V2G system cannot be utilized due to regulatory barriers, sector standards or both. See also below. Regulation and standardization: • Similarly, regulation must be adapted to enable better use of the current and future electricity system possibilities such as use of ﬂexibility and congestion management. 3. Results Due to current regulations and sector standards, certain capabilities of both the VPP and V2G system cannot be utilized. Examples are the use of the V2G system for emergency power and the self-consumption of energy using the VPP by connecting the battery and solar panels of a household to the same phase. • Implementation of a variable tariﬀsystem in the NL in which energy taxes are coupled to the energy price and network tariﬀ. Changing the Dutch taxation tariﬀsystem can pave the way for using (local) ﬂexibility and prevent more grid reinvestments than necessary. This can be accomplished with, e.g., variable network tariﬀs. Coupling the energy tax not to the energy amount but the price of energy and network tariﬀs can have positive eﬀects. Further research is needed in order to determine which tariﬀsystem is best applicable. • Finding and retaining participants for VPP and V2G projects is a time-consuming task. Active participation and a substantial time investment are required which is not always possible or desired. In addition, participants sometimes move or retract from the project due to life events. The promoted deﬁnitions of CEC and REC from the EU Green Energy Package can provide a much-needed boost on this area. 9 of 16 Energies 2020, 13, 5811 Table 3. Mapping Évora. Table 3. Mapping Évora. Implementation Factor Explanation Level 1 realization and output Hardware Pre-deployment assessment • Deﬁnition of objectives for the ﬂexibility control algorithms. • Characterization of controllable devices providing energy ﬂexibility. • Preliminary tests using software tools. Technology assessment • Flexibility control algorithms were developed based on knowledge collected during the participation on the working group “Annex 67” of the Energy in Buildings and Communities (EBC) program from the International Energy Agency (IEA). • The deployment of ﬂexibility control algorithms has been considered to (i) improve PV self-consumption and buildings’ self- suﬃciency; (ii) explore the existing energy ﬂexibility available in water heaters to decrease electricity costs; or (iii) characterize and use of the energy ﬂexibility provided by water pumping and storage systems to reduce electricity costs and support power systems. Impact on energy grid • The impact related to the deployment of ﬂexibility control algorithms on existing grids should be considered before the respective real-world implementations. • This impact assessment should consider diﬀerent scenarios (e.g., seasons of the year) and can be conducted using simulation tools and speciﬁc information about the grids under consideration. Data and data ownership • In the pre-pilot, data are private but available for authorized third parties during the assessment phase. 3. Results • The acquisition of information about the grids under consideration can be challenging if the operator is not included in the process. Software Privacy • Data privacy issues in relation to the framework for data exchange and related roles and responsibilities were not considered in the pre-pilots of ﬂexibility control algorithms, however technical issues supporting the exchange of data in a secure and interoperable manner are easy to integrate, respecting Portuguese law. Security • Data security in relation to the framework for data exchange and related roles and responsibilities were not considered in the pre-pilots, however technical issues supporting the exchange of data in a secure and interoperable manner are easy to integrate. Smart grid ICT • All ﬂexibility-related data are available on the cloud and can be shared with authorized third parties. User interfaces • User interfaces were developed for the pre-pilot installations and they can be easily integrated with high-level SCADA (or similar) solutions. Orgware Business models • Depending on the objectives of the considered DSM measures, the ﬂexibility control algorithms have resulted in PV self-consumption improvements up to 30% or in peak load reduction up to 10%. • Flexibility control algorithms have also been used to take advantage of time-of-use tariﬀs in order to shift energy consumption to less expensive periods, resulting in savings up to 40%. • New revenue schemes based on ﬂexibility usage provide grid operation cost reduction. Data and data ownership • In the pre-pilot, data are private but available for authorized third parties during the assessment phase. 10 of 16 Energies 2020, 13, 5811 Table 3. Cont. Implementation Factor Explanation Level 2 embedded outcomes of multiple smart solutions Hardware Communicating infrastructure • As a software solution, ﬂexibility control algorithms will not have a physical presence in the Positive Energy Blocks (PEBs). This solution will run in servers located at partners’ facilities, communicating with the devices installed at the demonstration sites. Robustness of the system • Servers will have a backup, in case of hardware failure. • On-site devices will have a distributed software solution in order to increase the robustness of the overall solution. • Backup procedures will be considered. 4. Analysis The RUGGEDISED framework allows for conducting an analysis of important implementation conditions in the cities of Alkmaar and Évora. This section presents the respective main observations. • Energy ﬂexibility management relies on both energy ﬂexibility characterization and its respective use. This means that the energy ﬂexibility system is based on two main steps. First, it is necessary to get insights (mainly related to ﬂexibility characterization) on the ﬂexibility usage potential and application. Secondly, this ﬂexibility potential will be applied while managing and controlling the energy system. • Complex energy services like peer-to-peer (P2P) energy trading and VPP, management aggregation services and an energy ﬂexibility marketplace are built upon diﬀerent individual components. These lower layer(s) components must be functioning and deliver their output in order to provide, possibly stacked, smart (energy) services. As such, Level 1 functioning of such individual components is a prerequisite for smart energy services to exist and be deployed. • P2P platforms are deemed important from the abstract scenario perspective. However, development mostly starts from a technical solution perspective, while end-user and other stakeholder requirements are paramount in development. Citizen perspectives are often overlooked. Citizens mostly experienced P2P as a burden or a complex, technical solution. According to Alkmaar’s experience, relevant stakeholders were not interested, and they lacked knowledge and information, while developers assumed citizens were eager for these technologies. Therefore, engaging all stakeholders and especially end-users already from the conceptualization phase of energy flexibility systems will benefit end-user satisfaction. To achieve this, it is important to bring developers and end-users together so that requirements and needs of the local community are considered and in the long term to ensure widespread and sustainable uptake. Most devices cannot provide flexibility without the involvement of users. Therefore, they are crucial in generating flexibility in energy systems. It is undeniable that the perspective of end-users is important in every phase from conceptualization to development, deployment and operations. • Transition from Level 1 to Level 2 is normally funded by subsidies. However, subsidized projects in which energy ﬂexibility technologies are piloted have diﬀerent timeframes that do not always match with the daily business of industrial and business partners. Next to this, the research or development objectives of subsidized energy projects do not per se align with business goals • Transition from Level 1 to Level 2 is normally funded by subsidies. 3. Results Table 3. Cont. Implementation Factor Explanation Level 3 upscaling and replication Orgware Integrated planning • High-level analysis will be needed in order to choose the best locations for upscaling the installation of controllable devices, combined with global ﬂexibility control algorithms. Innovation platforms • Demonstration partners will have preferential access to all developments/features of the innovative elements and the opportunity to shape them according to their needs. Conditions for upscaling: ﬁnance, regulation (including standardization), access to information and social aspects • Mobilization of (and networking with) key stakeholders to create strong links and foster engagement of the diﬀerent target groups at the local, national and international levels, which also represent the enablers for further upscaling and replication of the POCITYF solutions. • Adapt regulations and market conditions in order to fully use the potential of ﬂexibility, providing market-oriented building ﬂexibility services. • Find attractive ways of engaging citizens. 3. Results Software Interoperability • Besides the building level control of the available energy ﬂexibility, ﬂexibility control algorithms can be used at district level to support the coordinated operation of other technologies providing energy ﬂexibility to achieve building-level objectives while targeting improvements at the district level (e.g., peak load reduction). • The deployment of the ﬂexibility control algorithms requires the interaction with existing controllable devices to perform the required monitoring and control activities. In POCITYF, this interaction will be supported by APIs made available by the controllable device itself (e.g., direct interaction with energy routers [14] or by other intermediate systems (e.g., indirect interaction with 2nd life batteries through energy management systems). • Usage of standard protocols. Dashboards • Dashboards will be provided by outside solutions that oﬀer a graphical interface for the users. Foreseen devices include: energy routers, 2nd life batteries, electrical water heaters or freezing storage systems. Orgware Integrated vision on the smart city • An assessment at district level should be carried out to deﬁne higher-level objectives to be considered by the ﬂexibility control algorithms in order to support the achievement of a speciﬁc smart city vision. Smart governance • Smart governance would increase the usage of ﬂexibility control algorithms at the municipality scale (and citizen engagement would be enlarged) in order to increase sustainability and climate change mitigation. Windows of opportunity • Deployment of ﬂexibility control algorithms (considering the energy ﬂexibility provided by, e.g., existing residential batteries and other controllable devices such as electric water heaters) instead of investments in grid reinforcement. Stakeholder management • Local communities’ engagement will be a key element in the deployment and proper operation of ﬂexibility control algorithms. Ownership • Residents and municipality will have access to the systems on their premises. Business models and split incentives • Speciﬁc business models and incentives should be developed to accommodate potential conﬂicts between building- and district-level objectives. • Besides the building level control of the available energy ﬂexibility, ﬂexibility control algorithms can be used at district level to support the coordinated operation of other technologies providing energy ﬂexibility to achieve building-level objectives while targeting improvements at the district level (e.g., peak load reduction). • Dashboards will be provided by outside solutions that oﬀer a graphical interface for the users. Foreseen devices include: energy routers, 2nd life batteries, electrical water heaters or freezing storage systems. 11 of 16 Energies 2020, 13, 5811 Table 3. Cont. 4. Analysis However, subsidized projects in which energy ﬂexibility technologies are piloted have diﬀerent timeframes that do not always match with the daily business of industrial and business partners. Next to this, the research or development objectives of subsidized energy projects do not per se align with business goals 12 of 16 12 of 16 Energies 2020, 13, 5811 (in time, budget and purpose). This results in the delivery of ﬂexibility technologies that function (Level 1) but will not necessarily graduate to achieve Level 2 and Level 3 impact; as such, upscaling and replication are not induced. Single ﬂexibility technologies are delivered but this does not result in energy ﬂexibility as a whole or ﬂexibility in smart city systems. At the community or neighbourhood level, subsidies are eﬀective in reaching goals, but in order to achieve large-scale impact (Level 2 and Level 3), combined strategies and widespread collaboration are needed. (in time, budget and purpose). This results in the delivery of ﬂexibility technologies that function (Level 1) but will not necessarily graduate to achieve Level 2 and Level 3 impact; as such, upscaling and replication are not induced. Single ﬂexibility technologies are delivered but this does not result in energy ﬂexibility as a whole or ﬂexibility in smart city systems. At the community or neighbourhood level, subsidies are eﬀective in reaching goals, but in order to achieve large-scale impact (Level 2 and Level 3), combined strategies and widespread collaboration are needed. • Energy ﬂexibility usage at higher levels should take into consideration the control objectives of previous levels. This is the case when the energy ﬂexibility of a speciﬁc device is being used at Level 1 to, e.g., reduce electricity costs and then it is used to, e.g., reduce electricity consumption CO2-related emissions at Level 2. Therefore, the considered business models should properly address the transition to upper limits in terms of objectives of energy ﬂexibility use. Following the previous comment, one can infer that each level may have diﬀerent objectives which may not only be disconnected from each other but also in some cases directly conﬂicting. Business models need to be designed in order to address the diﬀerent strategies that can be applied to the same individual devices at diﬀerent levels. • User engagement is key in exploiting the energy ﬂexibility provided by several types of controllable devices as the comfort needs should always be respected. 4. Analysis Additionally, the characterization of the energy ﬂexibility provided by some devices (e.g., white appliances) might require the direct user interaction to deﬁne the respective operation boundaries. This constitutes a constraint to be considered at all levels. • It is of upmost importance to regard investments in ﬂexibility usage as win–win situations. For eﬀective upscaling to take place, all involved stakeholders (e.g., DSO, municipalities, end-users) must have a clear idea about the advantages of investing in ﬂexibility. • It is of upmost importance to regard investments in ﬂexibility usage as win–win situations. For eﬀective upscaling to take place, all involved stakeholders (e.g., DSO, municipalities, end-users) must have a clear idea about the advantages of investing in ﬂexibility. • There is no possibility to have accurate modeling without data, and data cannot be used if privacy and security issues are not considered. Agile data management should be considered in order to take the best proﬁt from the considered technologies. A good example is the smart metering data, where often end-users do not take advantage of their full potential. 5. Conclusions A feedback 13 of 16 Energies 2020, 13, 5811 loop enables deeper understanding of the sources of error, that can either be found in the control (use) or in the characterization function. For acceptance of energy ﬂexibility, it is important to close this loop. Insights delivered via tracking key performance indicators (KPIs) are needed both on rational arguments (economic and energy metrics) and on perception arguments (e.g., satisfaction of citizens). • A legal framework will have to be updated to realize the full-scale potential of ﬂexibility, because the current legal framework does not allow for using and implementing ﬂexibility. We may apply it as a local, pilot-based tool. However, if one wishes to exploit its potential in full, a consistent and encompassing legal framework is necessary. • A legal framework will have to be updated to realize the full-scale potential of ﬂexibility, because the current legal framework does not allow for using and implementing ﬂexibility. We may apply it as a local, pilot-based tool. However, if one wishes to exploit its potential in full, a consistent and encompassing legal framework is necessary. • In order to implement energy ﬂexibility systems in the built environment and to eventually scale up at district and city levels, gaps between daily practice and subsidized projects should be closed. Goals of all partners must coincide with the aims of pilot projects. This is crucial for realizing Level 3 outcomes but also for Level 2. • The absence of an adequate legal framework is a very important issue and not only for speciﬁc energy ﬂexibility technologies. Systemic hiccups introduced by a lack of appropriate or mature legal instruments should be overcome beyond pilot project boundaries which in any case are funded by subsidies whose power to inﬂuence directly relevant legislation is limited (both in scope and time). The experiences of Alkmaar and Évora result in an easy-to-use methodology for cities to deal with energy ﬂexibility technologies and create optimal conditions for their successful implementation and integration. In the coming period, demonstration of the related energy ﬂexibility technologies is being carried out in both cities at the block and district levels. Starting from the analysis, progress is to be tracked especially focused on achieving Level 1 and moving forward to higher impact levels. 5. Conclusions This eﬀort will also support the replication process not only in other areas within Alkmaar and Évora but also the so-called “fellow” cities participating in the POCITYF project. 5. Conclusions In this paper, the RUGGEDISED framework was applied as the starting point to evaluate the implementation conditions of energy ﬂexibility solutions within the POCITYF project. Indeed, the selected framework can be leveraged to provide a ﬁrst analysis of both the success and hindering factors for the implementation of smart technologies while taking a holistic approach that includes not only technological perspectives but also organizational and systemic ones. The main recommendations and ﬁndings for implementing energy ﬂexibility-related technologies at the PED level are summarized below. • Advanced innovative services and new business models, such as P2P trading and VPP, are quite complex technologies and procedures. They can only be implemented at Level 1 if the individual necessary components such as storage devices (for example, batteries) have passed Level 1. • Advanced innovative services and new business models, such as P2P trading and VPP, are quite complex technologies and procedures. They can only be implemented at Level 1 if the individual necessary components such as storage devices (for example, batteries) have passed Level 1. • Services like P2P can only be introduced when user needs have been considered and user requirements are met. Therefore, involving end-users in the development of the services is key to reach goals and ensure continuous uptake from a bigger group of clients. It is the developer’s responsibility to consult end-users and the entire stakeholder value chain. • Services like P2P can only be introduced when user needs have been considered and user requirements are met. Therefore, involving end-users in the development of the services is key to reach goals and ensure continuous uptake from a bigger group of clients. It is the developer’s responsibility to consult end-users and the entire stakeholder value chain. • The two main functions of the energy ﬂexibility system must be connected. Data insights will have to feed back into the management and control process. The results of the control process will have to be monitored and provide insight on the impact of the introduced control mechanism. Without this feedback loop, the installed energy ﬂexibility system will not be able to deliver the potential optimal ﬂexibility. Ergo, if there is impact, it is by coincidence and therefore it cannot be sustained in the long term. Feedback can change the characterization and the use. 6. Practitioners Review by Roel Massink MSc The authors of this paper asked me to provide my view on the methods and results of this research paper in my capacity as Project Coordinator for the H2020 Smart City Lighthouse project IRIS and innovation manager for the Municipality of Utrecht. I am happy to fulﬁl this task and I thank the authors for the carefully presented research results based on actual demonstration. This review is based on the experiences collected in the IRIS Smart Cities project, a similar project to RUGGEDISED and POCITYF. In IRIS, an integrated project approach is demonstrated: in the Kanaleneiland district (Utrecht, The Netherlands), near zero-energy-eﬃcient building retroﬁtting is connected to the development of a smart energy and mobility system integrating PV panels with stationary and V2G storage. The smart solutions in IRIS Smart Cities Utrecht mostly all fall within Level 2 of the RUGGEDISED implementation framework. Below, some general reﬂections based on these experiences toward the RUGGEDISED implementation framework and the conclusions of the paper can be found. The structure of the RUGGEDISED framework is very much in line with what is happening in practice in the IRIS Smart Cities project and further development of the framework in practice is appreciated. Within the IRIS Smart Cities project, the smart solutions are implemented along transition tracks and structured through a maturity assessment in the following categories: (1) pre-pilots: solutions that have been tested on a small scale in a pilot project; (2) integrated solutions: multiple smart solutions that are demonstrated as integrated solutions at a larger scale at the demonstration site of the project; and (3) replicated solutions: through either the copying of a successful integrated solution to other sites or by upscaling of the integrated solution in the same city or region. This categorization provides the IRIS partners and stakeholders a framework and a roadmap for all smart solutions to be developed, demonstrated and scaled-up. The RUGGEDISED framework is valuable and will 14 of 16 14 of 16 Energies 2020, 13, 5811 oﬀer practitioners at public authorities, grid operators, solution providers and knowledge institutes a coherent overview of what is important for implementation. However, below I have some suggestions in view of the practical applicability of the RUGGEDISED framework. First, smart solutions are integrated solutions by nature. 6. Practitioners Review by Roel Massink MSc To ensure that the smart solutions (that are realized in isolation; Impact Level 1) meet the requirements of an embedded outcome of multiple smart solutions (Impact Level 2), it is suggested that an integrated approach with all implementation factors is taken into account. To ensure such an integrated (and systemic) assessment throughout the development of smart solutions, an iterative execution of the implementation framework could be proposed. This means that at Level 1, the implementation factors of Level 2 and Level 3 are also considered. Assessment of implementation factors in Level 2 and Level 3 includes more detailed and speciﬁc information once the development of smart solutions progresses. The following three reﬂections are supportive of including higher-level implementation factors at the start of the pilot projects. The paper highlights the importance of aligning partner goals with pilot project aims. This should be seen as a conditional factor for achieving progress at the diﬀerent impact levels. An example from IRIS Smart Cities Utrecht is the development of a bidirectional charging ecosystem for grid ﬂexibility and mobility services. Currently, this integrated solution consists of multiple smart solutions (bidirectional charging infrastructure, bidirectional enabled electric vehicles, stationary battery storage, energy management system) that are being connected to provide embedded outcomes (Level 2). The current state of development is a result of careful stakeholder management from the start of the pilot projects (already pre-IRIS). Companies, knowledge institutes, grid operators and public authorities aligned their roadmaps and activities in pilot projects under the lead of an ambitious SME (Level 1) and now this is leading to embedded outcomes in the IRIS demonstration project (Level 2). Furthermore, scaled adoption in public procurement documents of electric charging infrastructure now paves the way for city-wide ﬂexibility services of electric vehicles (Level 3). An important success factor was/is stakeholder alignment from the start of the pilot projects. Based on this experience, it is argued that stakeholder management (or rather stakeholder alignment) should take position already at Level 1 to ensure that partner roadmaps are aligned to move the smart solution to Level 2 and 3. The paper also points towards the requirement of end-user involvement (or co-creation) in the development of smart solutions and could argue for the inclusion of a new implementation factor in the RUGGEDISED framework. This could be “end-user satisfaction” or similar. 6. Practitioners Review by Roel Massink MSc This is a valid point; often smart solutions are hampered because end-user needs were not considered well enough in the original approach. A design-thinking (or another systemic) approach could oﬀer smart solution developers tools to better involve end-users. A practical notion that requires attention here is the use of grant subsidies in this innovation framework (as the authors also refer to in moving from Level 1 to Level 2). It is recommended that subsidy programs, grant applicants and consortia put more attention into making end-user involvement more explicit in call texts and subsequently also allow more ﬂexibility in the implementation of the grant project based on changes oﬀered by end-users. Next to this, the paper explains the requirement of a legal framework that supports the transition from pilot projects to scaled solutions. This is true as well for the IRIS Smart Cities project. Especially within the ﬁeld of smart energy projects or services like peer-to-peer, a supportive legal framework is needed for scaled adoption. The exploitation of grid ﬂexibility services is hampered by legal constraints. Research from the IRIS project shows that bidirectional charging services are discouraged because of double energy taxation (for each charging and discharging cycle, energy tax needs to be paid on either the stored or consumed kWh). This signiﬁcantly hampers the realization of proﬁtable business models and commercial scaling of these integrated smart solutions. Therefore, it could be argued that regulation/the legal framework is already introduced as an implementation factor in Level 2 of the framework to ensure that, in time, legal constraints are targeted in a concerted action by smart cities. Energies 2020, 13, 5811 15 of 16 Finally, what the paper does not explain but what is highly valuable for practitioners is guidance on how the implementation framework leads into an implementation process. The presented implementation framework provides an assessment of implementation factors related to smart solutions but does not directly translate the assessment into implementation guidelines Further guidance on the implementation how the implementation framework leads into an implementation process. The presented implementation framework provides an assessment of implementation factors related to smart solutions but does not directly translate the assessment into implementation guidelines. Further guidance on the implementation pathway resulting from the assessment could support practitioners in applying this framework more easily. Author Contributions: Conceptualization, N.M., V.G. and S.R.; methodology, N.M. and S.R.; validation, N.M., R.A.L. 6. Practitioners Review by Roel Massink MSc and J.M.; formal analysis, N.M., V.G., S.R., R.A.L. and J.M.; investigation, N.M., V.G., S.R., R.A.L., A.P. and J.M.; resources, S.R. and R.A.L.; writing—original draft preparation, N.M., V.G., S.R., R.A.L., A.P. and J.M.; writing—review and editing, N.M., V.G., S.R., R.A.L. and J.M.; visualization, V.G.; supervision, N.M. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by POCITYF, (FP7 grant agreement N◦864400). And The APC was funded by EERA Joint Program Smart Cities. Acknowledgments: This paper is based on information collected during the initial phase of EU H2020 Smart Cities and Communities project POCITYF (grant agreement N◦864400). The authors would like to express their gratitude to Roel Massink MSc, coordinator of the H2020 Smart City Lighthouse project IRIS, for reviewing the methods and results of this research and for all the suggestions provided. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. References 1. Mosannenzadeh, F.; Vettorato, D. Deﬁning smart city. A conceptual framework based on keyword analysis. TeMA-J. Land Use Mobil. Environ. 2014. [CrossRef] . Dixon, T.; Eames, M. Scaling up: The challenges of urban retroﬁt. Build. Res. Inf. 2013, 41, 499–503. [Cross g p g f 3. Shnapp, S.; Paci, D.; Bertoldi, P. 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https://openalex.org/W3085177178	https://journal.uinjkt.ac.id/index.php/edusains/article/download/11034/pdf	Indonesian	null	WEB MODUL IPA BERBASIS INKUIRI TERBIMBING UNTUK MENINGKATKAN THINKING SKILL	EDUSAINS	2,020	cc-by-sa	3,614	Tersedia online di EDUSAINS Website: http://journal.uinjkt.ac.id/index.php/edusains EDUSAINS,12(1), 2020, 47-53 Research Artikel WEB MODUL IPA BERBASIS INKUIRI TERBIMBING UNTUK MENINGKATKAN THINKING SKILL SCIENCE WEB MODULE BASED ON GUIDED INQUIRY TO IMPROVE THINKING SKILL Agnesi Sekarsari Putri, Nurfina Aznam Universitas Negeri Yogyakarta, Indonesia agnesi.sekar@gmail.com, nurfina_aznam@uny.ac.id Agnesi Sekarsari Putri, Nurfina Aznam Universitas Negeri Yogyakarta, Indonesia agnesi.sekar@gmail.com, nurfina_aznam@uny.ac.id Abstract The science web module is a systematically structured teaching material to achieve electronic learning objectives using website applications in which writing, animation, video, and navigation are exciting, interactive, active, and motivating learning that can facilitate students' thinking skills. Guided inquiry is a model that trains students to be actively involved in investigations with questions raised by teachers to direct students to find concepts. Guided inquiry-based web modules are used to improve students' thinking skills measured through written tests. This study uses a pretest-posttest control group design. Based on the results of the analysis using the Kruskal Wallis test, there are differences in the experimental class using the science web module based on guided inquiry and the control class using the science teaching materials commonly used by teachers. The results showed an increase in higher thinking skills in the experimental class than in the control class, resulting in the high effect size category so that the experimental class increased the thinking skills more effectively than the control class. Keywords: science web module; guided inquiry; thinking skill. nding author EDUSAINS, p-ISSN 1979-7281 e-ISSN 2443-1281 This is an open access article under CC-BY-SA license (https://creativecommons.org/licenses/by-sa/4.0/) PENDAHULUAN Abad 21 dikenal sebagai abad perkembangan ilmu pengetahuan dan teknologi yang berkembang pesat. Permasalahan yang terjadi pada abad ini hanya bisa diselesaikan dengan mengasah dan menguasai ilmu pengetahuan. Ilmu pengetahuan dapat ditingkatkan melalui suatu pendidikan (Smaldino, 2011). Paradigma pembelajaran abad 21 menuntut perubahan pendidikan agar dapat menghasilkan sumber daya manusia yang unggul. Oleh karena itu, pendidikan harus mampu mengembangkan potensi peserta didik agar peserta didik memiliki bekal memadai untuk dapat mengatasi tantangan dan persaingan dalam dunia global (Lee et al, 2014). Partnership for 21st Century Learning (P21) mengembangkan kerangka kerja pendidikan abad 21 di seluruh dunia. Salah satu keterampilan abad 21 dalam kerangka kerja tersebut keterampilan berpikir (thinking skill). Thinking skill merupakan kunci dalam pembelajaran untuk menghasilkan sumber daya manusia yang unggul dan kompetensi penting yang dibutuhkan dalam mengatasi persaingan dunia global (Smit, 2015). Model inkuiri terbimbing adalah model pembelajaran yang mendeskripsikan fenomena, mengidentifikasi masalah, merumuskan pemecahan masalah, mengkomunikasikan ide-ide ke orang lain, mengembangkan kreativitas dalam memilih solusi terbaik pemecahan masalah, mengembangkan pemahaman sains dengan menggabungkan pengetahuan ilmiah dengan penalaran dan keterampilan berpikir (Llewellyn, 2011). National Academy of Science (2000) menyatakan karakteristik model inkuiri yaitu peserta didik aktif terlibat dalam aktivitas hands-on and minds-on, tugas guru memilih masalah yang dilontarkan ke kelas untuk dipecahkan peserta didik dengan memberikan pertanyaan yang mengarahkan pada penemuan konsep dan pemecahan masalah serta menyediakan sumber belajar dalam rangka pemecahan masalah. Selain model pembelajaran, guru membutuhkan bahan ajar. Bahan ajar digunakan untuk menunjang proses pembelajaran serta mencapai kompetensi dasar (Susilowati, 2015). Hasil observasi di SMP Negeri 2 Gamping, bahan ajar yang digunakan guru berupa buku teks sumbangan pemerintah, buku teks pelajaran, dan Lembar Kerja Peserta Didik (LKPD) yang dibeli melalui penyalur di sekolah. Pelaksanaan pembelajaran dilakukan dengan membaca rangkuman materi dan mengerjakan soal di LKPD dengan sesekali guru menjelaskan materi. Hal ini perlu inovatif bahan ajar agar dapat meningkatkan kualitas pembelajaran. Kusuman et al (2016) menyatakan bahwa bahan ajar yang inovatif menciptakan pembelajaran menarik, menumbuhkan minat, motivasi, mengurangi ketergantungan dan mendapatkan kemudahan dalam mempelajari setiap indikator yang terdapat pada perangkat pembelajaran yang disusun oleh guru. Thinking skill merupakan kemampuan dasar yang melatih otak untuk berpikir kritis dan logis dalam memahami informasi, menggunakan kemampuan analitis, memecahan masalah, serta meningkatkan kemampuan pengambilan keputusan dengan tepat (Prajapati et al, 2017; Salonen et al, 2017; Butterworth & Thwaites, 2013). EDUSAINS. Volume 12 Nomor 01 Tahun 2020, 48-53 This is an open access article under CC-BY-SA license (https://creativecommons.org/licenses/by-sa/4.0/) Abstrak Web modul IPA merupakan bahan ajar yang disusun sistematis untuk mencapai tujuan pembelajaran berbentuk elektronik menggunakan aplikasi website yang didalamnya terdapat tulisan, animasi, video, serta navigasi yang menjadi pembelajaran yang menarik, interaktif, aktif, dan memotivasi belajar yang dapat memfasilitasi thinking skill peserta didik. Inkuiri terbimbing merupakan model yang melatih peserta didik terlibat aktif dalam penyelidikan dengan pertanyaan-pertanyaan yang diajukan guru untuk mengarahkan peserta didik menemukan konsep. Web modul berbasos inkuiri terbimbing digunakan untuk meningkatkan thinking skill peserta didik. Penelitian ini menggunakan pretest posttest control group design. Thinking skill diukur melalui tes tertulis. Berdasarkan hasil analisis menggunakan uji Kruskal Wallis terdapat perbedaan kelas eksperimen yang menggunakan web modul IPA berbasis inkuiri terbimbing dengan kelas kontrol yang menggunakan bahan ajar IPA yang biasa digunakan guru. Hasil penelitian menunjukkan bahwa terdapat peningkatan thinking skill yang lebih tinggi pada kelas eksperimen daripada kelas kontrol, dengan hasil effect size kategori tinggi, sehingga kelas eksperimen meningkatkan thinking skill yang lebih efektif daripada kelas kontrol. Kata Kunci: web modul IPA; inkuiri terbimbing; thinking skill Permalink/DOI: http://doi.org/10.15408/es.v12i1.11034 Kata Kunci: web modul IPA; inkuiri terbimbing; thinking skill Kata Kunci: web modul IPA; inkuiri terbimbing; thinking skill Permalink/DOI: http://doi.org/10.15408/es.v12i1.11034 Corresponding author Corresponding author nding author EDUSAINS, p-ISSN 1979-7281 e-ISSN 2443-1281 This is an open access article under CC-BY-SA license (https://creativecommons.org/licenses/by-sa/4.0/) Putri, A. S., Aznam, N. Putri, A. S., Aznam, N. pembelajaran yang dapat membantu peserta didik berperan aktif dalam pembelajaran. PENDAHULUAN Keterampilan berpikir penting agar peserta didik dapat mengoptimalkan kemampuan berpikir sehingga meningkatkan kemampuan pemecahan masalah dalam kehidupan sehari-hari, menganalisa pemikiran untuk memastikan bahwa peserta didik telah membuat keputusan dengan tepat, serta dapat menjadi bekal untuk bersaing di dunia global (Smit, 2015; Salih, 2010; Roekel, 2011). Hasil observasi dan wawancara di SMP Negeri 2 Gamping, proses pembelajaran berlangsung satu arah dengan dilakukan ceramah dan diskusi. Pembelajaran kurang melibatkan keterlibatan aktif peserta didik, sehingga thinking skill peserta didik kurang terlatih dengan baik. Oleh karena itu, perlu dilakukan inovasi model Salah satu bahan ajar yang dapat digunakan yaitu web modul IPA. Modul dipilih karena memiliki karakteristik self-instruction dan self- contained sehingga peserta didik dilatih mandiri untuk mengelola waktu belajarnya dan memahami materi pelajaran (Kaur et al, 2017). Web modul berbentuk elektronik menggunakan aplikasi website Web Modul Ipa Berbasis Inkuiri Terbimbing … yang dapat digunakan pada komputer, laptop, serta smartphone yang didalamnya terdapat tulisan, animasi, video, serta navigasi (Tambunan, 2013). Penggunaan modul menjadi pembelajaran alternatif dalam rangka interaksi guru dan peserta didik dengan modul sehingga pembelajaran menjadi menarik, interaktif, aktif, dan memotivasi belajar (Johar, A., et al., 2014). Penggunaan web modul dapat membantu peserta didik untuk mengidentifikasi masalah, menganalisis informasi, merumuskan pemecahan masalah, dan berdiskusi untuk menentukan solusi terbaik dalam pemecahan masalah (Kaur, R., et al., 2017). web modul IPA untuk meningkatkan thinking skill dapat diketahui dari analisis effect size (Cohen, et al, 2007). HASIL DAN PEMBAHASAN Penelitian dilaksanakan pada kelas eksperimen dan kelas kontrol. Kelas eksperimen melaksanakan pembelajaran menggunakan web modul IPA, sedangkan kelas kontrol melaksanakan pembelajaran dengan bahan ajar IPA yang biasa digunakan guru. Pelaksanaan penelitian diawali dengan memberikan pretest dengan hasil pretest yang hampir sama. Setelah selesai pretest, peserta didik kelas eksperimen melaksanakan pembelajaran dengan web modul IPA dengan inkuiri terbimbing menggunakan laptop atau smartphone. Berdasarkan penjelasan di atas, penelitian ini bertujuan untuk melihat ada atau tidaknya peningkatan thinking skill peserta didik setelah menggunakan web modul IPA berbasis inkuiri terbimbing. Pada kelas kontrol, pelaksanaan pembelajaran dengan bahan ajar IPA yang biasa digunakan guru serta proses pembelajaran yang biasanya diberikan guru. Setelah selesai seluruh rangkaian kegiatan pembelajaran, peserta didik melaksanakan posttest untuk mengetahui sejauh mana peningkatan thinking skill peserta didik. Soal thinking skill mencakup indikator mengidentifikasi permasalahan, menganalisis informasi, merumuskan pemecahan masalah, memilih solusi terbaik pemecahan masalah (Pasific Policy Research Center, 2010; Trilling & Fadel, 2009; McGuinness, et al, 2000). Hasil thinking skill peserta didik dapat dilihat pada Tabel 4. EDUSAINS. Volume 12 Nomor 01 Tahun 2020, 49-53 This is an open access article under CC-BY-SA license (https://creativecommons.org/licenses/by-sa/4.0/) METODE Penelitian ini dilaksanakan di SMP Negeri 2 Gamping tahun akademik 2018/2019. Teknik pengambilan sampel yang digunakan adalah cluster random sampling. Masing-masing kelas diasumsikan memiliki kemampuan yang sama, karena pengelompokan kelas sudah ditetapkan oleh sekolah atas dasar pengetahuan dan jenis kelamin yang telah tersebar secara seimbang. Sampel dalam penelitian ini adalah peserta didik dari dua kelas yang digunakan yaitu VII E sebagai kelas eksperimen dan VII F sebagai kelas kontrol. Penelitian ini menggunakan pretest posttest control group design (Creswell, 2012). Tabel 4. Data Nilai Thinking Skill Deskripsi Pretest Posttest Eksperimen 25,10 80,16 Kontrol 25,15 75,31 Tabel 4. Data Nilai Thinking Skill Teknik pengumpulan data menggunakan metode tes untuk mengukur thinking skill berupa soal essay sebanyak lima butir soal. Soal thinking skill disusun berdasarkan Trilling & Fadel (2009), Pasific Policy Research Center (2010), & McGuinness et.al (2000) yaitu mengidentifikasi permasalahan, menganalisis informasi, merumuskan pemecahan masalah, memilih solusi terbaik pemecahan masalah. Tabel 4 menunjukkan bahwa nilai thinking skill kelas eksperimen lebih baik dibandingkan dengan nilai thinking skill pada kelas kontrol. Nilai thinking skill peserta didik dapat dilihat lebih detail dengan melihat perbandingan data nilai thinking skill peserta didik kelas eksperimen dan kelas kontrol setiap indikator yang dapat dilihat pada Gambar 1. Teknik analisis data yang digunakan adalah uji K-Independent Sample Test/Kruskal Wallis dengan SPSS 22 menggunakan taraf signifikansi (α) 0,05 (Stanislaus, 2006). Besarnya efektivitas Putri, A. S., Aznam, N. Gambar 1. Perbandingan Rata-rata Thinking Skill Setiap Indikator gambar, video yang diberikan oleh guru. Peserta didik menyiapkan hasil diskusi yang telah dilakukan dan mempresentasikan hasil diskusi di depan kelas. Peserta didik menanggapi kelompook yang melakukan presentasi serta guru memberi umpan balik jawaban peserta didik yang kurang tepat dengan mengajukan pertanyaan yang dapat menggiring peserta didik menjawab dengan benar. Merumuskan pemecahan masalah tersebut merupakan indikator dati thinking skill. Guru meminta peserta didik untuk berdiskusi dalam rangka memilih solusi terbaik pemecahan masalah yang merupakan indikator thinking skill. Setelah menemukan solusi terbaik, guru dan peserta didik bersama-sama membuat kesimpulan hasil kegiatan pembelajaran. Pembelajaran web modul IPA memuat langkah inkuiri terbimbing dan materi pembelajaran yang dihubungkan dengan fenomena/permasalahan sekitar peserta didik sehingga dapat melatih mengidentifikasi konsep yang berkaitan dengan fenomena di lingkungan sekitar, melatih thinking skill, tertarik pada sains, serta menumbuhkan kepedulian terhadap lingkungan (Ilmiyah et al, 2019; Lia et al, 2016; Agung, 2015). Gambar 1. Perbandingan Rata-rata Thinking Skill Setiap Indikator Gambar 1 menunjukkan bahwa rata-rata thinking skill setiap indikator pada kelas eksperimen dan kelas kontrol berbeda. EDUSAINS. Volume 12 Nomor 01 Tahun 2020, 50-53 This is an open access article under CC-BY-SA license (https://creativecommons.org/licenses/by-sa/4.0/) UCAPAN TERIMA KASIH Terimakasih kepada Kepala Sekolah, guru, dan peserta didik SMP Negeri 2 Gamping yang telah memberikan kesempatan kepada peneliti untuk melakukan penelitian tentang web modul IPA terintegrasi potensi lokal batik terhadap thinking skill peserta didik. Bagi peneliti yang akan melakukan penelitian serupa, saran yang diberikan bagi peneliti selanjutnya diharapkan dapat meneliti lebih tentang pengaruh web modul IPA berbasis inkuiri terbimbing yang berbeda serta pengaruh web modul IPA terhadap variabel yang berbeda dengan latar permasalahan yang berbeda. PENUTUP meningkatkan thinking skill peserta didik menggunakan effect size diperoleh skor sebesar 0,8 dengan kategori tinggi sehingga dapat dikatakan bahwa web modul IPA berbasis inkuiri terbimbing mempunyai efek yang tinggi untuk meningkatkan thinking skill peserta didik. Hasil penelitian ini sejalan dengan penelitian yang telah dilakukan Anafidah et.al (2017) yang menyatakan bahwa modul pembelajaran dapat melatih peserta didik untuk aktif dalam pembelajaran dan mampu meningkatkan keterampilan berpikir. Penelitian lain yang dilakukan Pistanty et.al (2015) menyatakan bahwa modul dapat meningkatkan kemampuan berpikir. Web modul IPA berbasis inkuriri terbimbing efektif meningkatkan thinking skill peserta didik karena kegiatan pembelajaran dalam web modul IPA disusun dengan langkah-langlah pembelajaran inkuiri terbimbing sehingga peserta didik lebih aktif melakukan diskusi serta terlatih untuk mengidentifikasi masalah, analisis informasi, mengidentifikasi upaya pemecahan masalah, dan menemukan solusi terbaik pemecahan masalah (Hairida, 2016; Novana, et al, 2014). Web modul IPA membantu peserta didik aktif dalam kegiatan pembelajaran, sehingga peserta didik diajak untuk bertanya, mengemukakan pendapat, mendengarkan pendapat, mengerjakan tugas, mengerjakan latihan, sehingga thinking skill dapat muncul dalam kegiatan pembelajaran (Ash-Shiddieqy, et al, 2018). Web modul IPA dapat melatih thinking skill karena pembelajaran dengan web modul IPA membantu peserta didik memperoleh pengalaman belajar dan pengetahuan untuk menemukan dan merumuskan pengetahuan melalui eksplorasi informasi terkait materi secara bersama dengan peserta didik lain (Noel, 2014; Pattiwael, 2016). Peserta didik yang menggunakan web modul IPA lebih aktif dan tertarik dalam belajar IPA daripada peserta didik yang menggunakan bahan ajar IPA yang biasa digunakan guru, sehingga hasil penelitian menunjukkan bahwa ada peningkatan thinking skill peserta didik yang lebih tinggi dengan menggunakan web modul IPA. Berdasarkan penelitian diatas maka dapat disimpulkan bahwa web modul IPA berbasis inkuiri terbimbing dapat meningkatkan thinking skill peserta didik dengan hasil uji Kruskal Wallis, diperoleh nilai Asymp. Sig. (0,000) < α (0,05) serta effect size diperoleh skor sebesar 0,8 sehingga web modul IPA berbasis inkuiri terbimbing mempunyai efek yang tinggi untuk meningkatkan thinking skill peserta didik. Hasil penelitian ini dapat dimanfaatkan guru untuk meningkatkan thinking skill peserta didik dalam pembelajaran IPA SMP. METODE Penelitian lain yang dilakukan Pistanty et.al (2015) menyatakan bahwa modul dapat meningkatkan kemampuan berpikir. Web modul IPA berbasis inkuriri terbimbing efektif meningkatkan thinking skill peserta didik karena kegiatan pembelajaran dalam web modul IPA disusun dengan langkah-langlah pembelajaran inkuiri terbimbing sehingga peserta didik lebih aktif melakukan diskusi serta terlatih untuk mengidentifikasi masalah, analisis informasi, mengidentifikasi upaya pemecahan masalah, dan menemukan solusi terbaik pemecahan masalah (Hairida, 2016; Novana, et al, 2014). Web modul IPA membantu peserta didik aktif dalam kegiatan pembelajaran, sehingga peserta didik diajak untuk bertanya, mengemukakan pendapat, mendengarkan pendapat, mengerjakan tugas, mengerjakan latihan, sehingga thinking skill dapat muncul dalam kegiatan pembelajaran (Ash-Shiddieqy, et al, 2018). Web modul IPA dapat melatih thinking skill karena pembelajaran dengan web modul IPA membantu peserta didik memperoleh pengalaman belajar dan pengetahuan untuk menemukan dan merumuskan pengetahuan melalui eksplorasi informasi terkait materi secara bersama dengan peserta didik lain (Noel, 2014; Pattiwael, 2016). Peserta didik yang menggunakan web modul IPA lebih aktif dan tertarik dalam belajar IPA daripada peserta didik yang menggunakan bahan ajar IPA yang biasa digunakan guru, sehingga hasil penelitian menunjukkan bahwa ada peningkatan thinking skill peserta didik yang lebih tinggi dengan menggunakan web modul IPA. EDUSAINS. Volume 12 Nomor 01 Tahun 2020, 51-53 This is an open access article under CC-BY-SA license (https://creativecommons.org/licenses/by-sa/4.0/) METODE Kelas eksperimen memiliki nilai setiap indikator thinking skill yang lebih tinggi daripada nilai setiap indikator thinking skill kelas kontrol. Thinking skill yang dicapai peserta didik pada kelas eksperimen lebih baik daripada kelas kontrol karena pembelajaran kelas eksperimen menggunakan web modul IPA yang disajikan dengan memuat model inkuiri terbimbing, materi pencemaran yang dihubungkan dengan limbah industri batik yang ada dilingkungan sekitar peserta didik, setiap kegiatan memuat tulisan, gambar, video, download, pertanyaan yang membantu peserta didik melakukan diskusi, soal latihan dan soal evaluasi (Setiyadi et al, 2017; Fitri et al, 2013). Hasil nilai thinking skill peserta didik dilakukan analisis lebih lanjut dengan uji K- Independent Sample Test/Kruskal Wallis dengan SPSS 22 menggunakan taraf signifikansi 0,05 yang disajikan pada Tabel 5. Indikator identifikasi masalah, pada kelas eksperimen lebih baik daripada kelas kontrol karena pada kelas eksperimen pembelajaran menggunakan web modul IPA. Pada web modul IPA disajikan gambar dan video terkait pencemaran lingkungan, peserta didik diminta untuk mengidentifikasi permasalahan terkait gambar dan video yang disajikan. Peserta didik bergabung dalam kelompok untuk melakukan pengumpulan data untuk didiskusikan dengan peserta didik lain. Guru memberikan artikel, gambar, video, serta dan pertanyaan-pertanyaan diskusi yang terdapat dalam web modul IPA dalam rangka melatih analisis informasi terkait permasalahan yang ditemukan, analisis informasi ini merupakan bagian dari indikator thinking skill. Peserta didik melakukan diskusi dan mengumpulkan informasi untuk melakukan penyelesaian masalah terkait artikel, Tabel 5. Hasil Uji Kruskal Wallis Nilai Thinking Skill Kruskal Wallis Hasil Chi-Square 13.199 Df 1 Asymp. Sig. .000 Tabel 5. Hasil Uji Kruskal Wallis Nilai Thinking Tabel 5. Hasil Uji Kruskal Wallis Nilai Thinking Tabel 5 menunjukkan bahwa hasil uji Kruskal Wallis diperoleh nilai Asymp. Sig. sebesar 0,000 sehingga Asymp. Sig. < α dengan α sebesar 0,05 maka H0 ditolak. Oleh karena itu, dapat disimpulkan bahwa ada perbedaan thinking skill peserta didik yang menggunakan web modul IPA dengan thinking skill peserta didik yang menggunakan bahan ajar IPA yang biasa digunakan guru. Analisis selanjutnya yaitu untuk mengetahui besarnya efektivitas web modul IPA untuk Web Modul Ipa Berbasis Inkuiri Terbimbing … meningkatkan thinking skill peserta didik menggunakan effect size diperoleh skor sebesar 0,8 dengan kategori tinggi sehingga dapat dikatakan bahwa web modul IPA berbasis inkuiri terbimbing mempunyai efek yang tinggi untuk meningkatkan thinking skill peserta didik. Hasil penelitian ini sejalan dengan penelitian yang telah dilakukan Anafidah et.al (2017) yang menyatakan bahwa modul pembelajaran dapat melatih peserta didik untuk aktif dalam pembelajaran dan mampu meningkatkan keterampilan berpikir. DAFTAR PUSTAKA Agung, L. S. (2015). 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Addressing 21st Century Communication Skills: Some Emerging Issues From Eil Pedagody & Intercultural Communicative Competence. Indonesian Journal of English Education 3(2). Salonen, A., Hartikainen-Ahia, A., Hense, J., & Keinonen, T. (2017). Secondary school students’ perceptions of working life skills in science-related careers. International Journal of Science Education 39(10):2-7. Prajapati, R., Sharma, B., & Sharma, D. (2017). Significance of Life Skills Education. Journal Contemporary Issues in Education Research-First Quarter 10(1):1-6. Setiyadi, M. W., Ismail, & Gani, H. M. (2017). Pengembangan Modul Pembelajaran Biologi Berbasis Pendekatan Saintifik untuk Meningkatkan Hasil Belajar Siswa. Journal of Education Science and Technology 3(2):102-112. Roekel, D. V. (2011). Preparing 21st Century Students for a Global Society.USA: National Education Association. Susilowati. (2015). IPA dan Pembelajrannya. Yogyakarta: UNY Press. Stanislaus. (2006). Pedoman Analisis Data dengan SPSS. Yogyakarta: Graha Ilmu. Trilling, B., & Fadel, C. (2009). 21st Century Skills: Learning for Life in Our Times. San Francisco: Jossey-Bass. Salih, M. (2010). Developing Thinking Skills in Malaysian Science Via An Analogical Task. DAFTAR PUSTAKA Journal of Science and Mathematic 33(1):110. Tambunan, H. (2013). Pengembangan Pembelajaran Berbasis Website dalam Matakuliah Pengaturan Mesin Listrik. Jurnal Cakrawala Pendidikan 31(1):75. Smaldino, S. E. (2011). Preparing Students with 21st Century ICT Literacy in Math and EDUSAINS. Volume 12 Nomor 01 Tahun 2020, 53-53 This is an open access article under CC-BY-SA license (https://creativecommons.org/licenses/by-sa/4.0/) EDUSAINS. Volume 12 Nomor 01 Tahun 2020, 53-53 This is an open access article under CC-BY-SA license (https://creativecommons.org/licenses/by-sa/4.0/)
https://openalex.org/W4281711082	https://journal.umy.ac.id/index.php/di/article/download/7535/pdf	English	null	The Comparison between Black Cumin Extract and Betel Leaf Extract As Antifungal Potential to Candida Albicans on Acrylic Resin Denture Base	Insisiva Dental Journal	2,022	cc-by-sa	3,700	The Comparison between Black Cumin Extract and Betel Leaf Extract As Antifungal Potential to Candida Albicans on Acrylic Resin Denture Base Hastoro Pintadi, Urai Rifaldy Aryandi* Department of Prosthodontics, School of Dentistry, Faculty of Medicine and Health Sciences, Universitas Muhammadiyah Yogyakarta, Indonesia. Received date: November 27th, 2019; revised date: June 22th, 2021; accepted: January 19th, 2022 DOI: 10.18196/di.v10i1.7535 Abstract Porosity in acrylic resin denture base causes a rough surface and can absorb fluid, leading to plaque. Candida albicans can stick to a plaque on the denture base and engender denture stomatitis. Denture base submersion has to be carried out in traditional plant solutions to prevent denture stomatitis. Traditional plants such as black cumin and betel leaf are considered to have anti-fungal properties. This study aims to identify the comparison of antifungal properties between black cumin and betel leaf extract to Candida albicans on the acrylic resin denture base. This study is an experimental laboratory. Treatment group 1 used aquadest as a control negative, treatment group 2 used a black cumin extract with a concentration of 0.25%, while treatment group 3 used betel leaf extract with a concentrate of 50%. In every group, 9 acrylic resin pieces were soaked for 8 hours. The colony calculation was then conducted and analyzed using the Mann-Whitney test to compare the average difference level between the two tested groups. There was no significant difference level among a total colony of Candida albicans in the treatment group with a concentration of 0.25% black cumin and the treatment group with a 50% concentration of betel leaf. The result showed that the black cumin extract is no more effective in hindering the growth of Candida albicans on the acrylic resin denture base than betel leaf extract. Keywords: antifungal; black cumin; betel leaf; candida albicans; dentures Insisiva Dental Journal: Majalah Kedokteran Gigi Insisiva, 11(1), May 2022, 1-6 Insisiva Dental Journal: Majalah Kedokteran Gigi Insisiva, 11(1), May 2022, 1-6 1 Research Article The Comparison between Black Cumin Extract and Betel Leaf Extract As Antifungal Potential to Candida Albicans on Acrylic Resin Denture Base Hastoro Pintadi, Urai Rifaldy Aryandi* Department of Prosthodontics, School of Dentistry, Faculty of Medicine and Health Sciences, Universitas Muhammadiyah Yogyakarta, Indonesia. * Corresponding author, e-mail: filter34@gmail.com INTRODUCTION membrane mucosa of the oral cavity, causing denture stomatitis.3 Dentures are instrument imitations used to replace missing teeth and prevent negative impacts.1 Denture generally uses a polymer-based material, including polymethyl methacrylate (PMMA) or acrylic resin. Acrylic resin naturally has some weaknesses such as dimension instability, color change, and porosity that cause rough surface and liquid absorption.2 Acrylic resin absorbs water when contacting saliva and shapes plaque where microorganisms gather, such as Candida albicans. It can lead to cleaning difficulty and harm oral and dental hygiene. Plaque causes microorganisms and reacts to the Candida albicans is a commensal organism that can lead to opportunistic infection due to supportive predisposition factors. Frequent causes of local Candida albicans infection are dentures, especially poor denture cleaning procedures.6 Prevention of denture stomatitis, including infection Candida albicans, is highly essential. Denture base submersion in a cleaning solution is an effective method to prevent it. However, the cleaning solution circulating in the community, such as chlorhexidine, is less affordable. Thus, it needs affordable alternative materials for base denture cleaning. The alternative Hastoro Pintadi, Urai Rifaldy Aryandi \| The Comparison between Black Cumin Extract and Betel Leaf Extract As Antifungal Potential to Candida Albicans on Acrylic Resin Denture Base 2 Hastoro Pintadi, Urai Rifaldy Aryandi \| The Comparison between Black Cumin Extract and Betel Leaf Extract As Antifungal Potential to Candida Albicans on Acrylic Resin Denture Base 2 The acrylic resin denture base started with making a plate from modeling wax with a diameter of 10 mm and a thickness of 2 mm. The total was 27 plates using wax. The wax was used to create the unpolished plate sample of acrylic resin. material can be obtained from traditional plants that play as anti-fungal properties such as black cumin (Nigella sativa) and green betel leaves (Piper betel L.).4 The acrylic resin denture base started with making a plate from modeling wax with a diameter of 10 mm and a thickness of 2 mm. The total was 27 plates using wax. The wax was used to create the unpolished plate sample of acrylic resin. Black cumin seeds are often used as traditional medicine in the Middle East and Asian countries to handle many diseases such as microorganism infection. Black cumin contains anti-fungal properties such as thymoquinone, thymohydroquinone and thymol. INTRODUCTION Such materials are the main components of black cumin essential oil and have excellent antifungal properties in hindering Candida albicans.5 Furthermore, Saponin compounds have also been reported to have an ability as an antifungal property.2 Furthermore, the production of black cumin and green betel leaf extract was carried out using the maceration method. 600 grams of black cumin seeds and green betel leaves were cleaned and dried for 48 hours at a temperature of 45o C. The dried black cumin seed and green betel leaves were ground using a pollinator machine. The two kinds of powder were mixed with methanol and then were stirred for 30 minutes. It was left for 24 hours and filtered. The process was repeated three times. After that, the filtration was done and evaporated. The evaporation produced a thick extract. Next, the thick extract was poured into a porcelain cup and heated until the black cumin and green betel leave extract was obtained. The black cumin extract was then diluted with dimethyl sulfoxide to reach 0.25% concentration, while the green betel leave extract was diluted with dimethyl sulfoxide to reach 50% concentration. Moreover, green betel leaves are experimentally known to have various pharmacological properties, such as anti- microbial, anti-fungal, and others. 6 the antifungal property in green betel leaf is found in the hydroxychavicol compound.7 Furthermore, the green betel leaves also contain several bioactive compounds as an antifungal property, such as alkaloids and flavonoids, which can hinder the formation of pseudohyphae and disturb nucleic acid biosynthesis. Candida albicans colony was prepared by taking the Candida albicans using ose sterile and then planting it into Sabouraud ' dextrose agar. It was then incubated for 24 hours at 37o C. The suspension of Candida albicans was customized with a solution standard of McFarland. MATERIALS AND METHODS This research is an experimental laboratory study conducted in the Microbiology Faculty of Medicine and Health Sciences laboratory, Universitas Muhammadiyah Yogyakarta and Research and Testing Integrated Laboratory (LPPT) Gajah Mada University. This study employed 27 samples for 3 groups; 9 samples for each group. The treatment group utilized the acrylic resin denture base submersion in aquadest for 8 hours. Treatment group 2 utilized the acrylic resin denture base submersion in black cumin extract concentration of 0.25% for 8 hours. Meanwhile, treatment group 3 utilized the acrylic resin denture base submersion in green betel leaf extract concentration of 50% for 8 hours after being contaminated with Candida albicans for 24 hours. Acrylic resin disc with a diameter of 10 mm and a thickness of 2 mm totaling 27 pieces were soaked using aquadest for 48 hours and then sterilized using 70% alcohol. Acrylic resin disc was soaked in sterile saliva for 1 hour and then rinsed with phosphate-buffered saline (PBS) twice, respectively. It was put into a reaction tube containing Candida albicans suspension and incubated for 24 hours. It was then soaked in a reaction tube containing aquadest, black cumin extract of 0.25% concentration and green betel leave extract Insisiva Dental Journal: Majalah Kedokteran Gigi Insisiva, 11(1), May 2022, 1-6 3 that the data were normally distributed. Meanwhile, the treatment group of green betel leave extract of 50% concentration showed the significance value of 0.002 (p <0.05), indicating the data were not normally distributed. Thus, the Mann- Whitney test's non-parametric hypothesis was conducted to identify the comparison between black cumin and green betel leave extract. of 50% concentration previously containing Candida albicans suspension for 8 hours. After that, each acrylic resin disc was put into a reaction tube containing 10 ml aquadest, then was shaken with a vortex mixer for 1 minute and diluted up to 10-3. Furthermore, it was put into Sabouraud's dextrose agar and incubated for 48 hours at 37o C. The calculation of the total colony of Candida albicans was conducted using a calculator. Furthermore, the homogeneity test was conducted using Levene's test to identify whether the analyzed data were homogeneous or heterogeneous. The results showed that the total colony of Candida albicans on all three treatment groups had a significance value of 0.787 (p> 0.05), indicating that the data were homogeneous. Moreover, the non-parametric hypothesis using the Man Whitney test was conducted. RESULT The study on the comparison between black cumin and green betel leave extract as antifungal properties towards the growth of Candida albicans on the acrylic resin denture base has been carried out in the Microbiology laboratory Faculty of Medicine and Health Sciences, Universitas Muhammadiyah Yogyakarta with 27 subjects. The total and average data are as follows: The result of the Mann-Whitney test showed a significance value of 0.659 (p>0.05); thus, H0 was accepted. It can be concluded that there was no significant difference between the total colony of Candida albicans in the treatment group of black cumin extract of 0.25% concentration and the treatment group of the green betel leave extract of 50% concentration. Table 2. The results of the Mann Whitney test The Number of Candida albicans Asymp. Sig. (2-tailed) .659 Exact Sig. [2(1-tailed Sig.)] .666 b Table 2. The results of the Mann Whitney test The Number of Candida albicans Asymp. Sig. (2-tailed) .659 Exact Sig. [2(1-tailed Sig.)] .666 b Table 1. Data in the study Group n Amount Average Aquadest 9 708 78.6667 Black Cumin extract 9 595 61.1111 Green Betel Leave Extract 9 399 44.3333 Furthermore, a normality test was conducted to identify the data distribution. The total number of the data was 27 samples. Therefore, the Shapiro-Wilk test (small number of the sample) was conducted to identify whether the data distribution was normal. The Shapiro-Wilk column showed the data distribution of Candida albicans colony in aquadest and black cumin extract concentration of 0.25% revealed the significance value of 0.498 and 0.114 (p>0.05), respectively. It indicated MATERIALS AND METHODS The data were analyzed using Shapiro Wilk and Levene's test. Test data analysis using SPSS showed that distributed data were not normal and homogeneous; thus, the Mann-Whitney test was conducted. DISCUSSION The study shows no significant mean difference between the total colony of Candida albicans in the treatment group of black cumin extract and the green betel leave extract (p>0.05). Therefore, it is not in line with the hypothesis revealing that black cumin extract more effectively hinders the growth of Candida albicans on acrylic resin denture base compared to green betel leave extract. Hastoro Pintadi, Urai Rifaldy Aryandi \| The Comparison between Black Cumin Extract and Betel Leaf Extract As Antifungal Potential to Candida Albicans on Acrylic Resin Denture Base 4 Hastoro Pintadi, Urai Rifaldy Aryandi \| The Comparison between Black Cumin Extract and Betel Leaf Extract As Antifungal Potential to Candida Albicans on Acrylic Resin Denture Base 4 Hastoro Pintadi, Urai Rifaldy Aryandi \| The Comparison between Black Cumin Extract and Betel Leaf Extract As Antifungal Potential to Candida Albicans on Acrylic Resin Denture Base 4 Less effectiveness of the black cumin extract of 0.25% in hindering the growth of Candida albicans colony is influenced by the low-level concentration from the black cumin extract. It is in line with Ornay et al., 8, denoting that substance concentration influences the effectiveness of the antifungal agent. Low extract concentration results in low active ingredients working as an antifungal that can cause low antifungal ability. Less effectiveness of the black cumin extract of 0.25% in hindering the growth of Candida albicans colony is influenced by the low-level concentration from the black cumin extract. It is in line with Ornay et al., 8, denoting that substance concentration influences the effectiveness of the antifungal agent. Low extract concentration results in low active ingredients working as an antifungal that can cause low antifungal ability. environments will influence the plant response in nutrient absorption in the land as well as the bioactive production. Both statements align with research conducted by Mahfur14, revealing that black cumin originating from Indonesia did not contain essential oil, while those originating from Habasyah and India contained essential oil. In addition, this research also used green betel leaf originating from Indonesia. The betel leaves are mostly distributed in tropical and subtropical countries worldwide, including Indonesia.15 As all of the plants originated from Indonesia, they did not influence nutrient absorption and bioactive production response. Thus, it can be concluded that the different environments influence the active ingredient level of the plants. DISCUSSION This statement is in line with research conducted by Rahmawati et al.,9 revealing that black cumin extract with 10% concentration contains more fungal agents than black cumin extract with 20% concentration. Furthermore, another study conducted by Dharma and Subaryanti10 showed that on the examination of the diameter of inhibition activity, the black cumin extract with a concentration of 20% and 30% has no inhibition zone compared to the black cumin extract with concentrations of 40%, 50%, and 60%. Therefore, it can be concluded that low- level concentration of the black cumin extract results in low active ingredient as an antifungal agent. p The black cumin and green betel leave extract contain chemical compounds with the same antifungal agent, namely essential oil. The essential oil for the black cumin contains derivative compounds such as thymoquinone, thymohydroquinone, dithymoquinone, p-cymene, carvacrol, 4- terpineol, t-anethol, sesquiterpene longifolene, α-pinene, and thymol.16 Out of the 10 compounds, three compounds showed good antifungal properties, especially in hindering the growth of Candida albicans, namely thymoquinone, thymohydroquinone and thymol.5 It is in line with the study conducted by Halamova et al., 17 evaluating the antifungal activity of thymoquinone, thymohydroquinone and dithymoquinone in vitro used the microdilution method at 6 species of rotting milk yeast. It revealed that thymoquinone and thymohydroquinone showed antifungal properties that could hinder the development of Candida albicans. antifungal agent. The result of this study is not in line with research conducted by Nagham Adil Ghani11, revealing that the black cumin extract with 0.25% concentration showed the most effective ability in hindering the growth of the Candida albicans colony due to the different areas of the black cumin seeds. The difference in the effectiveness of the plants could be influenced by the origin of the plants, agro-climate factors, and phytochemical contents in the used extract.12 The research used black cumin seeds earned from Iraq, while this research used black cumin seeds from Indonesia. Furthermore, according to Dharma and Subaryanti,10 the difference in climate and land nutrition could influence active ingredient level from simplicia. Furthermore, according to Suryadi et al., 13 black cumin can grow in Jordan with a sub- tropical climate and Indonesia with a tropical climate. DISCUSSION Therefore, the different The result of this study is not in line with research conducted by Nagham Adil Ghani11, revealing that the black cumin extract with 0.25% concentration showed the most effective ability in hindering the growth of the Candida albicans colony due to the different areas of the black cumin seeds. The difference in the effectiveness of the plants could be influenced by the origin of the plants, agro-climate factors, and phytochemical contents in the used extract.12 The research used black cumin seeds earned from Iraq, while this research used black cumin seeds from Indonesia. Furthermore, according to Dharma and Subaryanti,10 the difference in climate and land nutrition could influence active ingredient level from simplicia. Furthermore, according to Suryadi et al., 13 black cumin can grow in Jordan with a sub- tropical climate and Indonesia with a tropical climate. Therefore, the different Thymoquinone is known to have an action mechanism as an antifungal activity by hindering the cell formation, destroying membrane cytoplasm, and changing the nucleus to amorphous (no shape).18 Furthermore, thymol, also known to have antifungal activity, also has an action Insisiva Dental Journal: Majalah Kedokteran Gigi Insisiva, 11(1), May 2022, 1-6 5 https://doi.org/10.1166/sam.2017.3023 4. Naeini A, Shayegh SS, Shokri H, Davati A, Khazaei A, Akbari A. In Vitro Antifungal Effect Of Herbal Mixture (Nigella Sativa, Foeniculum Vulgare And Camellia Sinensis) Against Candida Species Isolated From Denture Wearers. Journal of Herbmed Pharmacology. 2017; 6(2): 74-79. Other contents that can hinder the growth of Candida albicans include alkaloids and flavonoids. Alkaloids could hinder the growth of Candida albicans by obstructing the biosynthesis of nucleic acid. Meanwhile, flavonoids have an action mechanism by impeding pseudohyphae formation during pathogenesis. 5. Shokri H. A Review On The Inhibitory Potential Of Nigella Sativa Against Pathogenic And Toxigenic Fungi. Avicenna Journal of Phytomedicine. 2016; 6(1): 21-33. REFERENCES mechanism in hindering the growth of Candida albicans by poisoning protoplasm, destroying and penetrating the cell wall, as well as precipitating cell proteins. 9 Besides essential oil, saponin also can help hinder the growth of Candida albicans. The action mechanism of saponins works by conducting protein denaturation and breaking down the membrane cell that leads to the dead cell.2 1. Gosal AA, Siagian KV, Wowor VNS. Hubungan Kebiasaan Menyikat Gigi Dan Status Kesehatan Gingiva Pada Pengguna Gigi Tiruan Sebagian Lepasan Di Kelurahan Batu Kota. Pharmacon. 2015;4(4): 82-89. https://doi.org/10.35799/pha.4.2015.10196 2. Hanoem E, Imam B, Pranoto KP. The Effectiveness of Nigella Sativa Seed Extract In Inhibiting Candida Albicans On Heat Cured Acrylic Resin. Dental Journal (Majalah Kedokteran Gigi). 2011; 44(3): 137-140. https://doi.org/10.20473/j.djmkg.v44.i 3.p137-140 Green betel leave is also known to contain an essential oil that has a derivative compound in the form of hydroxychavicol, p-cymene, α-terpinol, terpinyl acetate, methyl eugenol, caryophyllenes, chavibetol, allylpyrocatechol diacetate, stearaldehyde, anethole, eugenol, safrole, ursolic acid, 3β-acetyl ursolic, and β- sitosterol. Hydroxychavicol is the only derivative compound of essential oil that plays a role as an antifungal agent. 7 Green betel leave is also known to contain an essential oil that has a derivative compound in the form of hydroxychavicol, p-cymene, α-terpinol, terpinyl acetate, methyl eugenol, caryophyllenes, chavibetol, allylpyrocatechol diacetate, stearaldehyde, anethole, eugenol, safrole, ursolic acid, 3β-acetyl ursolic, and β- sitosterol. Hydroxychavicol is the only derivative compound of essential oil that plays a role as an antifungal agent. 7 3. Albarrag AM, Alothman OY, Elsharawy MA, Al Rez MF, Fouad H, Hashem M, et al. Effect of Nigella Sativa Extracts on Candida Species Adhesion to Acrylic Denture Base Material And on Nanomechanical Properties of The Denture Material. Science of Advanced Materials. 2017; 9(5): 775-781. Ali et al., 19 stated that hydroxychavicol has an action mechanism in resisting Candida albicans by damaging the cell membrane structure that obstructs the permeability barrier. Furthermore, Singburaudom20 added that hydroxychavicol also has an action mechanism in hindering biofilm growth and reducing biofilm formation carried out by Candida albicans as well as preventing the formation of glucan, which is not water- soluble. Ali et al., 19 stated that hydroxychavicol has an action mechanism in resisting Candida albicans by damaging the cell membrane structure that obstructs the permeability barrier. REFERENCES Furthermore, Singburaudom20 added that hydroxychavicol also has an action mechanism in hindering biofilm growth and reducing biofilm formation carried out by Candida albicans as well as preventing the formation of glucan, which is not water- soluble. CONCLUSION 6. Bajpai V, Sharma D, Kumar B, Madhusudanan Kp. Profiling Of Piper Betle Linn. Cultivars By Direct Analysis In Real Time Mass Spectrometric Technique. Biomedical Chromatography. 2010; 24(12): 1283- 1286. Based on the results of this study, it can be concluded that the black cumin extract was not more effective in hindering the growth of Candida albicans colony on the acrylic resin denture base than the green betel leave extract. https://doi.org/10.1002/bmc.1437 Hastoro Pintadi, Urai Rifaldy Aryandi \| The Comparison between Black Cumin Extract and Betel Leaf Extract As Antifungal Potential to Candida Albicans on Acrylic Resin Denture Base 6 dengan Menggunakan Metode Kromatografi Gas. Surakarta; 2012. 7. Kumar N, Misra P, Dube A, Bhattacharya S, Dikshit M, Ranade S. Piper Betle Linn. A Maligned Pan- Asiatic Plant With An Array Of Pharmacological Activities And Prospects For Drug Discovery. Current Science. 2010; 99: 922-932. 15. Vikash C, Shalini T, Verma NK, Singh DP, Chaudhary SK, Asha R. Piper Betel: Phytochemistry, Traditional Use & Pharmalogical Activity-A Review. International Journal of Pharmaceutical Research and Development; 2012;4:216-23. 8. Ornay AKD, Prehananto H, Dewi ASS. Daya Hambat Pertumbuhan Candida Albicans Dan Daya Bunuh Candida Albicans Ekstrak Daun Kemangi (Ocimum Sanctum L.). Jurnal Wiyata. 2017; 4(1): 78-83. 16. Sultana S, Asif HM, Akhtar N, Iqbal A, Nazar H, Rehman RU. Nigella Sativa: Monograph. Journal of Pharmacognosy And Phytochemistry. 2015; 4: 103-106. 9. Rahmawati A, Al-Anwary N, Sasongkowati R. Pengaruh Pemberian Infusa Jintan Hitam (Nigella Sativa Linn) Terhadap Pertumbuhan Jamur Candida Albicans. Analis Kesehatan Sains. 2012; 1(1): 16-20. 17. Halamova K, Kokoska L, Flesar J, Sklenickova O, Svobodova B, Marsik P. In Vitro Antifungal Effect of Black Cumin Seed Quinones Against Dairy Spoilage Yeasts At Different Acidity Levels. Journal of Food Protection. 2010; 73 (12): 2291–2295. https://doi.org/10.4315/0362-028X- 73.12.2291 10. Dharma ST, Subaryanti. Uji Anti Jamur Ekstrak Biji Jintan Hitam (Nigella Sativa L.) Terhadap Candida Albicans. Sainstech Farma. 2015; 8(2): 28-32. https://doi.org/10.37277/sfj.v8i2.385 18. Işcan G, Işcan A, Demircia F. Anticandidal Effects of Thymoquinone: Mode of Action Determined By Transmission Electron Microscopy (Tem). Natural Product Communications. 2016; 11(7): 977-978. https://doi.org/10.1177/1934578X160 1100726 https://doi.org/10.37277/sfj.v8i2.385 11. Mashhadian, Naser Vahdati and Hassan Rakhshandeh. “Antibacterial and antifungal effects of Nigella sativa extracts against S. aureus, P. aeroginosa and C. albicans. Pakistan Journal of Medical Sciences;(2005); 21: 47-52. 19. Ali I, Khan FG, Suri KA, Gupta BD, Satti NK, Dutt P, et al. In Vitro Antifungal Activity of Hydroxychavicol Isolated From Piper Betle L. CONCLUSION Annals of Clinical Microbiology and Antimicrobials. 2010; 9(7): 1-9. https://doi.org/10.1186/1476-0711-9-7 12. Hasan NA, Nawahwi MZ, Malek HA. Antimicrobial Activity of Nigella Sativa Seed Extract. Sains Malaysiana. 2013; 42(2): 143-147. 13. Suryadi R, Ghulamahdi M, Kurniawati A. Respon Pertumbuhan Dan Produksi Jintan Hitam (Nigella Sativa L.) Dengan Pemupukan Nitrogen Dan Fosfor. J. Agron. Indonesia. 2015; 3(43): 227-234. https://doi.org/10.24831/jai.v43i3.11249 20. Singburaudom N. Hydroxychavicol From Piper Betel Leave Is An Antifungal Activity Against Plant Pathogenic Fungi. Jbiopest. 8(2):82- 92. 14. Mahfur. Perbandingan Profil Kromatogram Minyak Atsiri Jinten Hitam (Nigella Sativa L.) Yang Berasal Dari Habasyah, India, dan Indonesia
https://openalex.org/W4298184068	https://dspace.mit.edu/bitstream/1721.1/89530/1/Borodin-2013-Markov%20dynamics.pdf	English	null	Markov dynamics on the Thoma cone: a model of time-dependent determinantal processes with infinitely many particles	arXiv (Cornell University)	2,013	cc-by	26,522	MIT Open Access Articles The MIT Faculty has made this article openly available. Please share how this access benefits you. Your story matters. Terms of use: Creative Commons Attribution MIT Open Access Articles E l e c t r o n i c J o u r n a l o f P r o b a b i l i t y Electron. J. Probab. 18 (2013), no. 75, 1–43. ISSN: 1083-6489 DOI: 10.1214/EJP.v18-2729 Electron. J. Probab. 18 (2013), no. 75, 1–43. ISSN: 1083-6489 DOI: 10.1214/EJP.v18-2729 Electron. J. Probab. 18 (2013), no. 75, 1–43. ISSN: 1083-6489 DOI: 10.1214/EJP.v18-2729 Electron. J. Probab. 18 (2013), no. 75, 1–43. ISSN: 1083-6489 DOI: 10.1214/EJP.v18-2729 Abstract The Thoma cone is an inﬁnite-dimensional locally compact space, which is closely related to the space of extremal characters of the inﬁnite symmetric group S∞. In another context, the Thoma cone appears as the set of parameters for totally positive, upper triangular Toeplitz matrices of inﬁnite size. ′ The purpose of the paper is to construct a family {X(z,z′)} of continuous time Markov processes on the Thoma cone, depending on two continuous parameters z and z′. Our construction largely exploits speciﬁc properties of the Thoma cone re- lated to its representation-theoretic origin, although we do not use representations directly. On the other hand, we were inspired by analogies with random matrix the- ory coming from models of Markov dynamics related to orthogonal polynomial en- sembles. ( ′) We show that processes X(z,z′) possess a number of nice properties, namely: (1) every X(z,z′) is a Feller process; (2) the inﬁnitesimal generator of X(z,z′), its spec- trum, and the eigenfunctions admit an explicit description; (3) in the equilibrium regime, the ﬁnite-dimensional distributions of X(z,z′) can be interpreted as (the laws of) inﬁnite-particle systems with determinantal correlations; (4) the corresponding time-dependent correlation kernel admits an explicit expression, and its structure is similar to that of time-dependent correlation kernels appearing in random matrix theory. Keywords: determinantal processes; Feller processes; Thoma simplex; Thoma cone; Markov intertwiners; Meixner polynomials; Laguerre polynomials. Keywords: determinantal processes; Feller processes; Thoma simplex; Thoma cone; Markov intertwiners; Meixner polynomials; Laguerre polynomials. AMS MSC 2010: Primary 60J25; 60J27, Secondary 60G55; 60C05; 05E05. Submitted to EJP on April 9, 2013, ﬁnal version accepted on July 13, 2013. AMS MSC 2010: Primary 60J25; 60J27, Secondary 60G55; 60C05; 05E05. Submitted to EJP on April 9, 2013, ﬁnal version accepted on July 13, 2013. Markov dynamics on the Thoma cone: a model of time-dependent determinantal processes with inﬁnitely many particles Grigori Olshanski† Alexei Borodin∗ ∗Department of Mathematics, Massachusetts Institute of Technology, USA, and Institute for Information Transmission Problems, Moscow, Russia. E-mail: borodin@math.mit.edu. †Institute for Information Transmission Problems, Independent University of Moscow, National Research University Higher School of Economics, Moskow, Russia. E-mail: olsh2007@gmail.com. Keywords: determinantal processes; Feller processes; Thoma simplex; Thoma cone; Markov intertwiners; Meixner polynomials; Laguerre polynomials. 1.1 Preliminaries: Markov processes related to orthogonal polynomials It is well known that for each family of classical orthogonal polynomials p0, p1, p2, . . . , there exists a second order differential operator D, which preserves the space of poly- nomials and is diagonalized in the basis {pn}: Dpn = mnpn, n = 0, 1, 2, . . . , where 0 = m0 > m1 > m2 > . . . are the eigenvalues. Let W(x) be the weight function of {pn} and supp W be its support. Operator D determines a diffusion Markov pro- cess X on supp W with W(x)dx being a symmetrizing measure, hence also a stationary distribution. where 0 = m0 > m1 > m2 > . . . are the eigenvalues. Let W(x) be the weight function of {pn} and supp W be its support. Operator D determines a diffusion Markov pro- cess X on supp W with W(x)dx being a symmetrizing measure, hence also a stationary distribution. All these objects, family {pn}, operator D, and Markov process X, have multidimen- sional analogs: Namely, ﬁx N = 2, 3, . . . . From {pn} on can construct a family of symmetric polyno- mials in N variables indexed by partitions ν of length at most N, as follows: pν(x1, . . . , xN) := det[pνi+N−i(xj)] V (x1, . . . , xN) , where the determinant in the numerator is of order N and where the determinant in the numerator is of order N and V (x1, . . . , xN) := Y 1≤i<j≤N (xi −xj). These polynomials form a basis in the space of symmetric polynomials. Next, the role of D is played by the second order partial differential operator DN := 1 V (x1, . . . , xN) (Dx1 + · · · + DxN ) V (x1, . . . , xN) −constN, where Dxi denotes a copy of D acting on variable xi and constN = m0 + · · · + mN−1. Although the coefﬁcients of DN in front of the ﬁrst order derivatives have singular- ities on the diagonals xi = xj, the operator is well deﬁned on the space of symmetric polynomials and is diagonalized in the basis {pν}: DNpν = mνpν, mν := N X i=1 (mνi+N−i −mN−i). EJP 18 (2013), paper 75. 1.1 Prelimin It is well kn there exists a s nomials and is where 0 = m0 of {pn} and su cess X on supp distribution. All these ob sional analogs: Namely, ﬁx mials in N vari where the dete These polynom of D is played b DN where Dxi den Although th ities on the dia polynomials an Finally, one ﬁgurations con with density This constr polynomials ar can be found i orthogonal poly In the pres parameter fam which are rela believe that th X b Markov dynamics on the Thoma cone 1 Introduction The ﬁrst two subsections of the introduction contain short preliminary remarks and a few necessary deﬁnitions. Next we state the main results of the paper, Theorems 1.2 and 1.3. Then we describe the method of proof and make a comparison with some related works. 1.2 The inﬁnite-dimensional Laguerre differential operator and the z-measures The operator in question, denoted by D(z,z′), serves as the pre-generator of process X(z,z′). Initially, D(z,z′) is deﬁned in the algebra of symmetric functions, Sym, which replaces the algebra of N-variate symmetric polynomials. The elementary symmetric functions e1, e2, . . . are algebraically independent generators of Sym; we use them as independent variables and deﬁne D(z,z′) : Sym →Sym as a second order differential operator D(z,z′) = X n≥1 n−1 X k=0 (2n −1 −2k)e2n−1−kek ! ∂2 ∂e2n + 2 X n′>n≥1 n−1 X k=0 (n′ + n −1 −2k)en′+n−1−kek ! ∂2 ∂en′∂en + ∞ X n=1 −nen + (z −n + 1)(z′ −n + 1)en−1 ∂ ∂en (1.1) (1.1) depending symmetrically on two complex parameters z and z′. Recall that the classical Laguerre polynomials depend on a continuous parameter (the “Laguerre parameter”) and so does the N-variate Laguerre operator DN. The origin of operator D(z,z′) is explained in Olshanski [32]: it is obtained from DN by formal analytic continuation with respect to N and the Laguerre parameter. Operator D(z,z′) is diagonalized in a special basis of Sym formed by the so-called La- guerre symmetric functions. These functions, denoted by L(z,z′) ν , depend on parameters (z, z′) and are indexed by arbitrary partitions ν = (ν1, ν2, . . . ). One has D(z,z′)L(z,z′) ν = −\|ν\|L(z,z′) ν , \|ν\| := ν1 + ν2 + . . . . (1.2) (1.2) As shown in [32], the Laguerre symmetric functions form an orthogonal basis in a Hilbert L2 space. Let us explain brieﬂy this point (for more detail, see [32] and Section 8.4 below). So far we treated Sym as an abstract commutative algebra, freely generated by elements e1, e2, . . . , but now we embed it into the algebra of continuous functions on a topological space, called the Thoma cone and denoted by eΩ: eΩ:= n (α1, α2, . . . ; β1, β2, . . . ; δ) ∈R∞× R∞× R : α1 ≥α2 ≥· · · ≥0, β1 ≥β2 ≥· · · ≥0, X αi + X βi ≤δ o . Note that the space eΩis locally compact and has inﬁnite dimension in the sense that its points depend on countably many continuous parameters. The way of converting elements F ∈Sym into continuous functions F(ω) on eΩis described in Section 7.4. Next, we impose the following condition on the parameters: Condition 1.1. 1.1 Preliminaries: Markov processes related to orthogonal polynomials Finally, one can use DN to deﬁne a diffusion process XN on the space of N-point con- ﬁgurations contained in supp W ⊆R. Again, this process has a symmetrizing measure, with density N Y i=1 W(xi) · V 2(x1, . . . , xN). This construction is well known in random matrix literature. The case of Hermite polynomials arises from Dyson’s Brownian motion model [16]. Some other examples can be found in König [25]. The construction also works for some families of discrete orthogonal polynomials, only then XN is a jump process. In the present paper, we make a further step of generalization leading to a two- parameter family of inﬁnite-dimensional, continuous time Markov processes X(z,z′), which are related to the Laguerre polynomials. These words can bring the reader to believe that the processes X(z,z′) are obtained from the ﬁnite-dimensional Laguerre processes XN by a large-N limit transition, but this is not true. Actually, the connection between X(z,z′)’s and XN’s is of a different kind: informally, one can say that the former are related to the later by analytic continuation in two parameters, dimension N and the continuous parameter entering the deﬁnition of the classical Laguerre polynomials. Page 2/43 Markov dynamics on the Thoma cone EJP 18 (2013), paper 75. 1.2 The inﬁnite-dimensional Laguerre differential operator and the z-measures Either both parameters z and z′ are complex numbers with nonzero imaginary part and z′ = ¯z, or both parameters are real and contained in an open unit interval of the form (m, m + 1) for some m ∈Z. This is equivalent to requiring that (z + k)(z′ + k) > 0 for every k ∈Z. In particular, Condition 1.1 implies that zz′ and z + z′ are real, so that the coefﬁcients of operator D(z,z′) are real. It was shown in [32] that for every (z, z′) satisfying Condition 1.1, there exists a unique probability distribution M (z,z′) on eΩsuch that all elements of Sym produce square integrable functions on eΩwith respect to measure M (z,z′), and the Laguerre ejp.ejpecp.org Page 3/43 Page 3/43 Markov dynamics on the Thoma cone functions L(z,z′) λ are pairwise orthogonal with respect to the inner product of the Hilbert space L2(eΩ, M (z,z′)). In other words, M (z,z′) serves as the orthogonality measure for the Laguerre symmetric functions. The measures M (z,z′) appeared even earlier in connec- tion with the problem of harmonic analysis on the inﬁnite symmetric group; we call them the z-measures on the Thoma cone. ′ A difﬁculty of working with the Laguerre operator D(z,z′) is that its domain as deﬁned above consists of unbounded functions (more precisely, all the nonconstant functions from Sym are unbounded functions on eΩ). To overcome this difﬁculty we modify the domain of deﬁnition of the operator in the following way. e For a triple ω = (α, β, δ) ∈eΩ, write \|ω\| := δ. Let F stand for the space of functions on eΩspanned by the functions of the form e−r\|ω\|F(ω), F ∈Sym, r > 0. Such functions are bounded; even more, they vanish at inﬁnity. On the other hand, D(z,z′) operates on F in a natural way: here we use the fact that \|ω\| = e1(ω), so that each function from F is expressed through variables e1, e2, . . . . Such functions are bounded; even more, they vanish at inﬁnity. On the other hand, D(z,z′) operates on F in a natural way: here we use the fact that \|ω\| = e1(ω), so that each function from F is expressed through variables e1, e2, . . . . EJP 18 (2013), paper 75. 1.3 Main results Given a locally compact separable metrizable space E, denote by C0(E) the Banach space of real continuous functions on E, vanishing at inﬁnity, with the supremum norm. A Feller semigroup is a strongly continuous operator semigroup T(t) on C0(E) afforded by a transition function P(t; x, dy) (such that P(t; x, · ) is a probability measure), (T(t)f)(x) = Z y∈E P(t; x, dy)f(y), x ∈E, f ∈C0(E). A Feller semigroup gives rise to a Markov process on E with càdlàg sample trajectories, called a Feller process. A Feller semigroup gives rise to a Markov process on E with càdlàg sample trajectories, called a Feller process. Throughout the paper we assume that (z, z′) satisﬁes Condition 1.1. Theorem 1.2. (i) The differential operator D(z,z′), viewed as an operator on C0(eΩ) with domain F, is dissipative, and its closure serves as the generator of a Feller semigroup on C0(eΩ), which we denote by T (z,z′)(t). ′ (ii) The corresponding Feller Markov process X(z,z′) has a unique stationary distri- bution, which is the z-measure M (z,z′). Proof is given in Section 8. Claim (ii) shows that the z-measures M (z,z′) can be characterized as the stationary distributions of Markov processes X(z,z′). Taking as the initial distribution for Markov process X(z,z′) its stationary distribution we get a stationary in time stochastic process, which we denote by e X(z,z′). Theorem 1.2 is complemented by the following result, established in Section 9: Theorem 1.3. e X(z,z′) can be interpreted as a time-dependent determinantal point pro- cess whose correlation kernel can be explicitly computed. Let us explain this claim. Consider the punctured real line R∗:= R \ {0} and the space Conf(R∗) of locally ﬁnite point conﬁgurations on R∗. The stationary distribution M (z,z′) can be interpreted as a probability measure on Conf(R∗). More generally, for any ﬁnite collection t1 < · · · < tn of time moments, the corresponding ﬁnite-dimensional distribution M (z,z′)(t1, . . . , tn) of stochastic process e X(z,z′) can be interpreted as a prob- ability measure on the space Conf( R∗⊔· · · ⊔R∗ \| {z } n ). This makes it possible to describe ejp.ejpecp.org Page 4/43 Page 4/43 Markov dynamics on the Thoma cone M (z,z′)(t1, . . . , tn) in the language of correlation functions. 1.4 Method of Markov intertwiners The results stated above, together with those of [32], were announced without proofs in the note Olshanski [31]. The scheme of the initial proof of Theorem 1.2 was the following: • Start with the semigroup eT (z,z′)(t) in the Hilbert space L2(eΩ, M (z,z′)) generated by the closure of operator D(z,z′) and show that eT (z,z′)(t) is positivity preserving. • Show that eT (z,z′)(t) preserves functions from C0(eΩ). • Show that the topological support of M (z,z′) is the whole space eΩ. The third claim means that the natural map C0(eΩ) →L2(eΩ, M (z,z′)) is injective, so that restricting eT (z,z′)(t) to C0(eΩ) gives the desired Feller semigroup T (z,z′)(t). In the present paper, we use a different approach, based on the method of Markov intertwiners proposed in Borodin–Olshanski [13], combined with the main idea of an- other recent paper, Borodin–Olshanski [14]. To explain this approach, we have ﬁrst to brieﬂy review what we did in [13]. That paper deals with the Gelfand–Tsetlin graph GT describing the branching rule for the irreducible characters of unitary groups U(N). The graph is graded, and its Nth level GTN is a countable set, identiﬁed with the dual object to the unitary group U(N). The graph structure determines a a sequence of stochastic matrices Λ2 1, Λ3 2, . . . , where the Nth matrix ΛN+1 N has format GTN+1 ×GTN and is viewed as a “link” connecting the (N + 1)th and Nth levels of graph GT. The boundary of graph GT is deﬁned as the en- trance boundary for the inhomogeneous Markov chain with varying state spaces GTN, discrete time parameter ranging over {. . . , 3, 2, 1}, and transition function given by the links. The boundary serves as the space of parameters for the extremal characters of the inﬁnite-symmetric group U(∞); this space is a connected, inﬁnite-dimensional lo- cally compact space. Now, the idea is to ﬁnd a family {TN(t) : N = 1, 2, . . . } of Feller semigroups, acting on the spaces C0(GTN) and compatible with the links in the sense that TN+1(t)ΛN+1 N = ΛN+1 N TN(t), N = 1, 2, . . . , t ≥0 (here the operators TN+1(t) and TN(t) are viewed as matrices of format GTN+1×GTN+1 and GTN ×GTN, respectively). One can say that the links serve as Markov intertwiners for the semigroups TN(t). 1.3 Main results The determinantal property claimed in the theorem means that the correlations functions are given by n × n minors extracted from a certain kernel. The kernel in question, denoted by K(z,z′)(x, s; y, t), has as arguments two space-time variables, (x, s) and (y, t), where s ∈R and t ∈R are time moments, while x ∈R∗and y ∈R∗are space positions. y p p The kernel K(z,z′)(x, s; y, t) appeared ﬁrst in our paper [11], but there it was derived as the result of a formal limit transition, without reference to an inﬁnite-dimensional Markov process. We called K(z,z′)(x, s; y, t) the extended Whittaker kernel to emphasize a similarity with the well-known dynamical kernels from random matrix theory, the “extended” versions of the classical sine, Airy, and Bessel kernels (see Tracy-Widom [40]). EJP 18 (2013), paper 75. EJP 18 (2013), paper 75. Page 5/43 ejp.ejpecp.org Markov dynamics on the Thoma cone In the present paper we show that a similar approach works for the Thoma cone eΩ. A nontrivial point is what is a suitable substitute of the Gelfand–Tsetlin graph. As is well known, a natural analog of the Gelfand–Tsetlin graph is the Young graph, which is the branching graph of the symmetric group characters. The boundary of the Young graph is an inﬁnite-dimensional compact space Ω, called the Thoma simplex, and eΩappears as the cone built over Ω. Although harmonic analysis on the inﬁnite symmetric group deals with the Thoma simplex and probability measures thereof, things go simpler when objects living on Ωare “lifted” to eΩ; this was the main reason for working with the Thoma cone. However, eΩitself is not a boundary of a branching graph, which was an evident obstacle for extending the method of [13]. A solution was found due to the results of [14], where we showed that eΩcan be identiﬁed with the entrance boundary of a continuous time Markov chain on the set Y of all Young diagrams. This fact enabled us to apply the formalism of Markov intertwiners with appropriate modiﬁcations; in particular, the discrete index N = 1, 2, . . . is replaced by continuous index r ranging over the half-line R>0. In one direction, the present work goes further than [13], because for the processes related to the Gelfand–Tsetlin graph, a result similar to Theorem 1.3 is yet unknown. 1.5 Comments It is natural to compare the results of the present paper to those of Borodin–Olshanski [12], [13], and Borodin–Gorin [5]. In all four papers the authors construct a Feller Markov process on an inﬁnite-dimensional boundary of a “projective system”. The process of [12] can be obtained by a normalization of the one we construct here, much similar to the way the Brownian Motion on the sphere can be obtained from that in the Euclidian space. However, the stationary distribution of the normalized process does not deﬁne a determinantal point process. Also, in that case the state space is compact, which is much easier to deal with from the analytic viewpoint. On the other hand, the process of the present paper is a certain scaling limit of that from [13], but in the case of [13] the situation is more complicated and we were not able to prove there that the time-dependent correlation functions of the equilibrium process are determinantal (we prove such a statement in this work). We also do not dispose of an explicit eigenbasis for the generator there, in contrast to (1.2) above. The process considered in [5] was proven to have time-dependent determinantal structure but it does not possess a stationary distribution, unlike the three other ones. Also, the underlying state space is quite different as its coordinates live on a lattice, not on the real line. Overall, the Markov process we consider in the present paper is the only one so far that is proven to have all the nice properties one would like to carry over from the well-known ﬁnite dimensional analogs, i.e. Feller property, existence of a station- ary distribution, an explicit description of the (pre)generator and its eigenbasis, and determinantal formulas for the time-dependent correlations. To the best of our knowledge, such completeness of the picture was not achieved in the study of inﬁnite-particle versions of Dyson’s Brownian Motion Model that are also expected to have determinantal time-dependent correlations, see Jones [20], Katori– Tanemura [21], [22], [23], Osada [34], [35], Spohn [38]. 1.4 Method of Markov intertwiners Given such a family of semigroups, a simple (essentially formal) argument shows that it gives rise to a “limit” Feller semigroup T∞(t) generating a Feller process on the boundary. We showed in [13] that there is quite a natural way to construct requiring pre-limit semigroups TN(t) depending on four additional continuous parameters, and so we obtain a four-parameter family of limit Feller processes on the boundary. EJP 18 (2013), paper 75. ejp.ejpecp.org ejp.ejpecp.org Page 5/43 Page 5/43 EJP 18 (2013), paper 75. EJP 18 (2013), paper 75. Page 6/43 ejp.ejpecp.org 1.7 Organization of the paper In Section 2 we recall basic facts about Feller semigroups and their generators, and state a remarkable general theorem from Ethier–Kurtz [18], which gives a convenient sufﬁcient condition on a matrix of jump rates ensuring that it generates a Feller Markov chain. In Section 3 we review necessary deﬁnitions and facts concerning convergence of Markov semigroups, taken again from Ethier–Kurtz [18]. Sections 4 and 5 are devoted to the formalism of Markov intertwiners (here we present a minimal necessary material and refer to [13] for more details). In Section 6 we apply the method of Markov intertwiners to constructing a concrete one-dimensional diffusion process; our goal here is to present all the steps of the main construction in a simpliﬁed situation. Short Section 7 introduces the Thoma cone and some related objects. Long Section 8 is devoted to the proof of Theorem 1.2; the argument is developed in strict parallelism with that of Section 6. Section 9 contains the proof of Theorem 1.3. Section 9 contains the proof of Theorem 1.3. Finally, in Section 10 we brieﬂy describe a Plancherel-type degeneration of our main construction. Markov dynamics on the Thoma cone interaction. On the other hand, as shown in [13], the process on the boundary of GT is “covered” by a certain Markov process with local interaction, living on the path space of GT. In the companion note [15] we describe a curious model which conjecturally provides a similar “covering” process for X(z,z′). If the conjectural claims stated in [15] hold true, this model leads to an alternative approach to our processes X(z,z′), which looks simple and intuitively appealing. 1.6 Covering Markov process Informally, both the Markov process X(z,z′) on the Thoma cone and its relative, the Markov process on the boundary of the Gelfand–Tsetlin graph GT, studied in our paper [13], may be viewed as interacting particle processes with nonlocal (or long-range) ejp.ejpecp.org ejp.ejpecp.org Page 6/43 Markov dynamics on the Thoma cone • For any ﬁxed x ∈E and t ≥0, one has 2 Feller semigroups Let E be a locally compact, noncompact, metrizable separable space. Denote by C(E) the Banach space of real-valued continuous functions on E with the uniform norm ∥f∥= sup x∈E \|f(x)\|. ∥f∥= sup x∈E \|f(x)\|. Let C0(E) ⊂C(E) denote its closed subspace formed by the functions vanishing at inﬁnity, and let Cc(E) be the dense subspace of C0(E) consisting of compactly supported functions. If E is a discrete countable space, then the continuity requirement disappears, C0(E) becomes the space of arbitrary real functions on E vanishing at inﬁnity, and Cc(E) becomes the subspace of ﬁnitely supported functions. Deﬁnition 2.1. A Feller semigroup {T(t) : t ≥0} is a strongly continuous, positive, conservative contraction semigroup on C0(E), see [18, p. 166]. Note that in [18], the conservativeness condition is stated in terms of the semigroup generator. Here are two equivalent reformulations of this property (see also Liggett [27, Chapter 3]: • For any ﬁxed x ∈E and t ≥0, one has sup{(T(t)f)(x) : f ∈C0(E), 0 ≤f ≤1} = 1 (if E is compact, then this simply means that T(t) preserves the constant functio 1) (if E is compact, then this simply means that T(t) preserves the constant function 1). EJP 18 (2013), paper 75. Markov dynamics on the Thoma cone Markov dynamics on the Thoma cone • The semigroup admits a transition function, where we mean that a transition func- tion P(t \| x, · ) is a probability measure (not a sub-probability one!) for all t ≥0 and x ∈E. • The semigroup admits a transition function, where we mean that a transition func- tion P(t \| x, · ) is a probability measure (not a sub-probability one!) for all t ≥0 and x ∈E. • The semigroup admits a transition function, where we mean that a transition func- tion P(t \| x, · ) is a probability measure (not a sub-probability one!) for all t ≥0 and x ∈E. Assume now that E is a countably inﬁnite set and Q = [Q(a, b)] is a matrix of format E × E such that Q(a, b) ≥0 for all a ̸= b and −Q(a, a) = X b: b̸=a Q(a, b) < +∞for all a ∈E. 2 Feller semigroups (2.1) (2.1) Then there is a constructive way to deﬁne a semigroup {Pmin(t) : t ≥0} of substochastic matrices, which provides the minimal solution to Kolmogorov’s backward and forward equations, d d Then there is a constructive way to deﬁne a semigroup {Pmin(t) : t ≥0} of substochastic matrices, which provides the minimal solution to Kolmogorov’s backward and forward equations, d dt P(t) = QP(t), d dt P(t) = P(t)Q, see Feller [19] and Liggett [27, Chapter 2]. see Feller [19] and Liggett [27, Chapter 2]. Deﬁnition 2.2. One says that Q is regular if the matrices Pmin(t) from the minimal solution are stochastic. If the Q-matrix is regular, then Pmin(t) is a unique solution to both the backward and forward Kolmogorov equations. Qualitatively, regularity of the Q-matrix means that the Markov chain is non-exploding: one cannot escape to inﬁnity in ﬁnite time. Recall a few general notions (see Ethier–Kurtz [18, Chapter 1, Sections 1–3]). Any strongly continuous contractive semigroup on a Banach space is uniquely determined by its generator, which is a densely deﬁned closed dissipative operator. We will denote generators by symbol A (possibly with additional indices), and Dom A will denote the domain of A. A core of a generator A is a subspace F ⊆Dom A such that the closure of the operator A\|F (the restriction of A to F) coincides with A itself; thus A is uniquely determined by its restriction to a core. It often happens that an explicit description of Dom(A) is unavailable but one can write down the action of A on a core F, and then the pre-generator A\|F serves as a substitute of A. We will need a result from Ethier–Kurtz [18] which provides a convenient sufﬁcient condition of regularity together with important additional information: Theorem 2.3. Let E be a countably inﬁnite set and Q = [Q(a, b)] be a matrix of format E × E satisfying (2.1). 3.1 Convergence of semigroups Let I be one of the sets R>0 (strictly positive real numbers) or Z>0 (strictly positive integers). Assume that {Lr : r ∈I} is a family of real Banach spaces, L∞is one more real Banach space, and for every r ∈I we are given a contractive linear operator πr : L∞→Lr. If f is a vector of one of these spaces, then ∥f∥denotes its norm. Deﬁnition 3.1. We say that vectors fr ∈Lr approximate a vector f ∈L∞and write fr →f if Deﬁnition 3.1. We say that vectors fr ∈Lr approximate a vector f ∈L∞and write fr →f if lim ∥fr −πrf∥= 0. lim r→∞∥fr −πrf∥= 0. Deﬁnition 3.2. Let {T∞(t) : t ≥0} and {Tr(t) : t ≥0} be strongly continuous con- traction semigroups on L∞and Lr. We say that the semigroups Tr(t) approximate the semigroup T∞(t) and write Tr(t) →T∞(t) if lim r→∞sup 0≤t≤t0 ∥Tr(t)πrf −πrT∞(t)f∥= 0 for all f ∈L∞and any t0 > 0. (3.1) (3.1) Our aim is to check this condition using an appropriate convergence of semigroup generators. So let A∞and Ar denote the generators of the above semigroups and let Dom(A∞), Dom(Ar) be the domains of the generators. Deﬁnition 3.3. Fix a core F ⊆Dom(A). We say that the operator A∞\|F is approx- imated by the operators Ar if for any vector f ∈F one can ﬁnd a family of vectors {fr ∈Dom(Ar) : r ∈I} such that fr →f, and Arfr →A∞f as r →∞. In other words, this kind of operator convergence means that every vector from the graph of A∞\|F can be approximated by vectors from the graphs of the operators Ar. Theorem 3.4. Let T∞(t), Tr(t), A∞, Ar, and F be as above. If A∞\|F is approximated by the operators Ar, then Tr(t) →T∞(t) in the sense of Deﬁnition 3.2. Proof. For I = Z>0, this is part of Ethier–Kurtz [18, Chapter 1, Theorem 6.1]. The case I = R>0 is immediately reduced to the case I = Z>0, because condition (3.1) is equivalent to saying that the same limit relation holds along any sequence of positive real numbers tending to +∞. (ii) This semigroup induces a Feller semigroup {T(t) : t ≥0} on C0(E). (iii) Let A denote the generator of T(t); its domain Dom(A) consists of those func- (iii) Let A denote the generator of T(t); its domain Dom(A) consists of those func- tions f ∈C0(E) for which Qf ∈C0(E). Moreover, A = Q on Dom A. (iii) Let A denote the generator of T(t); its domain Dom(A) consists of those func- tions f ∈C0(E) for which Qf ∈C0(E). Moreover, A = Q on Dom A. ( ) ( ) tions f ∈C0(E) for which Qf ∈C0(E). Moreover, A = Q on Dom A. f ( ) Qf ( ) Q (iv) The subspace Cc(E) ⊂C0(E) of compactly supported functions is a core for A. f ( ) Qf ( ) Q (iv) The subspace Cc(E) ⊂C0(E) of compactly supported functions is a core for A. Proof. This is an adaptation of Theorem 3.1 in [18, Chapter 8], which actually holds under less restrictive assumptions. Proof. This is an adaptation of Theorem 3.1 in [18, Chapter 8], which actually holds under less restrictive assumptions. 2 Feller semigroups Assume additionally that Q has ﬁnitely many nonzero entries in every row and every column, and there exist strictly positive functions γ(a) and η(a) on E that tend to +∞at inﬁnity and are such that −Q(a, a) ≤Cγ(a), ∀a ∈E, (2.2) Q 1 γ ≤C γ pointwise (2.3) Qη ≤Cη pointwise (2.4) (2.2) (2.3) (2.4) where C is a positive constant and, for an arbitrary function f(a) on E, the notation Qf means the function where C is a positive constant and, for an arbitrary function f(a) on E, the notation Qf means the function (Qf)(a) = X b∈E Q(a, b)f(b) = X b∈E, b̸=a Q(a, b)(f(b) −f(a)), the sum being ﬁnite because of the row ﬁniteness condition. Under these hypotheses we have: (i) Q is regular and so determines a Markov semigroup P(t) (i) Q is regular and so determines a Markov semigroup P(t). (i) Q is regular and so determines a Markov semigroup P(t). EJP 18 (2013), paper 75. Page 8/43 ejp.ejpecp.org ejp.ejpecp.org EJP 18 (2013), paper 75. EJP 18 (2013), paper 75. Markov dynamics on the Thoma cone (ii) This semigroup induces a Feller semigroup {T(t) : t ≥0} on C0(E). Markov dynamics on the Thoma cone Markov dynamics on the Thoma cone Let E be a locally compact metrizable space and T(t) be a Feller semigroup on C0(E); then T(t) gives rise to a Markov process X(t) on E with càdlàg sample trajecto- ries, see Ethier–Kurtz [18, Chapter 4, Section 2]. The ﬁnite-dimensional distributions of X(t) are determined by the semigroup T(t) in the following way: For arbitrary functions g1, . . . , gk ∈C0(E), deﬁne recursively functions hk, . . . , h0 by hk = gk, hk−1 = gk−1 · (T(tk −tk−1)hk), . . . . . . , h1 = g1 · (T(t2 −t1)h2), h0 = T(t1)h1, (3.2) . . . , h1 = g1 · (T(t2 −t1)h2), h0 = T(t1)h1, (3.2) where dots mean pointwise product, so that hk−1 is obtained by applying operator T(tk− tk−1) to hk−1 and then multiplying the resulting function by gk−1, etc. Then ⟨g1 ⊗· · · ⊗gk, M(t1, . . . , tk)⟩= ⟨h0, M(0)⟩, (3.3) (3.3) where the angle brackets denote the canonical pairing between functions and mea- sures, and (g1 ⊗· · · ⊗gk)(x1, . . . , xk) = g1(x1) . . . gk(xk) for (x1, . . . , xk) ∈Ek (this is a function from C0(Ek)). Let Xr(t) and X(t) be Markov processes with state spaces Er and E, respectively (as before, r ranges over the index set I, which is either R>0 or Z>0). Assume that E is a locally compact metrizable separable space and each Er is realized as a discrete locally ﬁnite subset of E. Further, assume that as r →∞, Er becomes more and more dense in E; more precisely, we postulate that any probability measure P on E can be represented as the weak limit w-limr→∞Pr, where Pr is a probability measure supported by Er. Deﬁnition 3.5. Under these assumptions we say that the processes Xr(t) approximate the process X(t) and write Xr(t) →X(t) if whenever an initial distribution M(0) for the process X(t) is represented as a weak limit of a family {Mr(0)} of initial distributions of processes Xr(t), we have w- lim r→∞Mr(t1, . . . , tk) = M(t1, . . . , tk), meaning weak convergence on Ek of the ﬁnite-dimensional distributions corresponding to any given time moments 0 < t1 < · · · < tk, k = 1, 2, . . . . Corollary 3.6. Markov dynamics on the Thoma cone Under the above assumptions, assume additionally that the Markov processes Xr(t) and X(t) come from some Feller semigroups on the Banach spaces Lr = C0(Er) and L = C0(E), respectively. Further, let the projection πr : L →Lr be deﬁned as the restriction map from E to Er. If the hypotheses of Theorem 3.4 are satisﬁed, then Xr(t) →X(t) in the sense of Deﬁnition 3.5. Note that πr is well deﬁned as a map from C0(E) to C0(Er) because Er is assumed to be locally ﬁnite, so that if a sequence of points goes to inﬁnity along Er then it also goes to inﬁnity in E. 3.2 Convergence of Markov processes Below we use the term Markov process as a shorthand for a Markov family which may start from any given point of the state space or from any given initial probability distribution. We are dealing exclusively with processes stationary in time and with inﬁnite life time. Given an initial distribution M(0) of a Markov process on a space E, one may speak about its ﬁnite-dimensional distributions M(t1, . . . , tk) corresponding to any prescribed time moments 0 ≤t1 < · · · < tk, k = 1, 2, . . . . Every such distribution M(t1, . . . , tk) is a probability measure on the k-fold direct product Ek = E × · · · × E. ejp.ejpecp.org ejp.ejpecp.org Page 9/43 Proof. It sufﬁces to prove that lim r→∞⟨g1 ⊗· · · ⊗gk, Mr(t1, . . . , tk)⟩= ⟨g1 ⊗· · · ⊗gk, M(t1, . . . , tk)⟩ (3.4) any collection g1, . . . , gk ∈C0(E), because the functions of the form g1 ⊗· · · ⊗gk are nse in C0(Ek) lim r→∞⟨g1 ⊗· · · ⊗gk, Mr(t1, . . . , tk)⟩= ⟨g1 ⊗· · · ⊗gk, M(t1, . . . , tk)⟩ (3.4) (3.4) for any collection g1, . . . , gk ∈C0(E), because the functions of the form g1 ⊗· · · ⊗gk are dense in C0(Ek). for any collection g1, . . . , gk ∈C0(E), because the functions of the form g1 ⊗· · · ⊗gk are dense in C0(Ek). Let hk, . . . , h0 ∈C0(E) be deﬁned as in (3.2) and, for each r ∈I, let hk;r, . . . , h0;r ∈ C0(Er) be deﬁned in the same way, starting from the collection g1;r := πr(g1), g2;r := πr(g2), . . . , gk;r := πr(gk). EJP 18 (2013), paper 75. Page 10/43 ejp.ejpecp.org EJP 18 (2013), paper 75. Page 10/43 ejp.ejpecp.org ejp.ejpecp.org Page 10/43 Page 10/43 Markov dynamics on the Thoma cone By virtue of (3.3), the desired limit relation (3.4) is equivalent to lim r→∞⟨h0;r, Mr(0)⟩= ⟨h0, M(0)⟩ By virtue of (3.3), the desired limit relation (3.4) is equivalent to lim r→∞⟨h0;r, Mr(0)⟩= ⟨h0, M(0)⟩ lim r→∞⟨h0;r, Mr(0)⟩= ⟨h0, M(0)⟩ Since w-limr→∞Mr(0) = M(0) by assumption, it sufﬁces to prove that lim r→∞∥h0;r −πrh0∥= 0. To do this, we prove step by step that lim r→∞∥hi;r −πrhi∥= 0, for i = k, . . . , 0, where each transition i →i −1 is justiﬁed by making use of Theorem 3.4. This argument is patterned from the proof of Theorem 2.5 in [18, Chapter 4]. Note also that another kind of convergence is established in [18, Chapter 4, Theorem 2.11]. 4.1 Links Let E′ and E be two measurable spaces. Recall that a Markov kernel linking E′ to E is a function Λ( · , · ) in two variables, one ranging over E′ and the other ranging over measurable subsets of E, such that Λ is measurable with respect to the ﬁrst argument and is a probability measure relative to the second argument. We use the notation Λ : E′ 99K E and call Λ a link between E′ and E. If E is a discrete set, then, setting Λ(x, y) := Λ(x, {y}), we may regard Λ as a function on E′ × E. If both E′ and E are discrete, then Λ is simply a stochastic matrix of format E′ × E. The operation of composition of two links E′′ 99K E′ and E′ 99K E is deﬁned in a natural way: denoting the ﬁrst link by ΛE′′ E′ and the second one by ΛE′ E we have (ΛE′′ E′ ΛE′ E )(x, dz) = Z y∈E′ ΛE′′ E′ (x, dy)ΛE′ E (y, dz). (ΛE′′ E′ ΛE′ E )(x, dz) = Z y∈E′ ΛE′′ E′ (x, dy)ΛE′ E (y, dz). In the discrete case this operation reduces to conventional matrix product. In the discrete case this operation reduces to conventional matrix product. The possibility of composing links makes it possible to regard them as morphisms in a category whose objects are measurable spaces, see [14]. However, links are not ordinary maps; this is why we denote them by the dash arrow. y p y y A link Λ : E′ 99K E takes a probability measure M on E′ to a probability measure MΛ on E: Z (MΛ)(dy) = Z x∈E′ M ′(dx)Λ(x, dy). If both spaces are discrete then measures may be viewed as row-vectors and then the product MΛ becomes the conventional product of a row-vector by a matrix. If both spaces are discrete then measures may be viewed as row-vectors and then th product MΛ becomes the conventional product of a row-vector by a matrix. Dually, Λ determines a contractive linear map B(E) →B(E′) between the Banach spaces of bounded measurable functions, denoted as F 7→ΛF : (ΛF)(x) = Z y∈E Λ(x, dy)F(y). (ΛF)(x) = Z y∈E Λ(x, dy)F(y). In the discrete case, functions may be viewed as column-vectors and then ΛF becomes the conventional product of a matrix by a column-vector. for arbitrary couples r′ > r. (The above deﬁnition is applicable to more general ordered index sets but we would like to avoid excessive formalism. For the purpose of the present paper we need the continuous index set I = R+. Concrete projective systems with discrete index sets are considered in [13] and [14]. In some general considerations (see below) the case I = R>0 is readily reduced to that of I = Z>0.) ′ Following [14], we deﬁne the boundary E∞of a projective system {Er, Λr′ r } in the following way. Consider the projective limit space lim ←−M(Er), where M(Er) stands for the set of probability measures on Er and the limit is taken with respect to the projections M(Er′) →M(Er) induced by the links Λr′ r . Assuming that the projective limit space is nonempty, we take as E∞the set of its extreme points. We refer to [14] for more details. Note that M(Er) may be viewed as a simplex with vertex set Er, and every projection M(Er′) →M(Er) is an afﬁne map of simplices (that is, it preserves barycenters), so our projective limit space is a projective limit of simplices. By the very deﬁnition of projective limit, an element of lim ←−M(Er) is a family {Mr ∈ M(Er) : r ∈I} of probability measures satisfying the relation Mr′Λr′ r = Mr for every couple of indices r′ > r. Such a family is called a coherent system of measures. As explained in [14], there is a canonical bijection As explained in [14], there is a canonical bijection (4.1) M(E∞) ←→lim ←−M(Er), (4.1) where M(E∞) denotes the space of probability measures on E∞. This means that for every r ∈I there is a link Λ∞ r : E∞→Er such that the correspondence M∞7→{Mr : r ∈ I} given by Mr := M∞Λ∞ r establishes a one-to-one correspondence between probability measures on the boundary and coherent families of probability measures. We say that M is the boundary measure for the coherent system {M } M∞is the boundary measure for the coherent system {Mr}. Obviously, the links Λ∞ r are compatible with the links Λr′ r in the sense that Obviously, the links Λ∞ r are compatible with the links Λr′ r in the sense that Λ∞ r′ Λr′ r = Λ∞ r for any r′ > r. Λ∞ r′ Λr′ r = Λ∞ r for any r′ > r. for arbitrary couples r′ > r. Observe that in the case of I = R>0 the boundary does not change if in the above construction we will assume that the indices range along an arbitrary ﬁxed sequence of strictly increasing real numbers converging to +∞. This enables one to reduce the case I = R>0 to that of I = Z>0. For further reference, let us call this simple trick discretization of the index set. 4.2 Projective systems and boundaries Let, as above, I denote one of the two sets R>0 or Z>0. By a projective system with index set I we mean a family {Er : r ∈I} of discrete spaces together with a family of links {Λr′ r : Er′ 99K Er : r′ > r}, where every Er is ﬁnite or countably inﬁnite, and for any triple r′′ > r′ > r of indices one has Λr′′ r′ Λr′ r = Λr′′ r ; see [14]. If I = Z>0, then it sufﬁces to specify the links Λr′ r for neighboring indices r′ = r + 1 and then set Λr′ r := Λr′ r′−1 . . . Λr+1 r 4.1 Links In the discrete case, functions may be viewed as column-vectors and then ΛF becomes the conventional product of a matrix by a column-vector. We say that a link Λ : E′ →E between two locally compact spaces is a Feller link if the corresponding linear map B(E) →B(E′) sends C0(E) ⊂B(E) to C0(E′) ⊂B(E′). If E is discrete, then this condition means that for any ﬁxed y ∈E, the function Λ( ) Λ( { }) E′ li i C (E′) We say that a link Λ : E′ →E between two locally compact spaces is a Feller link if the corresponding linear map B(E) →B(E′) sends C0(E) ⊂B(E) to C0(E′) ⊂B(E′). p g p ( ) ( ) ( ) ( ) ( ) ( ) If E is discrete, then this condition means that for any ﬁxed y ∈E, the function x 7→Λ(x, y) := Λ(x, {y}) on E′ lies in C0(E′). EJP 18 (2013), paper 75. ejp.ejpecp.org Markov dynamics on the Thoma cone 4.4 Feller projective systems Let {Er, Λr′ r } be a projective system as deﬁned above. Equip the boundary E∞with the intrinsic topology — the weakest one in which all functions of the form x 7→Λ∞ r (x, y), r ∈I, y ∈Er, x 7→Λ∞ r (x, y), r ∈I, y ∈Er, are continuous. We say that {Er, Λr′ r } is a Feller system if the following three conditions are satisﬁed: (1) All li k Λr′ F ll are continuous. We say that {Er, Λr′ r } is a Feller system if the following three conditions are satisﬁed: (1) All links Λr′ r are Feller. (1) All links Λr′ r are Feller. (1) All links Λr′ r are Feller. (2) The boundary E∞is a locally compact Hausdorff space with respect to the in- trinsic topology. (2) The boundary E∞is a locally compact Hausdorff space with respect to the in- trinsic topology. (3) In this topology, all links Λ∞ r are Feller. (3) In this topology, all links Λ∞ r are Feller. (3) In this topology, all links Λ∞ r are Feller. Note that under condition (1), the deﬁnition of the intrinsic topology is not affected by discretization of the index set, which entails that the intrinsic topology is automati- cally metrizable with countable base. As an illustration, let us check that the binomial projective system from our running example (see Section 4.3 above) is a Feller system. Indeed, from the very deﬁnition of the “binomial” links Λr′ r and “Poissonian” links Λ∞ r it is clear that they are Feller links. It remains to check that the intrinsic boundary topology on R+ is the conventional topology and so is locally compact. By the very deﬁnition, the intrinsic topology is the weakest one in which all the functions x 7→Λ∞ r (x, m), where parameter r ranges over R+ and parameter m ranges over Z+, are continuous. We will prove a stronger claim: even if only m varies but r > 0 is chosen arbitrarily and ﬁxed, then the corresponding topology coincides with the conventional one. To do this, consider the map R+ →[0, 1]∞assigning to x ∈R+ the sequence {am(x) : m ∈Z+}, am(x) := Λ∞ r (x, m) = e−rx (rx)m m! . EJP 18 (2013), paper 75. 4.3 Running example: The binomial projective system B In this illustrative example taken from Borodin–Olshanski [14], the index set I is R>0; for every index r ∈R>0 the corresponding discrete set Er is a copy of Z+ := {0, 1, 2, . . . }; and for every two indices r′ > r the corresponding link Z+ 99K Z+ is given b BΛr′ r (l, m) = l! m!(l −m)! r r′ m 1 −r r′ l−m , l, m ∈Z+. EJP 18 (2013), paper 75. Page 12/43 Markov dynamics on the Thoma cone Note that Λr′ r (l, · ) is a binomial distribution on the set {m : 0 ≤m ≤l}. For this reason we call this system the binomial projective system. Note that Λr′ r (l, · ) is a binomial distribution on the set {m : 0 ≤m ≤l}. For this reason we call this system the binomial projective system. As shown in [14], its boundary E∞can be identiﬁed with the halﬂine R+ (the set of nonnegative real numbers) and the links Λ∞ r : R+ →Z+ are given by Poisson distribu- tions: BΛ∞ r (x, m) = e−rx (rx)m m! , x ∈R+, m ∈Z+. EJP 18 (2013), paper 75. 4.5 The density lemma If {Er, Λr′ r } is a Feller projective system with boundary E∞, then the subspace If {Er, Λr′ r } is a Feller projective system with boundary E∞, then the subspace [ r∈I Λ∞ r C0(Er) ⊂C0(E∞) is dense in the norm topology; see Borodin–Olshanski [13, Lemma 2.3]. Here Λ∞ r C0(Er) denotes the range of the operator Λ∞ r : C0(Er) →C0(E∞). For further reference we call this assertion the density lemma. Its proof is simple; it relies on the fact that for a locally compact space E, the vector space of (signed) measures on E with ﬁnite total variation is the Banach dual to C0(E). Since Cc(Er) is dense in C0(Er) and the operator Λ∞ r : C0(Er) →C0(E∞) is contrac- tive, the density lemma is equivalent to the assertion that the set of functions of the form x 7→Λ∞ r (x, y), r ∈I, y ∈Er, is total in C0(E∞) meaning that the linear span of these functions is dense. is total in C0(E∞) meaning that the linear span of these functions is dense. For our running example, the latter assertion means that the set of functions e−rxxn, r > 0, n ∈Z+ is total in C0(R+). But here a stronger claim holds: it is not necessary to take all r > 0, we may assume that r is ﬁxed. In other words, for any ﬁxed r > 0, the space of polynomials in x multiplied by the exponential e−rx is dense in C0(R+); see [14, Corollary 3.1.6] for a simple proof. Thus, in this situation, Λ∞ r C0(Er) ⊂C0(E∞) is dense for any ﬁxed r. However, this is a special property of the projective system under consideration; for instance, it does not hold in the context of [13]. 4.4 Feller projective systems This map is injective, for x is recovered from {am(x)} from the identity This map is injective, for x is recovered from {am(x)} from the identity ∞ X m=0 smam(x) = e(s−1)rx. By the very deﬁnition, the weakest topology on R+ making all the functions am(x) con- tinuous is exactly the topology induced by the embedding of R+ into the cube [0, 1]∞ equipped with the product topology. Observe now that the cube [0, 1]∞is compact and the above map extends by conti- nuity to the one-point compactiﬁcation R+ ∪{+∞} of R+ by setting am(+∞) = 0 for all m. Obviously, the extended map is injective, too. Therefore, it is a homeomorphism onto a closed subset of [0, 1]∞. This implies the desired claim. ejp.ejpecp.org ejp.ejpecp.org Page 13/43 Page 13/43 Markov dynamics on the Thoma cone Therefore, (4.3) follows from the fact that (see [14, Lemma 3.1.4]) Therefore, (4.3) follows from the fact that (see [14, Lemma 3.1.4]) Therefore, (4.3) follows from the fact that (see [14, Lemma 3.1.4]) lim r→+∞ 1 −s r rx xm = e−rxxm uniformly on x ∈R+. lim r→+∞ 1 −s r rx xm = e−rxxm uniformly on x ∈R+. For this example, Proposition 4.2 gives a speciﬁc recipe for approximating arbitrary probability measures on R+ by atomic measures supported by the grids r−1Z+. 5 Boundary Feller semigroups: general formalism 4.6 Approximation of boundary measures Our deﬁnition of the boundary measure M∞as a limit of a coherent system of mea- sures Mr was purely formal. Here we show that, under a suitable additional assumption, M∞is a limit of {Mr} in a conventional sense. Let, as above, {Er, Λr′ r } be a Feller projective system with boundary E∞, and adopt the following assumption: Condition 4.1. For every r ∈I there exists an embedding ϕr : Er ,→E∞such that: (i) The image ϕr(Er) is a discrete subset in E∞. (ii) For any ﬁxed s ∈I and any ﬁxed y ∈Es lim r→∞sup x∈Er \|Λr s(x, y) −Λ∞ s (ϕr(x), y)\| = 0. lim r→∞sup x∈Er \|Λr s(x, y) −Λ∞ s (ϕr(x), y)\| = 0. So far our measures lived on varying spaces. Now, using the maps ϕr, we can put all them on one and the same space, the boundary E∞. Namely, we simply replace Mr with its pushforward ϕr(Mr), which is a probability measure on E∞. A natural question is whether the resulting measures converge to M∞, and the next proposition gives an afﬁrmative answer. Proposition 4.2. Assume that Condition 4.1 is satisﬁed. Let {Mr : r ∈I} be a coherent system of probability distributions and M∞be the corresponding boundary measure. As r →∞, the measures ϕr(Mr) converge to M∞in the weak topology. Note that for this proposition, part (i) of the condition is not relevant, but it will be used in the sequel (see Section 5.2). EJP 18 (2013), paper 75. ejp.ejpecp.org ejp.ejpecp.org Page 14/43 Page 14/43 Markov dynamics on the Thoma cone Proof. We have to show that for any bounded continuous function F ⟨F, ϕr(Mr)⟩→⟨F, M∞⟩. Since all the measures in question are probability measures, we may replace the weak convergence by the vague convergence, that is, we may assume that F lies in the space C0(E∞). Next, we apply the density lemma (see Section 4.5), which enables us to further assume that F has the form F(x) = Λ∞ s (x, y) for some ﬁxed s ∈I and y ∈Es. Then we get Z ⟨F, M∞⟩= Z x∈E∞ M∞(dx)Λ∞ s (x, y) = Ms(y). On the other hand, ⟨F, ϕr(Mr)⟩= ⟨F ◦ϕr, Mr⟩. (4.2) (4.2) (F ◦ϕr)(x) = F(ϕr(x)) = Λ∞ r (ϕr(x), y) = Λr s(x, y) + ε(r, x), where, by virtue of Condition 4.1, the remainder term ε(r, x) tends to 0 uniformly on x, as r →∞. 4.6 Approximation of boundary measures Therefore, (4.2) equals where, by virtue of Condition 4.1, the remainder term ε(r, x) tends to 0 uniformly on x, as r →∞. Therefore, (4.2) equals ⟨Λr s( · , y), Mr⟩+ . . . = Ms(y) + . . . , where the dots denote a remainder term converging to 0. This completes the proof. where the dots denote a remainder term converging to 0. This completes the proof. Example 4.3. Consider the projective system B introduced in Section 4.3. Recall that then the index set I is R>0, Er = Z+ for all r > 0, and the boundary E+ is R+. Deﬁne the map ϕr : Er →E∞as ϕr(l) = r−1l, l ∈Z+, ϕr(l) = r−1l, l ∈Z+, d let us check that Condition 4.1 is satisﬁed. and let us check that Condition 4.1 is satisﬁed. Indeed, in our situation it means that that fo and let us check that Condition 4.1 is satisﬁed. Indeed, in our situation it means that that for ﬁxed s > 0 and m ∈Z+ lim r→∞sup l∈Z+ \|BΛr s(l, m) −BΛ∞ s (r−1l, m)\| = 0. (4.3) (4.3) The explicit expressions for the links in question are (see Section 4.3): BΛr s(l, m) = l! m!(l −m)! s r m 1 −s r l−m , l, m ∈Z+, BΛ∞ s (x, m) = e−sx (sx)m m! , x ∈R+, m ∈Z+. In (4.3), set x = r−1l and note that l! (l −m)!rm = xm 1 + O(r−1) , 1 −s r l−m = 1 −s r rx 1 + O(r−1) . l! (l −m)!rm = xm 1 + O(r−1) , 1 −s r l−m = 1 −s r rx 1 + O(r−1) . Markov dynamics on the Thoma cone Markov dynamics on the Thoma cone 5 Boundary Feller semigroups: general formalism In this section, {Er, Λr′ r } is a Feller projective system with index set I equal to R>0 or Z>0, and boundary E∞. EJP 18 (2013), paper 75. Page 15/43 5.1 Intertwining of semigroups Let E′ and E be two locally compact metrizable spaces, T ′(t) and T(t) be Feller semigroups on C0(E′) and C0(E), respectively, and Λ : E′ 99K E be a Feller link. Let us say that Λ intertwines the semigroups T ′(t) and T(t) if T ′(t)Λ = ΛT(t), t ≥0, (5.1) (5.1) where both sides are interpreted as operators C0(E) →C0(E′). Proposition 5.1. Assume that for every r ∈I we are given a Feller semigroup {Tr(t) : t ≥0} on C0(Er). Assume further that the links Λr′ r intertwine the corresponding semi- groups, that is, for any two indices r′ > r Tr′(t)Λr′ r = Λr′ r Tr(t). (5.2) (5.2) Then the there exists a unique Feller semigroup {T∞(t) : t ≥0} on E∞such that Λ∞ r intertwines T∞(t) and Tr(t) for every r ∈I, T∞(t)Λ∞ r = Λ∞ r Tr(t), t ≥0. (5.3) (5.3) Proof. In the case I = Z>0 this assertion was established in [13, Proposition 2.4]. The same argument works in the case I = R>0. Proof. In the case I = Z>0 this assertion was established in [13, Proposition 2.4]. The same argument works in the case I = R>0. We call the semigroup T∞(t) constructed in the above proposition the boundary semigroup. Now we are going to describe its generator. We start with the simple observation that relation (5.1) has an inﬁnitesimal analog: namely, denoting by A′ and A the generators of the semigroups T ′(t) and T(t) from (5.1), one has Λ : Dom(A) →Dom(A′) Proof. See [13, Section 6.2]. We will use this result to check condition (5.2) from Proposition 5.1. Markov dynamics on the Thoma cone Markov dynamics on the Thoma cone Take now as F the linear span of the union of the subspaces Λ∞ r Fr. We already know that F is contained in Dom(A∞). By the criterion above, it sufﬁces to prove that (c−A∞)F is dense in C0(E∞) for any c > 0. We have (c −A∞)F = span [ r∈I (c −A∞)Λ∞ r Fr ! = span [ r∈I Λ∞ r (c −Ar)Fr ! , where the last equality follows from (5.5). On the other hand, we know that for every r ∈I, (c −Ar)Fr is dense in C0(Er), because Fr is a core for Ar. Therefore, the closure of (c −A∞)F coincides with the closure of the subspace S r∈I Λ∞ r C0(Er). But the latter subspace is dense by Proposition 5.1. Therefore, (c −A∞)F is dense, too. Let us return to the basic intertwining relation (5.1). Under suitable assumptions, one can check it on the inﬁnitesimal level, as seen from the next proposition. and A′Λ = ΛA. (5.4) (5.4) In words, if a Feller link intertwines two Feller semigroups, then it also intertwines their generators. Indeed, this is an immediate consequence of the very deﬁnition of the semigroup generator. In words, if a Feller link intertwines two Feller semigroups, then it also intertwines their generators. Indeed, this is an immediate consequence of the very deﬁnition of the semigroup generator. Proposition 5.2. Let the semigroups Tr(t) be as in the above proposition, T∞(t) be the corresponding boundary semigroup, and Ar and A∞denote the generators of these semigroups. Take for each r ∈I an arbitrary core Fr ⊆Dom(Ar) for the operator Ar; then the linear span of the vectors of the form Λ∞ r f, where r ranges over I and f ranges over Fr, is a core for A∞. Note that the action of A∞on such a core is determined according to (5.4), that is A∞Λ∞ r f = Λ∞ r Arf, f ∈Dom(Ar). (5.5) (5.5) Proof. We will apply a well-known characterization of cores based on Hille–Yosida’s theorem: Let A be the generator of a strongly continuous contraction semigroup on a Banach space; a subspace F ⊆Dom(A) is a core for A if and only if, for any constant c > 0, the subspace (c−A)F is dense. The proof is simple (cf. [18, Chapter 1, Proposition 3.1]). Indeed, ﬁx an arbitrary c > 0. By Hille–Yosida’s theorem, the operator (c −A)−1 is deﬁned on the whole space and bounded. Next, the closure of A\|F coincides with A if and only if the closure of (c −A\|F)−1 coincides with (c −A)−1, and this in turn just means that (c −A)F, which is the domain of (c −A\|F)−1, is dense. EJP 18 (2013), paper 75. Page 16/43 ejp.ejpecp.org Page 16/43 ejp.ejpecp.org EJP 18 (2013), paper 75. ejp.ejpecp.org ejp.ejpecp.org Page 16/43 Page 16/43 Then Q′Λ = ΛQ implies that T ′(t)Λ = ΛT(t) for all t ≥0. Then Q′Λ = ΛQ implies that T ′(t)Λ = ΛT(t) for all t ≥0. Note that the assumptions on Λ, Q′, and Q imply that the products Q′Λ and ΛQ are well deﬁned and, moreover, these two matrices have ﬁnitely many nonzero entries in every row. Proof. See [13, Section 6.2]. Proposition 5.3. Assume that: • E′ and E are two ﬁnite or countably inﬁnite sets; • E′ and E are two ﬁnite or countably inﬁnite sets; • Λ : E′ 99K E is a stochastic Feller matrix with ﬁnitely many nonzero entries in every row; • Q′ and Q are two matrices of format E′ × E′ and E × E, respectively, satisfying the assumptions of Theorem 2.3; • {T ′(t)} and {T(t)} are the corresponding Feller semigroups afforded by that theo- rem. Then Q′Λ = ΛQ implies that T ′(t)Λ = ΛT(t) for all t ≥0. Markov dynamics on the Thoma cone Proof. Let A∞be the generator of the boundary semigroup T∞(t). By virtue of Theorem 3.4, it sufﬁces to prove that the restriction of A∞to some core F is approximated by the operators Ar. As F we take the linear span of the subspaces Λ∞ r Cc(Er) ⊂C0(E∞), where r ranges over I. The second condition postulated above says that Cc(Er) is a core of Ar; consequently, F is a core for A∞, by virtue of Proposition 5.2. According to Deﬁnition 3.3 we have to show that for any vector f ∈F one can ﬁnd a family of vectors fr ∈Dom(Ar) such that the following two limit relations hold: fr →f and Arfr →A∞f as r →∞. Without loss of generality we may assume that f ∈Λ∞ s g with g ∈Cc(Es) for some s ∈I. Next, for r > s we set fr := Λr sg and observe that it sufﬁces to prove the ﬁrst limit relation only. Indeed, once we know that fr →f with such a choice of {fr}, the second limit relation, Arfr →A∞f, follows simply by replacing g with Asg, because the links intertwine the generators. We also use the fact that g ∈Cc(Es) implies Asg ∈Cc(Es) (see the end of the second condition above). We proceed to the proof of the convergence fr →f. By Deﬁnition 3.1, it means that lim r→∞sup x∈Er \|fr(x) −f(ϕr(x))\| = 0. Without loss of generality we may assume that g is the delta-function at a point y ∈Es, but then the desired limit relation holds by virtue of Condition 4.1. Without loss of generality we may assume that g is the delta-function at a point y ∈Es, but then the desired limit relation holds by virtue of Condition 4.1. 6 A toy example: the one-dimensional Laguerre diffusion In this section we apply the abstract formalism described above to a construction of the Laguerre diffusion process on the halﬂine R+, generated by the differential operator x d2 dx2 + (c −x) d dx (here c > 0 is a parameter). This process is well known — it is related to the Bessel process in the same way as the Ornstein-Uhlenbeck process is related to the Wiener process, see, e.g. Eie [17]. Thus, the ﬁnal result is by no means new. However, the de- tailed exposition presented below will serve us as a preparation and a guiding example for Section 8, where we establish the main results. 5.2 Approximation of semigroups Here we are going to show that, under suitable additional assumptions, the bound- ary semigroup T∞(t) that is afforded by the construction of Proposition 5.1 is approxi- mated by semigroups Tr(t) in the sense of Deﬁnition 3.2. We keep to the hypotheses of Proposition 5.1. Next, we assume that Condition 4.1 is satisﬁed and one more condition holds: Condition 5.4. For every r ∈I, the space Cc(Er) of ﬁnitely supported functions is a core for the generator Ar of the semigroup Tr(t). Moreover, this space is invariant under the action of Ar. We set Lr = C0(Er), L∞= C0(E∞). Given a function f on E∞, we deﬁne the function πrf on Er by We set Lr = C0(Er), L∞= C0(E∞). Given a function f on E∞, we deﬁne th function πrf on Er by (πrf)(x) := f(ϕr(x)), x ∈Er. Since ϕr(Er) is assumed to be a locally ﬁnite subset of E∞(see part (i) of Condition 4.1), πr maps L∞into Lr. Obviously, the norm of πr is less or equal to 1. Proposition 5.5. Under the above assumptions, Tr(t) →T∞(t) in the sense of Deﬁni- tion 3.2. EJP 18 (2013), paper 75. ejp.ejpecp.org Page 17/43 Markov dynamics on the Thoma cone 6.1 The binomial projective system B Recall that B was introduced in Section 4.3. We will prove two technical propositions concerning the properties of the links of B. ′ Observe that every link BΛr′ r can be applied to an arbitrary function on Z+ (viewed as a column vector), because each row in BΛr′ r has ﬁnitely many nonzero entries. As for BΛ∞ r , it can be applied to functions on Z+ with moderate (say, at most polynomial) growth at inﬁnity. In the next two propositions we provide explicit formulas for the action of the links on functions of some special kind. Introduce a notation: Introduce a notation: y↓m = y(y −1) . . . (y −m + 1), m ∈Z+. Here y is assumed to range over R+ or Z+, depending on the context. By 1m, where m ∈Z+, we denote the function on Z+ equal to 1 at m and to 0 on Z+ \ {m}. The letter q always denotes a number from the open interval (0, 1). Note that Here y is assumed to range over R+ or Z+, depending on the context. By 1m, where m ∈Z+, we denote the function on Z+ equal to 1 at m and to 0 on Z+ \ {m}. The letter q always denotes a number from the open interval (0, 1). Note that lim q→+0 1 m!qm qyy↓m = 1m on Z+. (6.1) (6.1) EJP 18 (2013), paper 75. Page 18/43 ejp.ejpecp.org EJP 18 (2013), paper 75. Markov dynamics on the Thoma cone Proposition 6.1. Assume r′ > r > 0 and 0 < q < 1, and let l range over Z+. Regard BΛr′ r as an operator in the space of functions on Z+ transforming a function F(l) to a function G(l). Under this transformation Proposition 6.1. Assume r′ > r > 0 and 0 < q < 1, and let l range over Z+. Regard BΛr′ r as an operator in the space of functions on Z+ transforming a function F(l) to a function G(l). Under this transformation l↓m 7→ r r′ m l↓m l↓m 7→ r r′ m l↓m (6.2) 1m 7→1 m! 1 −q′ q′ m · (q′)ll↓m, q′ := 1 −r r′ (6.3) qll↓m 7→ qr q′r′ m (q′)ll↓m, q′ := 1 −(1 −q) r r′ (6.4) (6.3) (6.4) Proof. Let us prove (6.4). The function F(l) = qll↓m vanishes on {0, . . which leads to the desired result. Formula (6.2) can be checked in exactly the same way. Observe also that (6.2) is a limit case of (6.4) as q →1. Formula (6.3) is immediate from the very deﬁnition of BΛr′ r . On the other hand, (6.3) can also be obtained from (6.4) as a limit case: to see this, divide by m!qm, let q →0 and use (6.9). Proposition 6.2. Assume r > 0 and 0 < q < 1, and let l range over Z+ while x ranges over R+. Regard BΛ∞ r as an operator transforming a function F(l) on Z+ to a function G(x) on R+. Under this transformation G(x) on R+. Under this transformation l↓m 7→rmxm (6.5) 1m 7→rm m! · qx ∞xm, q∞:= e−r (6.6) qll↓m 7→qmrm · qx ∞xm, q∞:= e−(1−q)r. (6.7) (6.5) (6.6) (6.7) Proof. We may argue exactly as in the proof of Proposition 6.1, replacing the binomial distribution by the Poisson distribution. Alternatively, one can use (4.3) and pass to the limit l →∞, r′ →∞, l/r′ →x in the formulas of Proposition 6.1. 6.1 The binomial projective system B . , m −1} and the same holds for BΛr′ r F , because the matrix BΛr′ r is lower triangular. Therefore, it sufﬁces to compute (BΛr′ r F)(l) for y ≥m. We have (BΛr′ r F)(l) = l X k=m 1 −r r′ l−k r r′ k l! (l −k)! k! qk k! (k −m)!. Setting k′ = k −m and l′ = l −m we rewrite the right-hand side as Setting k′ = k −m and l′ = l −m we rewrite the right-hand side as qr r′ m l↓m l′ X k′=0 1 −r r′ l′−k′ qr r′ k′ l′! (l′ −k′)! k′!. The latter sum equals The latter sum equals (q′)l′ = (q′)−m(q′)l, which leads to the desired result. which leads to the desired result. Markov dynamics on the Thoma cone where k ranges over Z+. Let us regard Q(c) r as a difference operator acting on functions on Z+, which are interpreted as column vectors: (Q(c) r F)(l) = r(c + l)F(l + 1) + (r + 1)lF(l −1) −[(2r + 1)l + rc]F(l), (6.8) (6.8) where l ∈Z+. As is seen from the next proposition, this difference operator is re- lated to the classical Meixner orthogonal polynomials. Recall the deﬁnition of the these polynomials (see, e.g., Koekoek–Lesky–Swarttouw [24] and references therein): The Meixner polynomials are orthogonal with respect to the negative binomial dis- tribution on Z+, X l∈Z+ (1 + r)−c (c)l l! r 1 + r l δl, where (c)l := c(c + 1) . . . (c + l −1) is the Pochhammer symbol and δl denotes the delta measure at l. The explicit expression for the monic Meixner polynomial of degree n = 0, 1, 2, . . . is Mn(l; c, r) = (c)n n X m=0 (−r)n−m n↓m (c)mm!l↓m. (6.9) (6.9) Proposition 6.3. The Meixner difference operator (6.8) preserves the space of poly- nomials. We have Q(c) r : l↓m →−ml↓m + rm(m + c −1)l↓(m−1) (6.10) (6.10) and Q(c) r : Mn(l; c, r) →−nMn(l; c, r). (6.11) (6.11) Thus, the Meixner difference operator is diagonalized in the basis of the Meixner polynomials. Proof. All claims can be veriﬁed directly. For (6.11), see also [24]. roof. All claims can be veriﬁed directly. For (6.11), see also [24]. Proposition 6.4. For arbitrary r′ > r > 0, we have Proposition 6.4. For arbitrary r′ > r > 0, we have Q(c) r′ BΛr′ r = BΛr′ r Q(c) r . Q(c) r′ BΛr′ r = BΛr′ r Q(c) r . Proof. Because the Q-matrices in question have a simple tridiagonal form and the en- tries of BΛr′ r are given by a simple expression, a direct check is possible. However, we prefer to give another proof, which has the advantage of being more conceptual and well suited for the generalization that we need. Observe that Q(c) r′ BΛr′ r F = BΛr′ r Q(c) r F for any polynomial F . Indeed, it sufﬁces to check this for F = Mm( · ; c, r). EJP 18 (2013), paper 75. 6.2 The Meixner and Laguerre semigroups Introduce a Q-matrix of format Z+ × Z+, depending on parameters c > 0 and r > 0, with the entries Q(c) r (k, k + 1) = r(c + k), Q(c) r (k, k −1) = (r + 1)k, Q(c) r (k, k) = −[r(c + k) + (r + 1)k] = −[(2r + 1)k + rc], Q(c) r (k, k′) = 0, \|k −k′\| ≥2, JP 18 (2013), paper 75. Page 19/43 ejp.ejpecp.org EJP 18 (2013), paper 75. ejp.ejpecp.org Page 19/43 Markov dynamics on the Thoma cone 6.3 A family of cores for Markov semigroup generators For any ﬁxed q ∈(0, 1), the functions qxxm, m = 0, 1, 2, . . . , span a dense subspace in C0(R+), see Borodin–Olshanski [14, Corollary 3.1.6]. This also implies that the func- tions qllm, where m = 0, 1, 2, . . . and l ranges over Z+, span a dense subspace in C0(Z+). These facts are used in the next proposition. Proposition 6.8. (i) For any r′ > r > 0, the operator BΛr′ r : C0(Z+) →C0(Z+) has a dense range. (ii) Likewise, for any r > 0, the operator BΛ∞ r : C0(Z+) →C0(R+) has a dense range. Proposition 6.8. (i) For any r′ > r > 0, the operator BΛr′ r : C0(Z+) →C0(Z+) has a dense range. (ii) ik i f 0 h BΛ∞ C (Z ) C (R ) h d (ii) Likewise, for any r > 0, the operator BΛ∞ r : C0(Z+) →C0(R+) has a dense range. Proof. (i) Take an arbitrary q ∈(0, 1). By (6.4), BΛr′ r maps the linear span of functions qllm, m = 0, 1, 2, . . . onto the linear span of functions (q′)llm, with some other q′ ∈(0, 1), see (6.4). Since these spans are dense, we get the desired claim. Proof. (i) Take an arbitrary q ∈(0, 1). By (6.4), BΛr′ r maps the linear span of functions qllm, m = 0, 1, 2, . . . onto the linear span of functions (q′)llm, with some other q′ ∈(0, 1), see (6.4). Since these spans are dense, we get the desired claim. (ii) The same argument, with reference to (6.7). (ii) The same argument, with reference to (6.7). Recall that A(c) r denotes the generator of semigroup T (c) r (t) (Corollary 6.6). Likewise, let A(c) denote the generator of semigroup T (c)(t). Recall that A(c) r denotes the generator of semigroup T (c) r (t) (Corollary 6.6). Likewise, let A(c) denote the generator of semigroup T (c)(t). Proposition 6.9. Fix an arbitrary number q ∈(0, 1). (i) For every r > 0, the linear span of functions qllm, m = 0, 1, 2, . . . , where argument l ranges over Z+, is a core for A(c) r . (ii) Likewise, the linear span of functions qxxm, m = 0, 1, 2, . . . , where argument x ranges over R+, is a core for A(c). Proof. Proof. Easy direct check. This proposition makes it possible to apply Theorem 2.3, which in turn entails the following assertions. Corollary 6.6. (i) The Q-matrix Q(c) r gives rise to a Feller semigroup T (c) r (t) on C0(Z+) whose generator A(c) r is implemented by Q(c). ( ) (ii) The subspace C0(Z+) is a core for generator A(c) r . (ii) The subspace C0(Z+) is a core for generator A(c) r . We call T (c) r (t) the Meixner semigroup. It determines a continuous time Markov chain on Z+ which we call the Meixner chain and denote by X(c)(t). Proposition 6.7. For every c > 0 there exists a unique Feller Markov process X(c)(t) on R+ such that the corresponding Feller semigroup, denoted by T (c)(t), is consistent with the Meixner semigroups T (c) r (t), r > 0, in the sense that T (c)(t) BΛ∞ r = BΛ∞ r T (c) r (t), t ≥0, r > 0. Proof. We know that the Q-matrices with various values of parameter r are consistent with the links (Proposition 6.4). It follows, by virtue of Proposition 5.3, that the semi- groups are also consistent with the links. Therefore, we may apply Proposition 5.1, which gives the desired result. We call X(c)(t) and T (c)(t) the Laguerre process and the Laguerre semigroup, re- spectively; this terminology is justiﬁed by the results of Section 6.4. Markov dynamics on the Thoma cone It follows from (6.2) and (6.9) that BΛr′ r Mm( · ; c, r) = r r′ m Mm( · ; c, r′), and then we use (6.11) to conclude that both Q(c) r′ BΛr′ r and BΛr′ r Q(c) r multiply Mm( · ; c, r) by −m(r/r′)m. ( / ) Further, both matrices Q(c) r′ BΛr′ r and BΛr′ r Q(c) r have ﬁnitely many nonzero entries in every row. Since the polynomials separate points on Z+, these two matrices coincide. Proposition 6.5. For any c, r > 0, the matrix Q(c) r satisﬁes the assumptions of Theorem 2.3 with functions γ(k) = η(k) = k + 1. Proposition 6.5. For any c, r > 0, the matrix Q(c) r satisﬁes the assumptions of Theorem 2.3 with functions γ(k) = η(k) = k + 1. Page 20/43 ejp.ejpecp.org EJP 18 (2013), paper 75. ejp.ejpecp.org ejp.ejpecp.org Markov dynamics on the Thoma cone EJP 18 (2013), paper 75. 6.4 The Laguerre differential operator Proposition 6.9 implies that the generator A(c) is uniquely determined by its action on functions of the form qxxm, m = 0, 1, 2, . . . , with an arbitrary ﬁxed q ∈(0, 1). This action can be readily computed from the basic relation A(c) BΛ∞ r = BΛ∞ r Q(c) r : Proposition 6.10. The action of A(c) on functions of the form qxxm, m = 0, 1, 2, . . . is implemented by the differential operator D(c) := x d2 dx2 + (c −x) d dx . (6.12) (6.12) Proof. Let r > 0 be related to q ∈(0, 1) by r = −log q. Consider the functions > 0 be related to q ∈(0, 1) by r = −log q. Consider the functions fm(x) := rm m! qxxm = rm m! e−rxxm, m = 0, 1, 2, . . . , x ∈R+. By (6.6), By (6.6), BΛ∞ r 1m = fm, m = 0, 1, 2, . . . . BΛ∞ r 1m = fm, m = 0, 1, 2, . . . . On the other hand, it is directly veriﬁed that the difference operator Q(c) r deﬁned in (6.8) acts on the delta functions 1m in the same way as the differential operator D(c) acts on the functions fm: Q(c) r 1m = r(c + m −1)1m−1 + (r + 1)(m + 1)1m+1 −[(2r + 1)m + rc]1m (6.13) D(c)fm = r(c + m −1)fm−1 + (r + 1)(m + 1)fm+1 −[(2r + 1)m + rc]fm, (6.14) where 1−1 := 0, f−1 := 0. 1−1 := 0, f−1 := 0. This concludes the proof. Consider the gamma distribution on R+ with parameter c: Consider the gamma distribution on R+ with parameter c: 1 Γ(c)xc−1e−xdx, x ∈R+, 1 Γ(c)xc−1e−xdx, x ∈R+, and let Ln(x; c) denote the monic Laguerre polynomials of degree n = 0, 1, 2, . . . , which are orthogonal with respect to this distribution: Ln(x; c) = (c)n n X m=0 (−1)n−m n↓m (c)mm!xm. (6.15) (6.15) The differential operator D(c) is diagonalized in the basis of the Laguerre polynomi- l The differential operator D(c) is diagonalized in the basis of the Laguerre polynomi- als: D(c)Ln( · ; c) = −nLn( · ; c), n = 0, 1, 2, . . . . (6.16) Note also that BΛ∞ r Mn( · ; c, r) = rnLn( · ; c). 6.3 A family of cores for Markov semigroup generators (i) Observe that if r2 > r1 > 0 and F1 is a core for A(c) r1 , then F2 := BΛr2 r1F1 is a core for A(c) r2 . Indeed, by virtue of claim (i) of Proposition 6.8, we may apply the argument of Proposition 5.2. Now take r2 = r and r1 = (1 −q)r. Then, as seen from (6.3), the linear span of functions qyym is just the image under BΛr2 r1 of the space Cc(Z+). By virtue of Proposition EJP 18 (2013), paper 75. Page 21/43 Page 21/43 Markov dynamics on the Thoma cone 6.5 and claim (iv) of Theorem 2.3, Cc(Z+) is a core for A(c) r1 . Therefore, its image is a core for A(c) r2 . Next, given q ∈(0, 1) we take r = −log q and F = C0(Z+). As mentioned above, F is a core for A(c) r . On the other hand, (6.6) shows that the linear span of functions qxxm coincides with BΛ∞ r F. EJP 18 (2013), paper 75. 7 A few deﬁnitions Here we collect some basic deﬁnitions that will be needed in the next section. For a more detailed information we refer to Sagan [36] and Stanley [39] (generalities on Young diagrams, Young tableaux, and symmetric functions); Olshanski–Regev–Vershik [33] (Frobenius–Schur symmetric functions); Borodin–Olshanski [14], [12] (Thoma’s simplex and Thoma’s cone). 6.5 Approximation We use the embedding ϕr : Z+ →R+ introduced in Example 4.3 and deﬁne the projection πr : C0(R+) →C0(Z+) as in Section 5.2. Proposition 6.11. Let c > 0 be ﬁxed. As r →+∞, the Meixner semigroups T (c) r (t) approximate the Laguerre semigroup T (c)(t) in the sense of Deﬁnition 3.2. Proof. Let us check all the hypotheses of Proposition 5.5. Then the desired result will follow from that proposition. In fact, the assumptions stated in Section 5.1 are satisﬁed: we know that B is a Feller system, the Meixner semigroups are consistent with the links of B, those are Feller links, and, by the very deﬁnition, the Laguerre semigroup is the boundary semigroup determined by the Meixner semigroups. Next, the fulﬁlment of Condition 4.1 was established in Example 4.3. It remains to check Condition 5.4. In our situation, it consists in the requirement that Cc(Z+) is a core for generator A(c) r and, moreover, is invariant under its action. The fact that Cc(Z+) is a core follows from Corollary 6.6, item (ii). Its invariance follows from item (i), because Cc(Z+) is obviously invariant under the action of Q(c) r . This completes the proof. Markov dynamics on the Thoma cone Markov dynamics on the Thoma cone in the poset Y. in the poset Y. More generally, for arbitrary two diagrams µ, λ ∈Y we deﬁne dim(µ, λ) as the num- ber of standard Young tableaux of skew shape λ/µ provided that µ ⊆λ; otherwise dim(µ, λ) = 0 (let us agree that dim(λ, λ) = 1). Obviously, dim λ = dim(∅, λ). If µ ⊂λ, then dim(µ, λ) equals the number of saturated chains with ends µ and λ. EJP 18 (2013), paper 75. 7.1 Young diagrams Recall that the Young poset is the set Y of all Young diagrams (including the empty diagram ∅) with the partial order determined by containment of one Young diagram in another. For λ ∈Y we denote by \|λ\| the number of boxes of λ and we set Yn = {λ ∈Y : \|λ\| = n}, n = 0, 1, 2, . . . . Yn = {λ ∈Y : \|λ\| = n}, n = 0, 1, 2, . . . . This makes Y a graded poset. It is actually a lattice, so it is often called the Young lattice. The dimension of a diagram λ ∈Y, denoted by dim λ, is the number of standard Young tableaux of shape λ, which is the same as the number of saturated chains ∅= λ(0) ⊂λ(1) ⊂· · · ⊂λ(n) = λ, n := \|λ\|, ∅= λ(0) ⊂λ(1) ⊂· · · ⊂λ(n) = λ, n := \|λ\|, 6.4 The Laguerre differential operator (6.17) (6.17) The proof is immediate: we compare the expansions of the Meixner and Laguerre poly- nomials in the bases {l↓m} and {xm}, respectively (see (6.9) and (6.15)), and then apply (6.5), which says that BΛ∞ r takes the factorial monomial l↓m to rmxm. EJP 18 (2013), paper 75. Page 22/43 ejp.ejpecp.org EJP 18 (2013), paper 75. on the Thoma cone introduced in Example 4.3 an ion 5.2. s r →+∞, the Meixner semig t) in the sense of Deﬁnition 3.2. Proposition 5.5. Then the desire n 5.1 are satisﬁed: we know that istent with the links of B, tho erre semigroup is the boundar Markov dynamics on the Thoma cone Sµ’s and FSµ’s is similar to the relationship between the one-variate monomials xm and their factorial counterparts x↓m. Observe that x↓m can be characterized as a unique polynomial in x with highest degree term xm and such that it vanishes at the integer points 0, 1 . . . , m −1. Likewise, one can realize Sym as a subalgebra in Fun(Y), the algebra of real-valued functions on Y with all operations deﬁned pointwise; see the next two paragraphs. Then FSµ can be characterized as a unique element of Sym that has top degree term Sµ and vanishes at all diagrams strictly contained in µ. Let p1, p2, . . . denote the power-sum symmetric functions. We turn them into func- tions on Y by setting pk(λ) := ∞ X i=1 (λr −i + 1 2)k −(−i + 1 2)k = d X i=1 ak r + (−1)k−1bk r , where λ ranges over Y, (λ1, λ2, . . . ) is the partition corresponding to λ, d is the number of boxes on the main diagonal of λ, and (a1, . . . , ad; b1, . . . , bd) is the collection of the modiﬁed Frobenius coordinates of λ: ar = λr −i + 1 2, br = λ′ r −i + 1 2, i = 1, . . . , d ar = λr −i + 1 2, br = λ′ r −i + 1 2, i = 1, . . . , d (here λ′ is the transposed diagram). One can easily prove that the resulting functions remain algebraically independent. Next, every element F ∈Sym is uniquely written as a polynomial in p1, p2, . . . ; then we deﬁne F(λ) as the same polynomial in numeric variables p1(λ), p2(λ), . . . . In this way we get the desired embedding of Sym into Fun(Y). y g g y ( ) A fundamental property of the Frobenius–Schur functions is the following identity (see [33, Section 2]) relating them to the dimension function in the poset Y: l↓m dim(µ, λ) dim λ = FSµ(λ), l := \|λ\|, m := \|µ\|. (7.1) (7.1) 7.2 Symmetric functions By Sym we denote the graded algebra of symmetric functions over the base ﬁeld R. We will need two bases in Sym, both indexed by arbitrary diagrams µ ∈Y: the Schur functions Sµ and the Frobenius–Schur functions FSµ. The relationship between EJP 18 (2013), paper 75. Page 23/43 ejp.ejpecp.org Page 23/43 Page 23/43 Markov dynamics on the Thoma cone 7.4 Two maps We embed Sym into the algebra of (non necessarily bounded) continuous functions on the Thoma cone by setting pk(ω) = (P∞ i=1 αk i + (−1)k−1 P∞ i=1 βk i , k = 2, 3, . . . \|ω\|, k = 1, where ω ranges over eΩ. We embed the set Y into eΩthrough the map λ 7→ωλ := ((a1, . . . , ad, 0, 0, . . . ), (b1, . . . , bd, 0, 0, . . . ), \|λ\|), λ 7→ωλ := ((a1, . . . , ad, 0, 0, . . . ), (b1, . . . , bd, 0, 0, . . . ), \|λ\|), where, as above, (a1, . . . , ad; b1, . . . , bd) is the collection of the modiﬁed Frobenius coor- dinates of a diagram λ ∈Y. Note that \|ωλ\| = \|λ\|. e \| \| \| \| For any F ∈Sym, the restriction of the function F(ω) to the subset Y ⊂eΩagrees with the previous deﬁnition of the function F(λ): F(ωλ) = F(λ), λ ∈Y. 7.3 The Thoma simplex and the Thoma cone The Thoma simplex is the subspace Ωof the inﬁnite product space R∞ + ×R∞ + formed by all couples (α, β), where α = (αi) and β = (βi) are two inﬁnite sequences such that α1 ≥α2 ≥· · · ≥0, β1 ≥β2 ≥· · · ≥0 (7.2) (7.2) and and ∞ X i=1 αi + ∞ X i=1 βi ≤1. (7.3) (7.3) We equip Ωwith the product topology inherited from R∞ + × R∞ + . Note that in this topology, Ωis a compact metrizable space. e We equip Ωwith the product topology inherited from R∞ + × R∞ + . Note that in this topology, Ωis a compact metrizable space. e The Thoma cone eΩis the subspace of the inﬁnite product space R∞ + × R∞ + × R+ formed by all triples ω = (α, β, δ), where α = (αi) and β = (βi) are two inﬁnite sequences and δ is a nonnegative real number, such that the couple (α, β) satisﬁes (7.2) and the modiﬁcation of the inequality (7.3) of the form ∞ X i=1 αi + ∞ X i=1 βi ≤δ. We set \|ω\| = δ. e We set \|ω\| = δ. e We set \|ω\| = δ. e We set \|ω\| = δ. e \| \| Note that eΩis a locally compact space in the product topology inherited from R∞ + × R∞ + × R+. The space eΩis also metrizable and has countable base. Every subset of the EJP 18 (2013), paper 75. Page 24/43 ejp.ejpecp.org EJP 18 (2013), paper 75. ejp.ejpecp.org Page 24/43 Markov dynamics on the Thoma cone form {ω ∈eΩ: \|ω\| ≤const} is compact. Therefore, a sequence of points ωn goes to inﬁnity in eΩif and only if \|ωn\| →∞. \| \| We will identify Ωwith the subset of eΩformed by triples ω = (α, β, δ) with δ = 1. The name “Thoma cone” given to eΩis justiﬁed by the fact that eΩmay be viewed as the cone with the base Ω: the ray of the cone passing through a base point (α, β) ∈Ωconsists of the triples ω = (rα, rβ, r), r ≥0. ( ) More generally, for ω = (α, β, δ) ∈eΩand r > 0 we set rω = (rα, rβ, rδ). 8 Construction of Feller processes on the Thoma cone 8.1 The projective system associated with the Young bouquet EJP 18 (2013), paper 75. 8.1 The projective system associated with the Young bouquet (8.3) l i (8.3) The links (8.1) satisfy the relation The links (8.1) satisfy the relation The links (8.1) satisfy the relation YBΛr′′ r′ YBΛr′ r = YBΛr′′ r , r′′ > r′ > r, YBΛr′′ r′ YBΛr′ r = YBΛr′′ r , r′′ > r′ > r, so that they do determine a projective system. We refer to [14] for more details. e so that they do determine a projective system. We refer to [14] for more details. e so that they do determine a projective system. We refer to [14] for more details. By [14, Theorem 3.4.7], the boundary of YB is the Thoma cone eΩtogether with a family of links eΩ99K Z+ indexed by positive real numbers r and given by By [14, Theorem 3.4.7], the boundary of YB is the Thoma cone eΩtogether with a family of links eΩ99K Z+ indexed by positive real numbers r and given by YBΛ∞ r (ω, µ) = e−r\|ω\| rm m! dim µ · Sµ(ω), ω ∈eΩ, µ ∈Y. (8.4) (8.4) Recall that Sµ is the Schur symmetric function and its value at ω ∈eΩis understood in accordance with the deﬁnition given in Section 7.4. Recall that Sµ is the Schur symmetric function and its value at ω ∈eΩis understood in accordance with the deﬁnition given in Section 7.4. Proposition 8.1. The projective system YB is Feller in the sense of the deﬁnition given in Section 4.3. Proof. The links YBΛr′ r and YBΛ∞ r are Feller: this immediately follows from the Feller property of the links BΛr′ r and BΛ∞ r . It remains to show that the product topology of the space eΩcoincides with that deﬁned by all the maps ω 7→Λ∞ r (ω, µ), where r ranges over R>0 and µ ranges over Y. Actually, this holds even if r is any ﬁxed number > 0, and the argument is similar to that given in Section 4.3. e e Namely, we extend the above maps to the one-point compactiﬁcation eΩ∪∞of eΩin a natural way: the value at inﬁnity is equal to 0 for any µ, which agrees with the Feller property of the links. Then we only have to check that any point of eΩ∪∞is uniquely determined by its images under the (extended) maps YBΛ∞ r ( · , µ), where µ ranges over Y. To do this, assume ﬁrst that ω ∈eΩand recall (8.4). 8.1 The projective system associated with the Young bouquet The representation theory of inductive limit groups provides two fundamental ex- amples of projective systems. One is related to the inﬁnite symmetric group S(∞) and comes from the Young graph Y, and the other one is related to the inﬁnite-dimensional unitary group U(∞) and comes from the Gelfand–Tsetlin graph GT. The boundaries of these two projective systems can be viewed as dual objects to S(∞) and U(∞), re- spectively. In attempt to explain a surprising similarity between the two boundaries, we introduced in [14] a new object which serves as a “mediator” between Y and GT. We called it the Young bouquet; it is a close relative of Y and at the same time it can be ob- tained as a degeneration of GT. Associated with the Young bouquet is a new projective system denoted by YB. Because GT is graded by discrete set Z+, the associated pro- jective system has Z+ as its index set, but under degeneration the index set becomes continuous. Here is a formal deﬁnition of YB: The index set of the projective system YB is the set R>0 and each set Er is a copy of the set Y. For every couple r′ > r of positive real numbers, the corresponding link Y 99K Y is the following stochastic matrix of format Y × Y: YBΛr′ r (λ, µ) = 1 −r r′ l−m r r′ m l! (l −m)! m! dim µ dim(µ, λ) dim λ , (8.1) (8.1) where l := \|λ\| and m := \|µ\|. where l := \|λ\| and m := \|µ\|. \| \| \| \| Note that (8.1) factorizes into a product of two links, which refer to two projective systems, the binomial system B and the Young graph Y: Note that (8.1) factorizes into a product of two links, which refer to two projective systems, the binomial system B and the Young graph Y: YBΛr′ r (λ, µ) = BΛr′ r (l, m) YΛl m(λ, µ), (8.2) (8.2) JP 18 (2013), paper 75. Page 25/43 ejp.ejpecp.org EJP 18 (2013), paper 75. Page 25/43 ejp.ejpecp.org Markov dynamics on the Thoma cone where YΛl m(λ, µ) := dim µ dim(µ, λ) dim λ . EJP 18 (2013), paper 75. Proposition 8.2 (cf. Proposition 6.1). Assume that: Proposition 8.2 (cf. Proposition 6.1). Assume that: • r′ > r > 0 and 0 < q < 1; • λ range over Y and l := \|λ\|; • µ ∈Y is ﬁxed and m = \|µ\|. • r′ > r > 0 and 0 < q < 1; • r′ > r > 0 and 0 < q < 1; • λ range over Y and l := \|λ\|; • λ range over Y and l := \|λ\|; • µ ∈Y is ﬁxed and m = \|µ\|. • µ ∈Y is ﬁxed and m = \|µ\|. Regard YBΛr′ r as linear map F 7→G transforming a function F(λ) on Y to another function G(λ). Under this transformation FSµ(λ) 7→ r r′ m FSµ(λ), (8.5) (dim µ)−11µ 7→ 1 m! 1 −q′ q′ m · (q′)lFSµ(λ), q′ := 1 −r r′ , (8.6) qlFSµ(λ) 7→ qr q′r′ m (q′)lFSµ(λ), q′ := 1 −(1 −q) r r′ . (8.7) (8.5) (8.7) Proof. Let us prove (8.7). The function F(λ) := qlFSµ(λ) vanishes unless λ ⊇µ, and the same holds for YBΛr′ r F , because the matrix YBΛr′ r is lower triangular with respect to the partial order on Y determined by the inclusion relation. Therefore, it sufﬁces to compute (YBΛr′ r F)(λ) for λ ⊇µ; in particular, l ≥m. We have (YBΛr′ r F)(λ) = l X k=m BΛr′ r (l, k) X κ∈Yk YΛl k(λ, κ)qkFSµ(κ). For ﬁxed k, YΛl k(λ, κ)qkFSµ(κ) = qk dim κ dim(κ, λ) dim λ k↓m dim(µ, κ) dim κ by virtue of (7.1) = qkk↓m dim(µ, κ) dim(κ, λ) dim λ , and summing the latter quantity over κ ∈Yk gives qkk↓m dim(µ, λ) dim λ . Therefore, Therefore, (YBΛr′ r F)(λ) = k↓m dim(µ, λ) dim λ · l X k=m BΛr′ r (l, k)qk = k↓m dim(µ, λ) dim λ · qr q′r′ m (q′)ll↓m by (6.4) = qr q′r′ m (q′)lFSµ(λ), 8.1 The projective system associated with the Young bouquet Keeping m ﬁxed and summing the quantity in the right-hand side over µ ∈Ym we get e−rx (rx)m m! , x := \|ω\|, X µ∈Ym dim µ Sµ = (p1)m because because and p1(ω) = \|ω\| = x. and p1(ω) = \|ω\| = x. p1( ) \| \| Observe that for r > 0 ﬁxed, the quantities e−rx (rx)m m! , where m ranges over Z+, determine x uniquely. It follows, in particular, that we can recognize whether we are dealing with an element of the Thoma cone eΩor the added point ∞, because the latter case corresponds to x = +∞. e Therefore, it sufﬁces to check that an element ω ∈eΩis uniquely determined by the quantities Sµ(ω), where µ ranges over Y. But this follows from the fact that the functions p1(ω), p2(ω), . . . separate the points of the Thoma cone. Note that YBΛr′ r (ν, µ) vanishes unless m ≤n and µ ⊆λ. This implies that each row of the matrix YBΛr′ r has ﬁnitely many nonzero entries, so that the link can be applied to an arbitrary function on Y. Below we denote by 1µ the delta function on Y concentrated at the point µ, that is, Below we denote by 1µ the delta function on Y concentrated a 1µ(λ) = ( 1, λ = µ, 0, λ ̸= µ. 1µ(λ) = ( 1, λ = µ, 0, λ ̸= µ. EJP 18 (2013), paper 75. Page 26/43 ejp.ejpecp.org EJP 18 (2013), paper 75. Page 26/43 ejp.ejpecp.org Markov dynamics on the Thoma cone Markov dynamics on the Thoma cone Markov dynamics on the Thoma cone Proposition 8.2 (cf. Proposition 6.1). Assume that: 8.2 Markov semigroups on Y and eΩ We are going to introduce a Q-matrix of format Y×Y depending on the triple (z, z′, r) of parameters, where r > 0 and (z, z′), as usual, is subject to Condition 1.1. For this we need some notation. Given λ ∈Y, let λ+ and λ−stand for the collections of boxes that can appended to, respectively, removed from λ. For a box 2, its content is deﬁned as the difference c(2) := j −i, where i and j are the row and column numbers of 2. The Q-matrix in question is denoted by Q(z,z′) r and its non-diagonal entries Q(z,z′) r (λ, κ), κ ̸= λ, vanish unless either κ = λ + 2 or κ = λ −2, meaning that κ is obtained from λ by appending a box 2 ∈λ+ or by removing a box 2 ∈λ−. In this notation, the entries are given by Q(z,z′) r (λ, λ + □) = r(z + c(□))(z′ + c(□)) dim(λ + □) (\|λ\| + 1) dim λ, □∈λ+, Q(z,z′) r (λ, λ −□) = (r + 1)\|λ\| dim(λ −□) dim λ , □∈λ−, −Q(z,z′) r (λ, λ) = (2r + 1)\|λ\| + rzz′. (8.11) (8.11) Note that each row of Q(z,z′) r has ﬁnitely many nonzero entries which sum to 0, and the constraints on the parameters imply that all off-diagonal entries are nonnegative (in particular, Q(z,z′) r (λ, λ + 2) > 0 because of Condition 1.1). ′ (For more detail about the deﬁnition of Q(z,z′) r , we refer to Borodin–Olshanski [11] and Olshanski [32]. Formula (8.11) coincides with that of [32, Proposition 4.25] and is a particular case of [11, (2.19)]. Note that parameter ξ ∈(0, 1) from those two papers is related to our parameter r > 0 by ξ = r(r + 1)−1. In [11, (2.19)], parameter ξ may vary with time; our setup corresponds to the particular case when ξ is ﬁxed, so that the time derivative ˙ξ equals 0. Then formula [11, (2.19)] simpliﬁes and reduces to (8.11).) ′ We can interpret Q(z,z′) r as an operator in the vector space Fun(Y) formed by arbi- trary real-valued functions on Y: (Q(z,z′) r F)(λ) = X κ∈Y Q(z,z′) r (λ, κ)F(κ), F ∈Fun(Y). (8.12) (8.12) As explained in [32], this operator should be viewed as a counterpart of the Meixner difference operator on Z+. as desired. as desired. as desired. Formula (8.5) can be checked in exactly the same way. Alternatively, it can be ob- tained a limit case of (8.7) as q →1. ′ Formula (8.6) is immediate from the very deﬁnition of YBΛr′ r and FSµ. Alternatively, (8.6) can also be obtained from (8.7) by a degeneration, like the derivation of (6.3) from (6.4), see the proof of Proposition 6.1. Proposition 8.3 (cf. Proposition 6.2). Assume r > 0 and 0 < q < 1; let λ range over Y and l = \|λ\|; let ω range over eΩand x = \|ω\|; let µ ∈Y be ﬁxed and m = \|µ\|. Regard EJP 18 (2013), paper 75. Page 27/43 ejp.ejpecp.org Page 27/43 Markov dynamics on the Thoma cone YBΛ∞ r as an operator transforming a function F(λ) on Y to a function G(ω) on eΩ. Under this transformation YBΛ∞ r as an operator transforming a function F(λ) on Y to a function G(ω) on eΩ. Under this transformation FSµ(λ) 7→rmSµ(ω), (8.8) (dim µ)−11µ 7→rm m! · qx ∞Sµ(ω), q∞:= e−r, (8.9) qlFSµ(λ) 7→qmrm · qx ∞Sµ(ω), q∞:= e−(1−q)r. (8.10) Proof. We may argue exactly as in the proof of the previous proposition. Proof. We may argue exactly as in the proof of the previous proposition. 8.2 Markov semigroups on Y and eΩ The next step is to introduce counterparts of the Meixner polynomials. According to [32, Deﬁnition 4.21], these are elements of Sym called the Meixner symmetric functions and denoted by Mν, where the index ν ranges over Y. They depend on the triple (z, z′, r) and are given by the following expansion in the basis of the Frobenius–Schur symmetric functions (cf. (6.9)): M(z,z′,r) ν = X µ: µ⊆ν (−1)\|ν\|−\|µ\|r\|ν\|−\|µ\| dim ν/µ (\|ν\| −\|µ\|)! × Y □∈ν/µ (z + c(□))(z′ + c(□)) · FSµ. (8.13) (8.13) EJP 18 (2013), paper 75. Page 28/43 ejp.ejpecp.org age 28/43 ejp.ejpecp.org EJP 18 (2013), paper 75. ejp.ejpecp.org Page 28/43 Markov dynamics on the Thoma cone Proposition 8.4 (cf. Proposition 6.3). Under the action of Q(z,z′) r in Fun(Y), FSµ →−\|µ\|FSµ + r X 2∈µ− (z + c(2))(z′ + c(2))FSµ\2, (8.14) M(z,z′,r) µ →−\|µ\|Mµ. (8.15) µ \| \| µ X 2∈µ− ( ( ))( ( )) µ\ M(z,z′,r) µ →−\|µ\|Mµ. (8.15) M(z,z′,r) µ →−\|µ\|Mµ. (8.15) Proof. See [32, Section 4.8]. Proof. See [32, Section 4.8]. Proof. See [32, Section 4.8]. Proof. See [32, Section 4.8]. Proposition 8.5 (cf. Proposition 6.4). For arbitrary r′ > r > 0, we have Qz,z′ r′ YBΛr′ r = YBΛr′ r Qz,z′ r . Qz,z′ r′ YBΛr′ r = YBΛr′ r Qz,z′ r . Proof. We literally follow the argument in the proof of Proposition 6.4. From the deﬁni- tion of the Meixner symmetric functions and (8.5) it is readily seen that YBΛr′ r M(z,z′,r) ν = r r′ \|ν\| M(z,z′,r′) ν and then (8.15) implies that the both sides of the operator equality in question give the same result when applied to M(z,z′,r) ν . Therefore, the equality holds on all elements of Sym. As these elements separate points of Y, this concludes the proof. Proposition 8.6 (cf. Proposition 6.5). The matrix Q(z,z′) r satisﬁes the assumptions of Theorem 2.3 with functions γ(λ) = η(λ) = \|λ\| + 1, λ ∈Y. Proof. As seen from the description of the Q-matrix given in [11, Section 2.5] (see the sentence just before [11, Proposition 2.11]), for any λ ∈Y one has X 2∈λ± Q(z,z′) r (λ, λ ± 2) = Q(c) r (\|λ\|, \|λ\| ± 1), c := zz′ (note that c > 0 because of Condition 1.1). EJP 18 (2013), paper 75. 8.2 Markov semigroups on Y and eΩ This implies that the action of Q(z,z′) r pre- serves the subspace in Fun(Y) formed by those functions in variable λ ∈Y that depend only on \|λ\|, and in that subspace, the action reduces to that of the difference operator Q(c) r with c = zz′. Therefore, the claim of the proposition reduces to that of Proposition 6.5. Combining this proposition with Theorem 2.3 we get Corollary 8.7 (cf. Corollary 6.6). (i) The Q-matrix Q(z,z′) r gives rise to a Feller semi- group T (z,z′) r (t) on C0(Y) whose generator A(z,z′) r is implemented by Q(z,z′) r . ( ′) Corollary 8.7 (cf. Corollary 6.6). (i) The Q-matrix Q(z,z′) r gives rise to a Feller semi- group T (z,z′) r (t) on C0(Y) whose generator A(z,z′) r is implemented by Q(z,z′) r . ( ′) (ii) The subspace C0(Y) is a core for generator A(z,z′) r . Proposition 8.8 (cf. Proposition 6.7). For every couple (z, z′) of parameters subject to Condition 1.1 there exists a unique Feller semigroup T (z,z′)(t) such that for every r > 0, Proposition 8.8 (cf. Proposition 6.7). For every couple (z, z′) of parameters subject to Condition 1.1 there exists a unique Feller semigroup T (z,z′)(t) such that for every r > 0, T (z,z′)(t) is consistent with the T (z,z′) r (t), r > 0, in the sense that ( ) T (z,z′)(t) is consistent with the T (z,z′) r (t), r > 0, in the sense that T (z,z′)(t) is consistent with the T (z,z′) r (t), r > 0, in the sense that T (z,z′)(t) YBΛ∞ r = YBΛ∞ r T (z,z′) r (t), t ≥0, r > 0. Proof. The argument is exactly the same as in Proposition 6.7: We know that the Q- matrices with various values of parameter r are consistent with the links (Proposition 8.5). It follows, by virtue of Proposition 5.3, that the semigroups are consistent with the links, too. Therefore, we may apply Proposition 5.1, which gives the desired result. Deﬁnition 8.9. For r > 0, we denote by X(z,z′) r the Feller Markov process on Y de- termined by the semigroup T (z,z′) r (t). Likewise, we denote by X(z,z′) the Feller Markov process on eΩdetermined by the semigroup T (z,z′)(t). EJP 18 (2013), paper 75. ejp.ejpecp.org ejp.ejpecp.org Page 29/43 Page 29/43 Markov dynamics on the Thoma cone 8.3 A family of cores for Markov semigroup generators The following two claims are used in the proposition below. First, let ω range over the Thoma cone eΩand µ range over Y. For any ﬁxed q ∈(0, 1), the functions q\|ω\|Sµ(ω) span a dense subspace in C0(eΩ), see [14, Corollary 3.4.6]. Second, let λ range over Y. Recall that in Section 7.4 we deﬁned an embedding Y ,→eΩvia the map λ 7→ωλ. Observe that \|λ\| = \|ωλ\|; this implies that a sequence {λ} of diagrams goes to inﬁnity in the discrete set Y if and only if its image {ωλ} goes to inﬁnity in the locally compact space eΩ. Combining this with the ﬁrst claim we conclude that for any ﬁxed q ∈(0, 1), the functions q\|λ\|Fµ(λ) span a dense subspace in C0(Y). Proposition 8.10 (cf. Proposition 6.8). (i) For any r′ > r > 0, the operator YBΛr′ r : C0(Y) →C0(Y) has a dense range. Proposition 8.10 (cf. Proposition 6.8). (i) For any r′ > r > 0, the operator YBΛr′ r : C0(Y) →C0(Y) has a dense range. Proposition 8.10 (cf. Proposition 6.8). (i) For any r′ > r > 0, the operator YBΛr′ r : C0(Y) →C0(Y) has a dense range. (ii) Likewise, for any r > 0, the operator YBΛ∞ r : C0(Y) →C0(eΩ) has a dense range. (ii) Likewise, for any r > 0, the operator YBΛ∞ r : C0(Y) →C0(eΩ) has a dense range. Proof. (i) Fix an arbitrary q ∈(0, 1) and let µ range over Y. By (8.7), YBΛr′ r maps the linear span of functions q\|λ\|Sµ(λ) onto the linear span of functions (q′)\|λ\|Sµ(λ) with some other q′ ∈(0, 1). Since these spans are dense, we get the desired claim. Proof. (i) Fix an arbitrary q ∈(0, 1) and let µ range over Y. By (8.7), YBΛr′ r maps the linear span of functions q\|λ\|Sµ(λ) onto the linear span of functions (q′)\|λ\|Sµ(λ) with some other q′ ∈(0, 1). Since these spans are dense, we get the desired claim. (ii) The same argument, with reference to (8.10). Denote by A(z,z′) r and A(z,z′) the generators of the semigroups T (z,z′) r (t) and T (z,z′)(t), respectively. Proposition 8.11 (cf. Proposition 6.9). Fix an arbitrary number q ∈(0, 1) and let µ range over Y. (i) For every r > 0, the linear span of functions q\|λ\|Sµ(λ), where argument λ ranges over Y, is a core for A(z,z′) r . EJP 18 (2013), paper 75. 8.4 The inﬁnite-variate Laguerre differential operator Following [32, Theorem 4.10], we introduce the following partial differential opera- tor in countably many formal variables e1, e2, . . . : D(z,z′) = X n≥1 n−1 X k=0 (2n −1 −2k)e2n−1−kek ! ∂2 ∂e2n + 2 X n′>n≥1 n−1 X k=0 (n′ + n −1 −2k)en′+n−1−kek ! ∂2 ∂en′∂en + ∞ X n=1 −nen + (z −n + 1)(z′ −n + 1)en−1 ∂ ∂en (8.16) with the agreement that e0 = 1. We call it the inﬁnite-variate Laguerre differential operator. Since all coefﬁcients of D(z,z′) are given by ﬁnite sums, D(z,z′) is applicable to any polynomial in e1, e2, . . . . This means that it is well deﬁned on Sym provided that we interpret our formal variables as the elementary symmetric functions (here we use the fact that {e1, e2, . . . } is a system of algebraically independent generators of Sym). But D(z,z′) is also applicable to more general cylinder functions, in particular, to the func- tions of the form qe1F , where q ∈(0, 1) and F ∈Sym. Note that e1(ω) = \|ω\|, so that these are just the functions considered in claim (ii) of Proposition 8.11. By virtue of this claim, for any ﬁxed q = e−r ∈(0, 1), the functions of the form qe1F with F ∈Sym enter the domain of the generator A(z,z′), and A(z,z′) is uniquely determined by its action on these functions. Proposition 8.12 (cf. Proposition 6.10). For any r > 0, the action of the generator A(z,z′) on the functions of the form exp(−re1)F with F ranging over the algebra Sym = R[e1, e2, . . . ] is implemented by the inﬁnite-variate Laguerre differential operator D(z,z′) deﬁned by (8.16). Proof. Let µ range over Y and m := \|µ\|. Recall that 1µ denotes the delta function on Y concentrated at the point µ. We also set e1µ = (dim µ)−11µ and and fµ = rm m! exp(−re1) Sµ. By virtue of (8.9), YBΛ∞ r e1µ = fµ. Recall also that µ+ and µ−denote the sets of boxes that can be appended to or removed from µ, respectively. Recall also that µ+ and µ−denote the sets of boxes that can be appended to or removed from µ, respectively. from µ, respectively. 8.3 A family of cores for Markov semigroup generators (i) For every r > 0, the linear span of functions q\|λ\|Sµ(λ), where argument λ ranges over Y, is a core for A(z,z′) r . (ii) Likewise, the linear span of functions q\|ω\|Sµ(ω), where argument ω ranges over eΩ, is a core for A(z,z′). Proof. (i) Observe that if r2 > r1 > 0 and F1 is a core for A(z,z′) r1 , then F2 := YBΛr2 r1F1 is a core for A(z,z′) r2 . Indeed, by virtue of claim (i) of Proposition 8.10, we may apply the argument of Proposition 5.2. Now take r2 = r and r1 = (1 −q)r. Then, as is seen from (8.6), the linear span of functions q\|λ\|Sµ(λ) is just the image under YBΛr2 r1 of the space Cc(Y). By virtue of Proposition 8.6 and claim (iv) of Theorem 2.3, Cc(Y) is a core for A(z,z′) r1 . Therefore, its image is a core for A(z,z′) r2 . (ii) We argue as above. First, application of claim (ii) of Proposition 8.10 allows us to conclude that if F ⊂C0(Y) is a core for A(z,z′) r for some r > 0, then YBΛ∞ r F is a core for A(z,z′). Next, given q ∈(0, 1) we take r = −log q and F = C0(Y). As pointed above, F is a core for A(z,z′) r . On the other hand, (8.9) shows that the linear span of functions q\|ω\|Sµ(ω) coincides with YBΛ∞ r F. Page 30/43 ejp.ejpecp.org ejp.ejpecp.org Page 30/43 Page 30/43 Markov dynamics on the Thoma cone Markov dynamics on the Thoma cone 8.4 The inﬁnite-variate Laguerre differential operator We are going to prove the following analogs of formulas (6.13) and (6.14): We are going to prove the following analogs of formulas (6.13) and (6.14): Q(c) r e1µ = −[(2r + 1)m + rzz′]e1µ + (r + 1)(m + 1) X 2∈µ+ e1µ+2 + r m X 2∈µ− (z + c(2))(z′ + c(2))e1µ−2 (8.17) EJP 18 (2013), paper 75. Page 31/43 EJP 18 (2013), paper 75. Page 31/43 ejp.ejpecp.org EJP 18 (2013), paper 75. ejp.ejpecp.org Page 31/43 Markov dynamics on the Thoma cone and and D(z,z′)fµ = −[(2r + 1)m + rzz′]fµ + (r + 1)(m + 1) X 2∈µ+ fµ+2 + r m X 2∈µ− (z + c(2))(z′ + c(2))fµ−2 (8.18) (8.18) (for the empty diagram µ, the set µ−is empty and the corresponding sum disappears). These formulas show that the operator Q(z,z′) r acts on the functions e1µ in exactly the same way as the operator D(z,z′) acts on the functions fµ, which implies the claim of the proposition. (for the empty diagram µ, the set µ−is empty and the corresponding sum disappears). These formulas show that the operator Q(z,z′) r acts on the functions e1µ in exactly the same way as the operator D(z,z′) acts on the functions fµ, which implies the claim of the proposition. The proof of (8.17) is trivial: this formula directly follows from the very deﬁnition of Q(z,z′) r , see (8.11). The proof of (8.18) is a bit more complicated. Observe that if a second order partial differential operator D, symbolically written as D = X i,i cij∂i∂j + ﬁrst order terms, is applied to a product of two functions, GF , then the result can be written as the sum of three expressions: is applied to a product of two functions, GF , then the result can be written as the sum of three expressions: D(GF) = (DF)G \| {z } 1 + G(DF) \| {z } 2 + X i,j cij[(∂iG)(∂jF) + (∂jG)(∂iF)] \| {z } . (8.19) (8.19) Let us apply this general formula to Let us apply this general formula to D := D(z,z′), G := rm m! e−re1, F := Sµ and examine the corresponding three expressions arising from (8.19). h ﬁ l and examine the corresponding three expressions arising from (8.19). 1. The ﬁrst expression is equal to and examine the corresponding three expressions arising from (8.19). 1. 8.4 The inﬁnite-variate Laguerre differential operator The ﬁrst expression is equal to rm m! D(z,z′)e−re1 Sµ = rm m! e1 d2 de2 1 + (−e1 + zz′) d de1 e−re1 Sµ = rm m! (r2 + r)e−re1e1Sµ −rm m! rzz′e−re1Sµ. It is well known that It is well known that e1Sµ = X 2∈µ+ Sµ+2. e1Sµ = X 2∈µ+ Sµ+2. It follows that the ﬁrst expression in question is equal to It follows that the ﬁrst expression in question is equal to (r + 1)(m + 1) rm+1 (m + 1)!e−re1 X 2∈µ+ Sµ+2 −rzz′ rm m! e−re1Sµ = (r + 1)(m + 1) X 2∈µ+ fµ+2 −rzz′fµ. (8.20) = (r + 1)(m + 1) X 2∈µ+ fµ+2 −rzz′fµ. (8.20) 2. The second expression in (8.19) takes the form 2. The second expression in (8.19) takes the form 2. The second expression in (8.19) takes the form rm m! e−re1(D(z,z′)Sµ). EJP 18 (2013), paper 75. EJP 18 (2013), paper 75. Page 32/43 EJP 18 (2013), paper 75. Page 32/43 ejp.ejpecp.org Markov dynamics on the Thoma cone t follows from [32, Theorem 4.1 and Deﬁnition 4.7] that D(z,z′)Sµ = −mSµ + X 2∈µ− (z + c(2))(z′ + c(2))Sµ−2, m = \|µ\|. It follows from [32, Theorem 4.1 and Deﬁnition 4.7] that D(z,z′)Sµ = −mSµ + X 2∈µ− (z + c(2))(z′ + c(2))Sµ−2, m = \|µ\|. This implies that the second expression is equal to −mfµ + r m X 2∈µ− (z + c(2))(z′ + c(2))fµ−2. (8.21) (8.21) 3. The only relevant part of our differential operator D = D(z,z′) that contributes to the third expression in (8.19) is 3. The only relevant part of our differential operator D = D(z,z′) that contributes to the third expression in (8.19) is 3. The only relevant part of our differential operator D = D(z,z′) that contributes to the third expression in (8.19) is e1 ∂2 ∂e2 1 + 2 X n′>1 n′en′ ∂2 ∂en′∂e1 , e1 ∂2 ∂e2 1 + 2 X n′>1 n′en′ ∂2 ∂en′∂e1 , because the remaining terms in D(z,z′) are either of the ﬁrst order or do not contain the partial derivative in variable e1 while our function G depends on e1 only. It follows that the third expression has the form 2rm m! d de1 e−re1 e1 ∂ ∂e1 Sµ + 2rm m! d de1 e−re1 X n′>1 n′en′ ∂ ∂en′ Sµ = −2rrm m! e−re1 X n≥1 nen ∂ ∂en Sµ. Observe that the operator Observe that the operator X n≥1 nen ∂ ∂en is the “Euler operator”; its action on the homogeneous function Sµ amounts to mul- tiplication by its degree m. Using this fact we see that the third expression is equal to is the “Euler operator”; its action on the homogeneous function Sµ amounts to mul- tiplication by its degree m. EJP 18 (2013), paper 75. 8.5 Approximation Recall that in Section 7.4 we introduced an embedding λ 7→ωλ of the set Y into the Thoma cone eΩ. Now let us introduce a family of embeddings ϕr : Y ,→eΩdepending on parameter r > 0: ϕr(λ) = r−1ωλ, λ ∈Y, where multiplication by constant factor r−1 in the right-hand side means that all co- ordinates of ωλ are multiplied by that constant — a natural homothety on the cone. Obviously, ϕ1 is the map λ 7→ωλ. where multiplication by constant factor r−1 in the right-hand side means that all co- ordinates of ωλ are multiplied by that constant — a natural homothety on the cone. Obviously, ϕ1 is the map λ 7→ωλ. The latter map should be viewed as a counterpart of the inclusion map Z+ ,→R+, while ϕr is a counterpart of the scaled embedding Z+ ∋l 7→r−1l ∈R+. e Note that ϕr(Y) is a discrete, locally ﬁnite subset of eΩ. Therefore, we may deﬁne the projection πr : C0(eΩ) →C0(Y) as in Section 5.2: (πrf)(λ) = f(ϕr(λ)), λ ∈Y. It is used in the proposition below to deﬁne the approximation procedure. )(t) Recall that in Corollary 8.7 and Proposition 8.8 we deﬁned Feller semigroups T (z,z′) r (t) and T (z,z′)(t) acting on the Banach spaces C0(Y) and C0(eΩ), respectively. Proposition 8.13 (cf. Proposition 6.11). Let (z, z′) be ﬁxed. As r →+∞, the semi- groups T (z,z′) r (t) approximate the semigroup T (z,z′)(t) in the sense of Deﬁnition 3.2. Proposition 8.13 (cf. Proposition 6.11). Let (z, z′) be ﬁxed. As r →+∞, the semi- groups T (z,z′) r (t) approximate the semigroup T (z,z′)(t) in the sense of Deﬁnition 3.2. Proof. As in the proof of Proposition 6.11, we only need to check all the hypotheses of Proposition 5.5. Again, the assumptions stated in Section 5.1 are satisﬁed: we know that YB is a Feller system, the semigroups T (z,z′) r (t) with varying parameter r > 0 are consistent with the links of YB, they are Feller links, and, by the very deﬁnition, the semigroup T (z,z′)(t) is the boundary semigroup determined by the pre-limit semigroups T (z,z′) r (t). Next, we have to check Conditions 4.1 and 5.4. The second condition consists in the requirement that Cc(Y) is a core for generator A(z,z′) r and, moreover, is invariant under its action. Markov dynamics on the Thoma cone Markov dynamics on the Thoma cone It follows that the ﬁrst expression in question is equal to Using this fact we see that the third expression is equal to (8.22) −2rmfµ. (8.22) (8.22) Finally, summing up (8.20), (8.21), and (8.22) we get the desired formula (8.18) The Laguerre symmetric functions, introduced in Olshanski [32], are elements of Sym depending on parameters z and z′, and indexed by Young diagrams ν ∈Y: L(z,z′) ν = X µ: µ⊆ν (−1)\|ν\|−\|µ\| dim ν/µ (\|ν\| −\|µ\|)! (z)ν/µ(z′)ν/µSµ. (8.23) As shown in [32], they form a basis in Sym diagonalizing operator D(z,z′): D(z,z′)L(z,z′) ν = −\|ν\|L(z,z′) ν , ν ∈Y. (8.24) (8.24) The above formula is similar to (6.16), and the next formula is an analog of (6.17): YBΛ∞ r M(z,z′,r) ν = r\|ν\|L(z,z′) ν , r > 0, ν ∈Y. (8.25) (8.25) The proof of (8.25) is easy and analogous to that of (6.17). Namely, we compare the expansions of the Meixner and Laguerre symmetric functions in the bases {FSµ} and {Sµ}, respectively (see (8.13) and (8.23)), and then apply (8.8), which says that YBΛ∞ r takes FSµ to r\|µ\|Sµ. EJP 18 (2013), paper 75. Page 33/43 ejp.ejpecp.org EJP 18 (2013), paper 75. Page 33/43 ejp.ejpecp.org 8.5 Approximation The fact that Cc(Y) is a core follows from Corollary 8.7, item (ii). Its invariance follows from item (i), because Cc(Y) is obviously invariant under the action of Q(z,z′) r . y Q Finally, the ﬁrst condition means that that for ﬁxed s > 0 and µ ∈Y ondition means that that for ﬁxed s > 0 and µ ∈Y lim r→∞sup λ∈Y YBΛr s(λ, µ) −YBΛ∞ s (ϕr(λ), µ) = 0, (8.26) lim r→∞sup λ∈Y YBΛr s(λ, µ) −YBΛ∞ s (ϕr(λ), µ) = 0, (8.26) (8.26) and this was established in the proof of [14, Theorem 3.4.7] (note only a slight diver- gence of notation: in [14], we wrote r′ and r instead of r and s, respectively). EJP 18 (2013), paper 75. Markov dynamics on the Thoma cone Markov dynamics on the Thoma cone Proposition 8.14. M (z,z′) r serves as a unique stationary distribution for the Markov process X(z,z′) r determined by the Feller semigroup T (z,z′) r (t). Proof. The fact that M (z,z′) r is a stationary measure is a particular case of [11, Proposi- tion 2.12]. ( ′) ( ′) Next, from the structure of matrix Q(z,z′) r and the construction of X(z,z′) r it follows that X(z,z′) r is an irreducible Markov chain: all states λ ∈Y are communicating. Ac- cording to a general theorem (see Anderson [1, Chapter 5, Theorem 1.6]) this implies the uniqueness claim. As shown in [14, Proposition 3.5.3], the measures M (z,z′) r with varying parameter r are compatible with the links YBΛr′ r , that is, they form a coherent family. Therefore, they give rise to a boundary measure on eΩ, which we denote by M (z,z′) and call the z-measure on the Thoma cone. Proposition 8.15. M (z,z′) serves as a unique stationary distribution for the Markov process X(z,z′) determined by the Feller semigroup T (z,z′)(t). Proof. We have to prove that M (z,z′) satisﬁes the relation M (z,z′)T (z,z′)(t) = M (z,z′) and is a unique probability measure on eΩwith this property. By Proposition 8.14, a similar claim holds for measures M (z,z′) r . Because {M (z,z′) r : r > 0} is a coherent family, this immediately implies the desired claim: an easy formal argument can be found in [13, Section 2.8]. Proposition 8.16. M (z,z′) is the weak limit of measures ϕr(M (z,z′) r ) as r →+∞. Proposition 8.16. M (z,z′) is the weak limit of measures ϕr(M (z,z′) r ) as r →+∞. Proof. Indeed, as mentioned above (see the proof of Proposition 8.13), in our situation Condition 4.1 is satisﬁed. Therefore, we may apply Proposition 4.2 which gives the desired result. Remark 8.17. In Olshanski [32], the z-measures on the Thoma cone were deﬁned in a different way, see [32, Theorem 5.18]. However, the two deﬁnitions are equivalent, as can be seen from the comparison of Proposition 8.16 with [32, Theorem 5.28]. 8.6 The stationary distribution The so-called mixed z-measure on Y with parameters (z, z′) and r is deﬁned by he so-called mixed z-measure on Y with parameters (z, z′) and r is deﬁned by M (z,z′) r (λ) = (r + 1)−zz′ r r + 1 \|λ\| · Y 2∈λ (z + c(2))(z′ + c(2)) · dim λ \|λ\|! 2 , (8.27) (8.27) where λ ranges over Y. As before, we assume that r > 0 and (z, z′) satisﬁes Condition 1.1. Then the weights M (z,z′) r (λ) are strictly positive and sum to 1, so that M (z,z′) r is a probability measure on Y whose support is the whole set Y. Measures M (z,z′) r ﬁrst ap- peared in Borodin–Olshanski [7]; additional information can be found in Okounkov [29], Borodin–Olshanski [9], and Olshanski [32]. These measures are a particular case of Okounkov’s Schur measures introduced in [28]. (As mentioned above, in those papers, the third parameter, denoted by ξ, is related to our parameter r by ξ = r(1 + r)−1.) Page 34/43 ejp.ejpecp.org EJP 18 (2013), paper 75. ejp.ejpecp.org ejp.ejpecp.org Page 34/43 Page 34/43 Markov dynamics on the Thoma cone Markov dynamics on the Thoma cone Let M be a probability Borel measure on Conf(X). Then the functions NB become random variables. We will assume that every such function has ﬁnite moments of any order, EM((NB)k) < +∞, ∀k = 1, 2, . . . , ∀B, where EM means expectation relative to M. Under this assumption one assigns to M an inﬁnite collection {ρk : k = 1, 2, . . . } of measures, where ρk is a (usually inﬁnite) measure on the k-fold product space Xk, deﬁned as follows. First, given ω ∈Conf(X), we form a purely atomic measure ∆k(ω) on Xk by setting First, given ω ∈Conf(X), we form a purely atomic measure ∆k(ω) on Xk ∆k(ω) := X x1,...,xk ∆x1 ⊗· · · ⊗∆xk, where the sum is taken on arbitrary ordered k-tuples of distinct points extracted from ω. where the sum is taken on arbitrary ordered k-tuples of distinct points extracted from ω. Second, we interpret ∆k(ω) as a random measure driven by the probability distribu- tion M and average over M, Second, we interpret ∆k(ω) as a random measure driven by the probability distribu- tion M and average over M, ρk = ρM k := EM(∆k( · )). The measure ρM k is called the kth correlation measure of M, and the ﬁrst corre- lation measure ρM 1 is also called the density measure. Under mild hypotheses on the correlation measures, they determine the initial measure M uniquely, see Lenard [26]. M is said to be a determinantal measure if the following condition holds. Choose a “reference” measure σ on X, equivalent to the density measure (the condition stated below does not depend on the choice of σ). There should exist a function K(x, y) on X×X such that, for every k ≥1, the kth correlation measure ρM k is absolutely continuous with respect to σ⊗k, and the corresponding Radon-Nikodým density is given by a k × k principal minor extracted from kernel K: ρM k σ⊗k (x1, . . . , xk) = det[K(xi, xj)]. The quantity in the left-hand side is called the kth correlation function, and K(x, y) is called the correlation kernel of M. In contrast to correlation functions, the correlation kernel, if it exists, is not a canonical object: there are ways to modify it without affecting the correlation functions. 9.1 Generalities on correlation functions Let X be a locally compact metrizable separable space (we will actually take for X the punctured real line R∗:= R \ {0} or the one-dimensional lattice). A ﬁnite or countably inﬁnite collection of points in X without accumulation points is called a con- ﬁguration. We say “collection” and not “subset” because, in principle, multiple points are permitted; one could also use the term “multiset”. To a conﬁguration ω we assign the Radon measure ∆(ω) := X x∈ω ∆x, where ∆x denotes the delta-measure at x. This assignment establishes a one-to-one correspondence between all possible conﬁgurations in X and all sigma-ﬁnite Radon measures on X with the property that the mass of any compact subset is a nonnegative integer. The space of conﬁgurations will be denoted by Conf(X). We equip it with the topology inherited from the vague topology on the space of Radon measures. In particular, Conf(X) has a natural Borel structure. This structure is generated by the integer-valued functions NB, where B ⊂X is an arbitrary relatively compact Borel subset and where ∆x denotes the delta-measure at x. This assignment establishes a one-to-one correspondence between all possible conﬁgurations in X and all sigma-ﬁnite Radon measures on X with the property that the mass of any compact subset is a nonnegative integer. The space of conﬁgurations will be denoted by Conf(X). We equip it with the topology inherited from the vague topology on the space of Radon measures. In particular, Conf(X) has a natural Borel structure. This structure is generated by the integer-valued functions NB, where B ⊂X is an arbitrary relatively compact Borel subset and subset and NB(ω) := \|ω ∩B\|, ω ∈Conf(X). Page 35/43 ejp.ejpecp.org EJP 18 (2013), paper 75. ejp.ejpecp.org Page 35/43 EJP 18 (2013), paper 75. Markov dynamics on the Thoma cone On the other hand, any determinantal measure is uniquely determined by its correlation functions and hence by the correlation kernel. “Determinantal measure” is another name for “determinantal point process” (more precisely, for the law of such a point process). A standard reference is Soshnikov’s expository paper [37]. See also the more recent survey Borodin [4] and references therein. Theorem 9.1. ¯ M (z,z′) is a determinantal measure whose correlation kernel is the Whit- taker kernel K(z,z′)(x, y). This result was ﬁrst proved in [3]. Below we give in detail a different derivation, because it is well suited for the extension to the case of ﬁnite-dimensional distributions of processes X(z,z′). Note that a similar argument is contained in [8, Proposition 4.2]. Proof. Step 1. Let M be a probability measure on eΩand ¯ M be the corresponding measure on Conf(R∗). We will establish a simple estimate which, in particular, provides a convenient sufﬁcient condition for the existence of the correlation measures. For ε > 0, set Bε := R \ (−ε, ε) ⊂R∗. Bε := R \ (−ε, ε) ⊂R∗. Recall the notation \|ω\| = \|(α, β, δ)\| = δ. The basic constraint P(αi + βi) ≤\|ω\| implies the inequality 1 Recall the notation \|ω\| = \|(α, β, δ)\| = δ. The basic constraint P(αi + βi) ≤\|ω\| implies the inequality \|¯ω ∩Bε\| ≤ε−1\|ω\|, (9.1) (9.1) which in turn implies that E ¯ M((NBε)k) ≤ε−k Z eΩ \|ω\|kM(dω), k = 1, 2, . . . . Denote by \|M\| the measure on R+ that is the pushfoward of M under the projection ω 7→\|ω\|. The above inequality can be rewritten as Denote by \|M\| the measure on R+ that is the pushfoward of M under the projection ω 7→\|ω\|. The above inequality can be rewritten as E ¯ M((NBε)k) ≤ε−k Z R+ sk\|M\|(ds) k = 1, 2, . . . . This shows that if \|M\| has ﬁnite moments of all orders, then the left-hand side is ﬁnite for all k and hence the correlation measures of ¯ M are well deﬁned. (Here we tacitly used the evident fact that any compact subset of R∗is contained in subset Bε with ε small enough.) This shows that if \|M\| has ﬁnite moments of all orders, then the left-hand side is ﬁnite for all k and hence the correlation measures of ¯ M are well deﬁned. (Here we tacitly used the evident fact that any compact subset of R∗is contained in subset Bε with ε small enough.) Step 2. For r > 0, set Mr := MΛ∞ r . It is initially deﬁned as a probability distribution on Y, but it is convenient to transfer it to eΩusing the embedding ϕr : Y →eΩ. So, we will regard each Mr as a probability distribution on eΩ. EJP 18 (2013), paper 75. Theorem 9.1. ¯ M (z,z′) is a determinantal measure whose correlation kernel is the Whit- taker kernel K(z,z′)(x, y). By Proposition 4.2, Mr converges to M in the weak topology as r →+∞, meaning that lim r→+∞⟨Ψ, Mr⟩= ⟨Ψ, M⟩ (9.2) (9.2) for any continuous bounded function Ψ on eΩ. 9.2 Determinantal structure of the stationary distributions Set X = R∗and deﬁne a map eΩ→Conf(R∗) as follows: eΩ∋ω 7→¯ω := {αi : αi ̸= 0} ∪{−βi : βi ̸= 0} ∈Conf(R∗). eΩ∋ω 7→¯ω := {αi : αi ̸= 0} ∪{−βi : βi ̸= 0} ∈ Because of the constraint P αi + P βi ≤δ < +∞, ¯ω is indeed a conﬁguration on R∗. Clearly, the map is continuous and hence Borel. So it converts every probability Borel measure M on eΩto a probability Borel measure ¯ M on Conf(R∗). This makes it possible to speak about the correlation functions of M, referring to those of ¯ M. We ﬁx a pair of parameters (z, z′) satisfying Condition 1.1 and denote by ¯ M (z,z′) the measure on Conf(R∗) coming from the z-measure M (z,z′). In the next theorem, K(z,z′)(x, y) denotes the Whittaker kernel on R∗×R∗studied in Borodin [3] and Borodin– Olshanski [7], [11]. We will not use its exact form here. EJP 18 (2013), paper 75. ejp.ejpecp.org ejp.ejpecp.org Page 36/43 Page 36/43 Markov dynamics on the Thoma cone Theorem 9.1. ¯ M (z,z′) is a determinantal measure whose correlation kernel is the Whit- taker kernel K(z,z′)(x, y). Theorem 9.1. ¯ M (z,z′) is a determinantal measure whose correlation kernel is the Whit- taker kernel K(z,z′)(x, y). Markov dynamics on the Thoma cone Markov dynamics on the Thoma cone Fix k = 1, 2, . . . . By deﬁnition, the vague convergence of the kth correlation mea- sures, ρMr k →ρM k , means that lim r→+∞⟨F, ρMr k ⟩= ⟨F, ρM k ⟩ for any continuous, compactly supported function F on (R∗)k. By the very deﬁnition of the correlation measures, the latter relation is equivalent to fulﬁllment of relation (9.2), where Ψ = ΨF has the following form for any continuous, compactly supported function F on (R∗)k. By the very deﬁnition of the correlation measures, the latter relation is equivalent to fulﬁllment of relation (9.2), where Ψ = ΨF has the following form ΨF (ω) = ⟨F, ∆k(¯ω)⟩= X x1,...,xk F(x1, . . . , xk), (9.4) (9.4) where the sum is taken over ordered k-tuples of distinct points extracted from conﬁgu- ration ¯ω. Now, by virtue of step 2, it sufﬁces to check that ΨF is continuous and has moderate growth at inﬁnity. k Choose ε so small that the support of F is contained in Bk ε . By virtue of bound (9.1), \|ΨF (ω)\| ≤ε−k∥F∥\|ω\|k. Therefore, ΨF has moderate growth at inﬁnity. Therefore, ΨF has moderate growth at inﬁnity. Therefore, ΨF has moderate growth at inﬁnity. Therefore, ΨF has moderate growth at inﬁnity. To see that ΨF is continuous look at the right-hand side of (9.4) and observe that F(x1, . . . , xk) vanishes unless all quantities \|x1\|, . . . , \|xk\| are bounded from below by ε, which in turn entails that the k-tuple {x1, . . . , xk} is contained in the subset {α1, . . . , αm, −β1, . . . , −βm}, m := [ε−1\|ω\|]. {α1, . . . , αm, −β1, . . . , −βm}, m := [ε−1\|ω\|]. That is, only coordinates of ω with a few ﬁrst indices really contribute, and this ﬁnite set of possible indices depends only on \|ω\|. Together with the continuity of F this gives the desired claim. That is, only coordinates of ω with a few ﬁrst indices really contribute, and this ﬁnite set of possible indices depends only on \|ω\|. Together with the continuity of F this gives the desired claim. Step 4. Now we apply the above general arguments to M := M (z,z′) and the corre- sponding pre-limit measures Mr := M (z,z′) r . for any continuous bounded function Ψ on eΩ. for any continuous bounded function Ψ on eΩ. Assume now that we dispose of the following uniform bound on the tails of measures \|Mr\|: \|Mr\|: For every k = 1, 2, . . . , one has Z R+ sk\|Mr\|(ds) ≤Ck (9.3) (9.3) with a constant Ck independent on r. Then, evidently, (9.2) holds under weaker assumptions on Ψ: it sufﬁces to require that Ψ is continuous and has moderate growth at inﬁnity, meaning that \|Ψ(ω)\| ≤const(1+\|ω\|)k for some k. Then, evidently, (9.2) holds under weaker assumptions on Ψ: it sufﬁces to require that Ψ is continuous and has moderate growth at inﬁnity, meaning that \|Ψ(ω)\| ≤const(1+\|ω\|)k for some k. Step 3. Assume that condition (9.3) is satisﬁed. We claim that then the correlation measures of Mr vaguely converge to the respective correlation measures of M. Indeed, ﬁrst of all, by virtue of step 1, our assumption guarantees the very exis- tence of the correlation measures for measures Mr. Moreover, the inequalities (9.3) are inherited by the limit measure M, so that its correlation measures exist, too. ejp.ejpecp.org Page 37/43 Markov dynamics on the Thoma cone Markov dynamics on the Thoma cone Recall that, according to our convention, M (z,z′) r lives on ϕr(Yr) ⊂eΩ. Then we know exactly what is \|M (z,z′) r \|: it is a scaled negative binomial distribution living on the subset r−1Z+ ⊂R+: \|M (z,z′) r \|(r−1l) = (r + 1)−zz′ (zz′)↓l l! r r + 1 l , l ∈Z+. Condition (9.3) on the tails is readily checked (note that the limiting measure \|M (z,z′)\| is the Γ-distribution with parameter zz′). Therefore, all correlation functions exist, and we have the limit relation lim r→+∞⟨F, ρMr k ⟩= ⟨F, ρM k ⟩, Mr := M (z,z′) r , M := M (z,z′) for any continuous compactly supported function F on (R∗)k. for any continuous compactly supported function F on (R∗)k. Step 5. Finally, we apply the results of our papers [7] and [11]. As shown in those papers, the pre-limit measures Mr = M (z,z′) r are determinantal, with some correlation kernels K(z,z′) r (x, y), called discrete hypergeometric kernels, for which an explicit ex- pression is known. ( ′) In accordance with our deﬁnition of measure M (z,z′) r , it lives on the lattice r−1Z′ ⊂ R∗, where Z′ := Z + 1 2. As the reference measure σ, we take the counting measure on the lattice. Then one can write ⟨F, ρMr k ⟩= X (x1,...,xk)∈(r−1Z′)k F(x1, . . . , xk) det[K(z,z′) r (xi, xj)]. F(x1, . . . , xk) det[K(z,z′) r (xi, xj)]. Page 38/43 ejp.ejpecp.org EJP 18 (2013), paper 75. ejp.ejpecp.org ejp.ejpecp.org Page 38/43 Page 38/43 (Note that eΩ0 is a dense Borel subset of type Gδ.) ( ′) (Note that eΩ0 is a dense Borel subset of type Gδ.) (Note that eΩ0 is a dense Borel subset of type Gδ.) ′ This is just the case for M = M (z,z′), as can be proved using Olshanski [30, Theorem 6.1]. Therefore, M (z,z′) is completely speciﬁed by the correlation kernel K(z,z′)(x, y) of the measure ¯ M (z,z′). 9.3 Determinantal structure of equilibrium ﬁnite-dimensional distributions Starting Markov process X(z,z′) at time t = 0 from the stationary distribution we get a stationary in time stochastic process e X(z,z′). Given time moments 0 ≤t1 < · · · < tn, let M (z,z′)(t1, . . . , tn) stand for the corresponding ﬁnite-dimensional distribution of e X(z,z′). The distributions M (z,z′)(t1, . . . , tn) are invariant under simultaneous shift of all time moments by a constant; they can be called the equilibrium ﬁnite-dimensional distributions. For n = 1, we have M (z,z′)(t) ≡M (z,z′). Initially M (z,z′)(t1, . . . , tn) is deﬁned as a probability measure on the n-fold prod- uct space eΩn, but then we convert it to a probability measure ¯ M (z,z′)(t1, . . . , tn) on (Conf(R∗))n, just as we did above for the case n = 1. Observe that (Conf(R∗))n can be identiﬁed, in a natural way, with Conf( R∗⊔· · · ⊔R∗ \| {z } n ). This shows that we can in- terpret ¯ M (z,z′)(t1, . . . , tn) as a probability distribution on conﬁgurations, and the next theorem says that it is again in the determinantal class. This means that the correlation functions of ¯ M (z,z′)(t1, . . . , tn) are described by a “dynamical” (or “space-time”) kernel K(z,z′)(x, s; y, t) on (R∗× R) × (R∗× R) whose two arguments, couples (x, s) and (y, t), should be viewed as space-time variables ranging over space-time R∗× R. Given an arbitrary ﬁnite collection (x1, t1), . . . , (xk, tk), the k × k determinant det h K(z,z′)(xi, ti; xj, tj) i det h K(z,z′)(xi, ti; xj, tj) i multiplied by dx1 . . . dxk gives the probability of the event that at each prescribed moment ti (where i = 1, . . . , k), the conﬁguration ¯ω ∈Conf(R∗) corresponding to ω := X(z,z′)(ti) contains a point in the inﬁnitesimal neighborhood dxi about position xi, for every i = 1, . . . , k. ′ The kernel K(z,z′)(x, s; y, t) in question is the extended Whittaker kernel; we refer to Borodin–Olshanski [11] for its description. Theorem 9.3. The pushforwards ¯ M (z,z′)(t1, . . . , tn) of the equilibrium ﬁnite-dimensional distributions M (z,z′)(t1, . . . , tn) are determinantal measures described by the extended Whittaker kernel K(z,z′)(x, s; y, t). where K(z,z′)(x, y) is the Whittaker kernel. This completes the proof. Remark 9.2. The map M 7→ ¯ M converting a measure on eΩto that on Conf(R∗) is not injective, because the map ω 7→¯ω ignores parameter δ. However, M is uniquely determined by its pushforward ¯ M if it is known a priori that M is supported by the subset eΩ0 := {ω : X αi + X βi = δ} ⊂eΩ. Markov dynamics on the Thoma cone Markov dynamics on the Thoma cone On the other hand, the limiting behavior of kernels K(z,z′) r (x, y) was studied in [7, Theorem 5.4]. It follows that, as r →+∞, the right-hand side of the above relation converges to Z (x1,...,xk)∈(R∗)k F(x1, . . . , xk) det[K(z,z′)(xi, xj)]dx1 . . . dxk, where K(z,z′)(x, y) is the Whittaker kernel. This completes the proof. Markov dynamics on the Thoma cone This is a generalization of Theorem 9.1, which is a particular case of Theorem 9.3 for n = 1, because M (z,z′)(t) ≡M (z,z′), and K(z,z′)(x, s; y, t) reduces to the Whittaker kernel K(z,z′)(x, y) for s = t. Remark 9.4 (cf. Remark 9.2). Note that measure M (z,z′)(t1, . . . , tn) is supported by the subset eΩn 0 , because every its one-dimensional marginal coincides with M (z,z′) and the latter measure is supported by eΩ0. As in the case n = 1, this implies that the equilibrium ﬁnite-dimensional distributions are uniquely determined by the extended Whittaker kernel. Proof of Theorem 9.3. The argument for Theorem 9.1 extends smoothly, with a few mi- nor evident modiﬁcations only. Let M (z,z′) r (t1, . . . , tn) stand for the pre-limit equilibrium ﬁnite-dimensional distributions. Corollary 3.6 tells us that they approximate the distri- butions M (z,z′)(t1, . . . , tn). To bound the tails we use the fact, mentioned above, that the one-dimensional marginals coincide with the stationary distribution. The correla- tion functions of the pre-limit distributions are described by the extended version of the discrete hypergeometric kernel, which converges to the extended Whittaker kernel as r →+∞: this is established in [11]. 9.3 Determinantal structure of equilibrium ﬁnite-dimensional distributions Page 39/43 ejp.ejpecp.org EJP 18 (2013), paper 75. ejp.ejpecp.org ejp.ejpecp.org Page 39/43 Page 39/43 Markov dynamics on the Thoma cone EJP 18 (2013), paper 75. Markov dynamics on the Thoma cone Markov dynamics on the Thoma cone The Markov chains Xθ were studied in our paper [10]. As shown in that paper, Xθ admits a nice description in terms of the Poisson process in the quarter-plane and the Robinson–Schensted algorithm. The formalism of the present paper says that the family {Xθ : θ > 0} gives rise to a Feller Markov process X on the boundary eΩ, and X has a unique stationary distribution M := lim ←−Mθ, the boundary measure corresponding to the family of the Poissonized Plancherel measures. On the other hand, it is readily seen that this boundary measure is simply the Dirac measure at the point ω1 := (α = 0, β = 0, δ = 1) ∈eΩ, where 0 := (0, 0, . . . ) is the null sequence. At ﬁrst glance, this looks strange, but the key is that X is not a genuine Markov process, but a deterministic process. Its transition function P(t) degenerates to a semi- group of continuous maps eΩ→eΩwhich have the following form: P(t) : (α, β, δ) 7→(e−tα, e−tβ, e−tδ + (1 −e−t)), t ≥0. From this formula it is seen that, as t →+∞, P(t) contracts the whole space eΩto the point ω1. There is no contradiction, because such a deterministic process is formally a Markov process. On the algebraic level, this phenomenon is clearly seen when we compute the gen- erator of X as an operator in the algebra of symmetric functions: In contrast to the Laguerre operator (8.16) we get a ﬁrst order differential operator. This operator is best written in terms of the generators p1, p2, . . . (the power-sum symmetric functions, see Section 7.4), it has the form (1 −p1) ∂ ∂p1 + X n≥2 npn ∂ ∂pn . The above discussion shows that our abstract formalism of constructing boundary Markov processes via Markov intertwiners conceals a potential danger, as it may hap- pen that the boundary process degenerates to a deterministic process. Therefore, if one is interested in constructing interesting inﬁnite-dimensional Markov processes (as we do), one needs additional arguments guaranteeing that such a degeneration does not occur. We were fortunate that we were able to explicitly compute the generator of our process X(z,z′): from the fact that the generator is a second order operator it is easy to conclude that X(z,z′) cannot be a deterministic process. 10 Remarks on the Plancherel limit Let us return to the context of Section 8.2. So far the basic parameters z and z′ were ﬁxed, but here we take a limit transition in formulas (8.11) assuming that z and z′ go to inﬁnity while the third parameter r goes to 0 in such a way that the product rzz′ tends to a ﬁxed real number θ > 0. One may simply assume that r is related to the couple (z, z′) by r = θ(zz′)−1; recall that because of Condition 1.1, zz′ is strictly positive, so that the above relation is compatible with the fact that r should be a positive number. The quantity θ becomes our new parameter. It is not difﬁcult to verify that in this limit transition, all results of Section 8.2 survive. Namely, the Q-matrix Q(z,z′) r turns into the following matrix: Qθ(λ, λ + □) = θ dim(λ + □) (\|λ\| + 1) dim λ, □∈λ+, Qθ(λ, λ −□) = \|λ\| dim(λ −□) dim λ , □∈λ−, −Qθ(λ, λ) = \|λ\| + θ. (10.1) (10.1) An analog of Proposition 8.4 holds, with the Meixner symmetric functions being re- placed by the so-called Charlier symmetric functions, introduced in [32] (these are obtained from the Meixner functions via the same limit transition). A key observation is that the links YBΛr′ r depend on parameters r and r′ through their ratio r/r′, which remains intact under the limit (it translates into the ratio θ/θ′). Because of this fact, all other results of Section 8.2 are smoothly extended, too. We only have to change the notation r →θ. Finally, we get a family {Xθ : θ > 0} of continuous time Feller Markov chains on Y. Further, one can prove that Xθ has a unique stationary distribution, which is nothing else than the the well-known Poissonized Plancherel measure, ﬁrst introduced in Baik– Deift–Johansson [2]: Mθ(λ) = e−θθ\|λ\| dim λ \|λ\|! 2 . Mθ(λ) = e−θθ\|λ\| dim λ \|λ\|! 2 It is a degeneration of the mixed z-measure (8.27), which played an important role in Borodin–Okounkov–Olshanski [6]. It is a degeneration of the mixed z-measure (8.27), which played an important role in Borodin–Okounkov–Olshanski [6]. EJP 18 (2013), paper 75. Page 40/43 ejp.ejpecp.org Page 40/43 ejp.ejpecp.org EJP 18 (2013), paper 75. ejp.ejpecp.org ejp.ejpecp.org Page 40/43 Page 40/43 Markov dynamics on the Thoma cone ′ Finally, note that the existence of a nontrivial stationary distribution, M (z,z′), makes it possible to prove the non-determinism of the boundary process in a different way. Markov dynamics on the Thoma cone [5] Borodin, A. and Gorin, V.: Markov processes of inﬁnitely many nonintersecting random walks. Probab. 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A. and Widom, H.: Differential equations for Dyson processes. Commun. Math. Phys. 252 (2004), no. 1-3, 7–41, arXiv:math/0309082. MR-2103903 [40] Tracy, C. A. and Widom, H.: Differential equations for Dyson processes. Commun. Math. Phys. 252 (2004), no. 1-3, 7–41, arXiv:math/0309082. MR-2103903 Acknowledgments. A. B. was partially supported by NSF-grant DMS-1056390. G. O. 1OJS: Open Journal Systems http://pkp.sfu.ca/ojs/ 2IMS: Institute of Mathematical Statistics http://www.imstat.org/ 3BS: Bernoulli Society http://www.bernoulli-society.org/ 4PK: Public Knowledge Project http://pkp.sfu.ca/ 5LOCKSS: Lots of Copies Keep StuﬀSafe http://www.lockss.org/ 6IMS Open Access Fund: http://www.imstat.org/publications/open.htm Markov dynamics on the Thoma cone was partially supported by a grant from Simons Foundation (Simons–IUM Fellowship), the RFBR-CNRS grant 10-01-93114, and the project SFB 701 of Bielefeld University. Acknowledgments. A. B. was partially supported by NSF-grant DMS-1056390. G. O. was partially supported by a grant from Simons Foundation (Simons–IUM Fellowship), the RFBR-CNRS grant 10-01-93114, and the project SFB 701 of Bielefeld University. 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https://openalex.org/W2059357478	https://openaccess.uoc.edu/bitstream/10609/92467/1/observacion.pdf	Spanish; Castilian	null	La observación participante y la deriva: dos técnicas móviles para el análisis de la ciudad contemporánea. El caso de Barcelona	EURE	2,013	cc-by	10,949	119 VOL 39 \| N O 116 \| ENERO 2013 \| pp. 119-139 \| ARTÍCULOS \| ©EURE La observación participante y la deriva: dos técnicas móviles para el análisis de la ciudad contemporánea. El caso de Barcelona Isabel Pellicer. Universidad Autónoma de Barcelona, Barcelona, España. Pep Vivas-Elias. Universidad Oberta de Catalunya, Barcelona, España. Jesús Rojas. Universidad Autónoma de Barcelona, Barcelona, España. resumen \| Somos territoriantes, radicantes, transumers, viajeros en la ciudad global. Con estas fguras damos forma y signifcado al espacio urbano contemporáneo. Dicho espacio y las prácticas que le dan lugar se relacionan con los constantes movimientos, fujos e interconexiones de personas, objetos, datos, ideas y mercancías, vinculándose directamente con el paradigma de la movilidad y con la lógica radicante. En este contexto es necesario el uso de la metodología móvil, ya que es la que mejor se adapta al movimiento de las ciudades contemporáneas y de sus habitantes. Este artículo trata de esta cuestión. Se defne la metodología móvil, y se muestra, mediante dos técnicas móviles concretas: la observación participante y la deriva, cómo esta metodología es especialmente útil para estudiar el contexto urbano actual y las prácticas que en él se dan, ya que comparte la lógica constitutiva de los fenómenos estudiados. palabras clave \| ciudad global, movilidad, sociología urbana. E-mail: Isabel Pellicer, pellicer.isabel@gmail.com \| Pep Vivas-Elias, pvivasi@uoc.edu \| Jesús Rojas, jrojasar@uoc.edu issn impreso 0250-7161 \| issn digital 0717-6236 Recibido el 15 de octubre de 2010, aprobado el 25 de julio de 2012 Recibido el 15 de octubre de 2010, aprobado el 25 de julio de 2012 E-mail: Isabel Pellicer, pellicer.isabel@gmail.com \| Pep Vivas-Elias, pvivasi@uoc.edu \| Jesús Rojas, jrojasar@uoc.edu ©EURE \| VOL 39 \| N O 116 \| ENERO 2013 \| pp. 119-139 ©EURE \| VOL 39 \| N O 116 \| ENERO 2013 \| pp. 119-139 120 Ryan Bingham (caracterizado por George Clooney en Up in the air, 2010) reco- rre toda Norteamérica transitando por aeropuertos, hoteles y coches de alquiler llevando todo lo que necesita en una simple maleta de ruedas. Este personaje se ha convertido en un territoriante, un habitante consumidor de la metrópolis global a tiempo parcial (Muñoz, 2008); una persona que no se ancla a un lugar concreto y que se “acomoda” en el “estar en tránsito continuo y permanente”; un habitante de la ciudad global que, a la vez que se desplaza, usa diferentes dispositivos móviles para comunicarse, para interactuar y para moverse más rápidamente. Ryan no sólo ejemplifca perfectamente a aquellas personas que se dedican a viajar por motivos de trabajo en la época actual. También es símbolo, mediante las prácticas sociales que desarrolla en la película, de la colectividad social actual y del papel que desem- peña la movilidad en la construcción de las identidades personales y colectivas. Este vivir y “apropiarse” de lo móvil es lo que caracteriza las dinámicas urbanas y sociales de las ciudades contemporáneas. Con este artículo pretendemos alcanzar diferentes objetivos. El primero, deli- near algunas de las prácticas características de la ciudad contemporánea y la forma en que, al ponerlas en funcionamiento, por un lado, las personas ejercen el rol de territoriantes, radicantes, transumers, viajeros, etcétera; y, por otro, dan forma y construyen colectivamente el espacio. Así, pues, tanto dicho espacio como dichas prácticas están imbricados con los constantes movimientos, fujos e interconexio- nes de personas, cosas, datos, ideas, mercancías, etcétera, que se dan en la urbe ac- tual; y, también, están estrechamente relacionados con el paradigma de la movili- dad (Urry, 2007; Sheller & Urry, 2006). El segundo objetivo, y como efecto del primero, es destacar el uso de la metodología móvil en las ciencias sociales y su importancia para el estudio de la ciudad global, una ciudad en la que la movilidad y las transformaciones urbanas adquieren especial relevancia. El tercero, presentar y profundizar en dos técnicas de investigación móviles concretas: la observación participante y la deriva. Ambas herramientas de recogida de datos están en sinto- nía con el movimiento y son sensibles a las necesidades que plantean los estudios sociales y urbanos actuales. ©EURE \| VOL 39 \| N O 116 \| ENERO 2013 \| pp. 119-139 Debido a que refexionamos ampliamente en el texto, el segundo y el tercer objetivo se convierten en los dos ejes centrales que articulan este trabajo. El cuarto, ejemplifcar, mediante dos casos concretos de estudio (el aeropuerto y el metro de Barcelona), cómo se usan estas dos técnicas de investi- gación en el contexto urbano actual; junto con esto, se presentan algunos datos sobre dinámicas y prácticas urbanas y sociales que se producen en la urbe, donde la movilidad y el intercambio (de datos, personas y mercancías), tanto interurbanos como intraurbanos, articulan el tejido urbano y social. La sociedad “en tránsito”: paisajes móviles, performativos e interconectados palabras clave \| ciudad global, movilidad, sociología urbana. abstract \| We are territoriants, radicants, transumers, travelers in the global city. With these fgures we give shape and meaning to the contemporary urban space. Such space and the practices that give rise to it are related to the constant movements, fows and interconnections of people, things, data, ideas and goods presently under way, thus being directly linked to the paradigm of mobility and the radicant logic. In this context, it is necessary to use mobile methodology, the best suited to the contemporary cities and their inhabitants. Tis paper deals with this issue. Mobile methodology is defned and its practices are shown through two specifc mobile techniques: participant observation and drif. Te paper also highlights how this methodology is especially useful for studying the current urban context and the practices that take place there, as it shares the constitutive logic of the phenomena studied. key words \| global city, mobility, urban sociology. Recibido el 15 de octubre de 2010, aprobado el 25 de julio de 2012 E-mail: Isabel Pellicer, pellicer.isabel@gmail.com \| Pep Vivas-Elias, pvivasi@uoc.edu \| Jesús Rojas, jrojasar@uoc.edu issn impreso 0250-7161 \| issn digital 0717-6236 La sociedad “en tránsito”: paisajes móviles, performativos e interconectados El mundo social, a lo largo de su historia, se ha construido gracias al movimiento, y ello pese a que “el concepto de sociedad presenta un grave defecto: reifca y cris- taliza los fenómenos sociales [y] su verdadero signifcado no reside en su solidez 121 121 Pellicer et al. \| La observación participante y la deriva… \| ©EURE sino precisamente en su fuidez, su carácter maleable” (Wallerstein, 1996, pp. 82– 83). De alguna manera, “la relación que establecemos entre nosotros, el espacio, el tiempo y el lugar, está mediatizada por nuestro movimiento en el mundo social y material” (Fincham, McGuinness & Murray, 2010, p. 1). Así pues, habitamos en un mundo caracterizado y mediatizado por las corrientes de fujos, de personas, ideas, bienes, imágenes, información, y un largo etcétera. No es difícil encontrar multitud de ejemplos cotidianos de desplazamientos físicos o virtuales: sólo hace falta refexionar un segundo para pensar o imaginar múltiples prácticas cotidianas que son posibles gracias a las distintas formas de interconexión que las personas tienen a su alcance. En consecuencia, la naturaleza de lo móvil está estrechamente imbricada con las interacciones, prácticas, formas y conexiones sociales. Como apunta Careri (2002), “el término ‘recorrido’ se refere al mismo tiempo al acto de atravesar (el recorrido como acción de andar), la línea que atraviesa el espacio (el recorrido como obje- to arquitectónico) y el relato del espacio atravesado (el recorrido como estructura narrativa)” (p. 25). Por tanto, son muchos, socialmente, los andares que se reali- zan por la ciudad y, por medio de ellos se trazan muchas trayectorias, se recopilan muchas historias sobre la cotidianidad de la urbe y, a la vez, mediante los desplaza- mientos, las personas escriben e inscriben acerca de la ciudad y sobre ella. Los andares y la marcha dan, así, forma al modo cómo los lugares son habitados y utilizados. Para Certeau (1999), mientras que un lugar, como una calle, por ejemplo, es estable y ordenado, un espacio existe sólo gracias a los movimientos, gracias a las velocidades. Se activa por el conjunto de los movimientos que se producen allí. El espacio, como tal, y el espacio urbano, en particular, son, pues, los lugares ‘performativos’ por excelencia (Morris, 1988, pp. 36–37, citado en Urry, 2000, p. 65). Por tanto, la característica fundamental de la ciudad actual, y a su vez de la so- ciedad, es su constante e interminable transformación. La sociedad “en tránsito”: paisajes móviles, performativos e interconectados Se desmorona, tan pronto, que las razones aleatorias de estar ensamblada desaparecen (p. 38). en el avión, el barco, el tren, el coche, etc., se comparte un espacio común: el tiempo de pasaje de un punto a otro. La cabina del vuelo, el pontón, el vagón, la banqueta, la cabina ofrecen ocasiones de proximidad, incluso de promiscuidad, que fuerzan la relación o constriñen la conversación. En este microcosmos comunitario se juega una intersubjetividad limitada en el tiempo. Desde la llegada del aeropuerto, del puerto, de la estación, del aparcamiento, esta sociedad se deshace la mayoría de las veces. Se desmorona, tan pronto, que las razones aleatorias de estar ensamblada desaparecen (p. 38). Pero el viaje, como se ha dicho, va mucho más allá: está en las experiencias coti- dianas de movilidad que posibilitan relaciones, desencuentros, apropiaciones y des- apropiaciones de los espacios “inter” e “intra” urbanos. Asimismo, la sociedad “en tránsito” está relacionada, íntimamente, con el paradigma de la movilidad (Urry, 2007; Sheller & Urry, 2006), que también enfatiza las complejas interdependencias entre esas diferentes movilidades, y los muchos, voluntarios o forzados, temporales o a largo plazo, agradables o problemáticos momentos de inmovilidad que supo- nen, que pueden construir y contingentemente mantener conexiones sociales por múltiples y variadas distancias (Urry, 2007). Además, ello hace que los “movimien- tos” que hacen esas (in)movilidades constitutivas de relaciones económicas, socia- les y políticas sean interesantes para el estudio, destacando cómo los discursos que pueden priorizar una u otra de dichas movilidades (como, por ejemplo, la creencia de que el negocio tiene que ser hecho ‘cara a cara’) simplifcan —más que explicar— dichas relaciones (Büscher & Urry, 2009, p. 102). Las relaciones, las interacciones, las prácticas sociales incorporan la lógica del cambio constante y del movimiento y, por tanto, los procesos sociales y urbanos también deben tener en cuenta la caracte- rística fundamental de lo móvil para analizar cómo se construyen dichos procesos. Así, esta sociedad “en tránsito” encuentra nuevas maneras de relacionarse con la ciudad, teñidas por la naturaleza móvil de la realidad social, determinada por la era de los fujos migratorios, de la interconexión y del desplazamiento propio o de familiares y amigos, que directa o indirectamente también inciden en la vida de las personas, a nivel tanto global como local. La sociedad “en tránsito”: paisajes móviles, performativos e interconectados Asimismo, la sociedad “en tránsito” se debe considerar como una realidad carpe diem, como una maraña de instantes y momentos inciertos, como una red fuida y fugaz, anclada en un presente que está en (re)construcción permanente. La exis- tencia y la experiencia social urbana de cada persona y de las colectividades pueden considerarse un viaje que se convierte en algo indesligable de la propia subsistencia humana. Así pues, la trayectoria, el desplazamiento, el movimiento, el viaje, el re- corrido, etcétera, no sólo constituyen una característica de lo “individual”, sino que también inciden en nuestra forma de ser seres sociales y colectivos. Algunas de las particularidades de esta sociedad “en tránsito” son la prolife- ración de nuevas formas, procesos y relaciones sociales, económicas, culturales y políticas que posibilitan no solamente que algunas personas habiten en más de un hogar, en un tiempo muy reducido; sino también la apropiación/desapropiación simultánea de multiplicidad de lugares a partir del trasladar con ellas algunas perte- nencias. Este fenómeno es defnido como ‘ubicuidad social’ (Muñoz, 2010). Según sus parámetros, las formas de relación social se dibujan desde los espacios que posi- ©EURE \| VOL 39 \| N O 116 \| ENERO 2013 \| pp. 119-139 122 bilitan nuestras trayectorias cotidianas: se producen en el instante en que se usa un lugar de forma fugaz e instantánea y mediante la proliferación de nodos y espacios que, en las ciudades contemporáneas, permiten la interconexión, posibilitando así la forma de vida actual. Entre tales nodos y espacios, que han aparecido o ganado protagonismo en la globalización, están, por ejemplo, los aeropuertos, los metros, los locutorios, etc. En consecuencia, se postula que la “teoría del viaje” está más vigente que nunca, porque aunque la persona no se “desplace físicamente”, el contexto social, en el que se encuentra, está en permanente movimiento. Como explica Onfray (2007), en el avión, el barco, el tren, el coche, etc., se comparte un espacio común: el tiempo de pasaje de un punto a otro. La cabina del vuelo, el pontón, el vagón, la banqueta, la cabina ofrecen ocasiones de proximidad, incluso de promiscuidad, que fuerzan la relación o constriñen la conversación. En este microcosmos comunitario se juega una intersubjetividad limitada en el tiempo. Desde la llegada del aeropuerto, del puerto, de la estación, del aparcamiento, esta sociedad se deshace la mayoría de las veces. La sociedad “en tránsito”: paisajes móviles, performativos e interconectados Como Bégout (2008) enuncia: “El sistema liberal en su desarrollo reclama con insistencia seres literalmente a la deriva, sin lazo ni vínculo, que puedan en cualquier momento, al cabo de una sola llamada telefónica, ser enviados al otro lado del mundo” (p. 74) o del país. A ello agrega que “todo el mundo está llamado a convertirse en un ser migratorio e itinerante, lanzado a las carreteras y al tránsito, en función del trabajo y sus oportunidades, 123 Pellicer et al. \| La observación participante y la deriva… \| ©EURE siguiendo el fujo de los capitales como los hobos seguían, a comienzos del siglo XX, las obras móviles a través de la inmensidad del territorio americano” (p. 75). p Es así como la sociedad contemporánea recupera viejas fguras literarias y socia- les de la ciudad moderna: el fâneur de Poe (“El hombre de la multitud”), de Hof- man (La atalaya del primo), de Baudelaire (El pintor de la vida moderna) y de Ben- jamin (París, capital del siglo XIX); el viajante de Cortázar (“Manuscrito hallado en un bolsillo”) y de Saramago (Viagem a Portugal); o también, el nómada de Genet (Diario de un ladrón). Figuras que emergían de las ciudades de fnales del siglo XIX y principios del XX y del movimiento que les dio forma. Figuras que se convertían en metáforas de las ciudades de tiempos pasados y que, a la vez, se usaban para des- cribir los problemas y las carencias de las urbes del momento. En la ciudad actual, el desplazamiento, la metamorfosis y la movilidad constante han provocado que los habitantes de las ciudades se hayan transformado en ciudadanos a tiempo parcial de múltiples emplazamientos urbanos. Las personas se han convertido en territo- riantes. “El territoriante no es sólo el habitante o residente de un lugar; también es usuario de otros lugares, visitante —intensivo o extensivo— de otros lugares; esta- blece su relación con el espacio metropolitano a partir de un criterio de movilidad (...); es habitante de geografías variables en ciudades de geometría variable; habita entre lugares más que habitante de un lugar” (Muñoz, 2008, p. 28). Y, también, los ciudadanos han adoptado las características de los radicantes. El radicante puede sin daño separarse de sus raíces primeras, volver a aclimatarse: no existe un único origen sino arraigamientos sucesivos, simultáneos o cruzados. 1 La lógica radicante, que subyace a la fgura del radicante de Bourriaud (2009), es aquella que tiene en cuenta los tra- yectos, las inestabilidades, la precariedad y las formas errantes propias de la época global. Con esta denominación se quiere resaltar el dinamismo y la manera de vivir y entender el mundo, enfatizando las trayectorias, la interconexión y el desplazamiento, resaltando la creación del cambio, la importancia del tener lugar como constructor del presente. En resumen, esta lógica pretende dar respuesta a la naturaleza móvil y dinámica de los fenómenos sociales actuales. La sociedad “en tránsito”: paisajes móviles, performativos e interconectados En este sentido, la sociedad “en tránsito” incorpora no sólo el movimiento formal o físico sino también el virtual, que, en cierta forma, determina al real y nos transforma en usuarios y consumidores de múltiples empla- zamientos de manera simultánea. Estas nuevas prácticas de la sociedad actual han convertido en obsoleta la anti- gua lógica que regía las tradicionales fronteras espacio y tiempo; se ha modifcado, ha cambiando la forma de entender y relacionarnos con los espacios. En el imagi- nario colectivo sobreviven algunas “supuestas” estabilidades, como elementos que dan una cierta coherencia e inmovilidad a las realidades sociales y urbanas. Estas estabilidades tienen tendencia a la desaparición. “Certezas”, como vivir siempre en el mismo sitio, tener la misma pareja toda la vida, jubilarse en la empresa en la que se empezó a trabajar (Sennett, 1999), y un largo etcétera, han dejado de serlo. El contexto actual se corresponde con un mundo cambiante, donde pocas cosas per- manecen estables, donde los cambios se suceden de una manera acelerada y donde todo es efímero. Las estabilidades, o elementos sociales que se mantenían más o menos invariables en el tiempo están desapareciendo, al ponerse en revisión cons- tantemente a causa de ciertos efectos colaterales de los procesos mundiales (como las políticas económicas y sociales, las nuevas estrategias de producción, la crisis y los desastres globales… que modifcan el panorama mundial y global en un tiempo muy reducido). Bauman (2006) nos habla de una sensación de ansiedad generaliza- da, ansiedad causada por lo que puede ocurrir en cualquier parte de manera inespe- rada. Si a esta ansiedad, este miedo líquido, le sumamos los múltiples cambios que a lo largo de nuestra vida experimentamos, tenemos algunos de los elementos que sustentan la forma de vida actual. En consonancia, hay que substituir la lógica de las estabilidades por la lógica radicante1: una lógica que se basa en los trayectos, en el proceso, en el camino, en el viaje, en las etapas, en cómo se produce y reproduce la cotidianidad y, por ende, lo social y la ciudad, desde lo movedizo y desde lo que ocurre en la metamorfosis permanente; una lógica que tenga en consideración el movimiento constante y que sea la alternativa a la estabilidad, a la reifcación y a la cristalización de lo social. Esa es justamente su condición de posibilidad. La sociedad “en tránsito”: paisajes móviles, performativos e interconectados (...) El radicante se pone en camino sin disponer de ningún espacio donde volver: no existe en su universo ni origen ni fn, excepto los que decida fjarse para sí mismo. Se puede llevar consigo fragmentos de identidad, a condición de que se los trasplante en otros suelos y que acepte su permanente metamorfosis (Bourriaud, 2009, pp. 57–58). El radicante puede sin daño separarse de sus raíces primeras, volver a aclimatarse: no existe un único origen sino arraigamientos sucesivos, simultáneos o cruzados. (...) El radicante se pone en camino sin disponer de ningún espacio donde volver: no existe en su universo ni origen ni fn, excepto los que decida fjarse para sí mismo. Se puede llevar consigo fragmentos de identidad, a condición de que se los trasplante en otros suelos y que acepte su permanente metamorfosis (Bourriaud, 2009, pp. 57–58). Las personas se han convertido en transumers; esto es, Las personas se han convertido en transumers; esto es, consumidores guiados por la adquisición de más experiencias en un universo no fjo, para la búsqueda de aquel entretenimiento o descubrimiento que anhele la sensación de aburrimiento. Consumidores con un estilo de vida progresivo y voluntariamente transitorio, en el que la ansiedad por la propiedad de objetos y posesiones no tiene lugar. El universo de lo fjo es remplazado, en cambio, por una nueva obsesión (...) donde el objetivo es más la acumulación de tantas experiencias y historias como sea posible (Muñoz, 2010, p. 86). Estas fguras, y otras muy parecidas (el inmigrante, el exiliado, el turista, el errante urbano), dominan la cultura urbana contemporánea (Bourriaud, 2009): se trata de fguras cuya característica común es la capacidad de convertir los desplaza- mientos en un estilo de vida, que se adapta a los mismos, integrándolos en la lógica de una trayectoria adaptativa. y p Las prácticas sociales de los ciudadanos no sólo han cambiado con relación a la movilidad física; también han sido modifcadas por las posibilidades que la tecno- logía ofrece. Ello ha modelado y propiciado nuevas formas de relaciones sociales, de consumo, de trabajo, posibilitando a la vez los desplazamientos, los trayectos y ©EURE \| VOL 39 \| N O 116 \| ENERO 2013 \| pp. 119-139 124 los movimientos virtuales. La sociedad “en tránsito”: paisajes móviles, performativos e interconectados Es desde el interior de la lógica radicante que se debe observar y comprender cómo se construye y se vive la ciudad actual. La investigación social no debe per- manecer ajena a esta lógica, debe ser sensible a los fenómenos que estudia y a sus procesos de naturaleza maleable y efímera. La investigación social tiene que, en consecuencia, incorporar el movimiento como característica principal de sus he- rramientas metodológicas, adaptándose a estas nuevas formas de relación. Es ne- cesario el uso de metodologías y técnicas de investigación que permitan vencer las 125 Pellicer et al. \| La observación participante y la deriva… \| ©EURE difcultades que presenta la movilidad. Como Urry (2007) apunta, para las ciencias sociales, acostumbradas a trabajar con “sociedades” y otras formas de arraigo per- manente o semipermanente, es complejo pasar a pensar procesos (y metodologías) que tienen que ver con el movimiento y el fujo constante de información. Esta necesidad también queda planteada en Fincham, McGuinness y Murray (2010): “No es apropiado usar métodos que, por su propia naturaleza, se empeñan en aco- modar el reto contextual que supone un mundo compuesto de prácticas móviles” (p. 7), sino que deben usarse métodos y técnicas que compartan la misma lógica, la misma forma de ver y entender la sociedad contemporánea que la produce (Sheller & Urry, 2006; Büscher & Urry, 2009, 2010; Urry, 2007; Delgado, 2002; Fincham, McGuinness & Murray, 2010). En defnitiva, en el contexto actual hay que optar por el uso de técnicas móviles que permitan experimentar y vivenciar la ciudad, formar parte de lo que ocurre en ella. De esta manera, el investigador no “verá” pasar las escenas, sino que se convertirá en un integrante más de ellas y las vivirá como tales (como ilustran las Figuras 1 y 2). Esta experiencia será esencial para el desarrollo de cualquier proyecto de investigación que pretenda acercarse a la me- tropolis actual. Figura 1 Formando parte del movimiento (Viviéndolo desde dentro) Figura 1 Formando parte del movimiento (Viviéndolo desde dentro) 126 ©EURE \| VOL 39 \| N O 116 \| ENERO 2013 \| pp. 119-139 fuente elaboración propia. Figura 2 Estando estáticos, y hasta cierto punto obstruyendo el fujo (Viviéndo- lo desde fuera) fuente elaboración propia. Defniendo la metodología móvil fuente elaboración propia. Defniendo la metodología móvil Defniendo la metodología móvil Como ya se ha especifcado, las personas son habitantes y practicantes de una so- ciedad “en tránsito”, regida por la lógica radicante. En la realidad urbana, meta- morfoseada, transformada, transmutada hasta la saciedad, sobresalen las fguras citadas. De la modernidad rescatamos la fgura del fâneur, y nos convertimos en viajeros que, con placer, buscamos observar minuciosamente la ciudad para dis- frutar —mientras investigamos— de las inacabables trayectorias cotidianas. De la posmodernidad destacamos que las personas se han convertido en territoriantes, radicantes y transumers. Las fguras y la lógica de lo móvil se relacionan directamen- 127 Pellicer et al. \| La observación participante y la deriva… \| ©EURE te con una metodología que tenga en cuenta el movimiento, es decir, con métodos de investigación que también necesitan estar en movimiento (Urry, 2007; Büscher & Urry, 2009; Fincham, Mcguinness & Murray, 2010; Büscher, Urry & Witchger, 2010). Se requieren métodos que permitan al investigador convivir con lo móvil, formar parte del fujo y de la lógica que los rige; observar el proceso desde su inte- rior y en el momento en que se produce para entenderlo y analizarlo. En defnitiva, se trata de acercarse al estudio de la vida urbana, a cómo se vive y cómo se construye la ciudad actual, teniendo presente la necesidad de usar técnicas de investigación que tengan en consideración, por un lado, la transformación física y social de los espacios; y por otro, la velocidad y la aceleración propia de la sociedad actual. Büscher y Urry (2009, 2010), Bruner (2005), y Hein, Evans y Jones (2008) subrayan la necesidad de potenciar la metodología móvil en las ciencias sociales. Proponen diferentes técnicas de investigación móvil, como, por ejemplo, “el segui- miento a las personas”, “el caminar con”, la etnografía móvil, diarios de tiempo y de espacio, imaginería de los espacios o los lugares, la descripción de lugares, la memo- ria de los lugares, “el seguimiento de los objetos”, etcétera. En este sentido, sostene- mos que hay aspectos importantes sobre la metodología móvil que hay que tener en cuenta y que vale la pena clarifcar: a) No hay que descuidar la tradición móvil de las técnicas de investigación social tradicionales. 2 Antes de empezar, un breve apunte sobre el método en el que se encuentran inscritas ambas técnicas de recogida de datos. Se trata de investigación etnográfca. Las etnografías “muestran las vidas de los actores sociales construyendo diferentes espacios y temporalidades, los diversos signos que no solamente organizan la percepción sino que contri- buyen, con diferenciado poder, a construir el mundo que nos rodea y establecen una especial relación con el exterior, caracterizando los lugares, a veces instituyéndolos” (Vergara, 2001, p. 8). Por la especifcidad del contexto en el que se realiza el contexto urbano, se trata de una etnografía urbana (Delgado, 2002). Defniendo la metodología móvil Cuadro 1 Técnicas móviles en las ciencias sociales entes Büscher y Urry (2009); Büscher, Urry y Witchger (2010). A continuación se muestran dos ejemplos de técnicas de investigación móviles, que permiten a los investigadores sociales y urbanos observar e interpretar la socie- dad contemporánea. Defniendo la metodología móvil A las técnicas móviles que están relacionadas directamente con la geografía, la sociología y la antropología, hay que añadir la recuperación o el uso de algunas técnicas móviles “clásicas” en las ciencias sociales, como, por ejemplo, la observación participante y la deriva, entre otras. b) Se debe incorporar el carácter intrínsecamente cualitativo de la metodología mó- vil: la característica que defne la realidad social es, justamente, su movilidad. Los investigadores de la ciudad y de lo social no pueden escapar a dicha característica, debido a que forman parte de lo móvil; por ende, tanto la metodología como las técnicas que usan incorporan, de forma intrínseca, esta cualidad. c) Hay que ofrecer una defnición clara y precisa de lo que entendemos por metodo- logía móvil. Autores como Büscher y Urry (2009, 2010), Fincham, McGuinness y Murray (2010) refexionan sobre la metodología móvil de una manera amplia y enumeran algunas técnicas concretas y algunos ejemplos interesantes. En el marco de las anteriores consideraciones, se propone que la metodolo- gía móvil es un tipo de aproximación cualitativa que capta la transformación de la sociedad y de lo urbano desde su interior, que indaga sobre el movimiento y la transformación de lo social. Y, también, que es aquella metodología cualitativa que capta la transformación de la ciudad en su proceso, que indaga sobre el movimiento cuando tanto la técnica como el investigador están en movimiento. En defnitiva, la metodología móvil es un tipo de metodología cualitativa que permite observar los diferentes fenómenos sociales y urbanos mientras se están produciendo, al mismo tiempo que se forma parte de ellos. ©EURE \| VOL 39 \| N O 116 \| ENERO 2013 \| pp. 119-139 128 Cuadro 1 Técnicas móviles en las ciencias sociales Técnicas que tienen por objeto el análisis del movimiento Técnicas en las cuales el investigador está en movimiento La entrevista semiestructurada, el grupo de discusión, la historia de vida, las fuentes documentales, la observación participante, los diarios de tiempo y de espacio, la imaginería de los espacios o los lugares, la descripción de lugares, la memoria de los lugares, la simulación, etcétera. La deriva, la observación participante, “el seguimiento a las personas”, “el caminar con”, la etnografía móvil, “el seguimiento de los objetos”, la entrevista en movimiento, la simulación en movimiento, la realidad aumentada, etcétera. fuentes Büscher y Urry (2009); Büscher, Urry y Witchger (2010). La observación participante y la deriva: dos ejemplos de técnicas móviles2 En este apartado se refexiona sobre dos técnicas para el estudio de la ciudad: la observación participante y la deriva. Nuestra intención es refexionar sobre ellas, mostrar aportes y debilidades de cada una, comparándolas y haciendo emerger los puntos en común y sus principales diferencias. Se profundiza en la deriva, debido a que su origen es crítico, lúdico y festivo, y en este artículo se presenta como una técnica móvil de investigación social. La observación participante implica, en el caso de los estudios urbanos, la in- mersión activa del investigador en la ciudad, con el objetivo de obtener el mayor número de datos sobre ella. La función principal de esta técnica es garantizar su ac- ceso privilegiado a los signifcados que los actores construyen y asignan a su mundo, por medio de un acercamiento a las prácticas que allí se realizan. En la observación participante se observa in situ y se busca compartir experiencias en el ambiente que se estudia (Platt, 1982). Esta técnica, en el contexto urbano actual, permite al in- vestigador formar parte y estar en contacto con los diversos procesos y los trayectos que se producen en ese ámbito. El investigador que adopta esta técnica requiere de una doble perspectiva. Por un lado, debe mirar y actuar como participante; y por otro, como observador. Para lograr esa doble experiencia, el observador participante de la ciudad debe intervenir simultáneamente como miembro de ella y como “extraño” a ella. De esta forma, el 129 Pellicer et al. \| La observación participante y la deriva… \| ©EURE investigador no se limita a observar el espacio, sino que también se propone formar parte de lo que ocurre en él: se genera así una participación interna o activa, en per- manente proceso lanzadera, que funciona como observación sistematizada natural de grupos reales o comunidades en su vida cotidiana, y que emplea la estrategia empírica y las técnicas de registro cualitativas. Como señalan Velasco y Díaz de Rada (1997, p. 103), “el investigador nunca trabaja sólo como investigador; trabaja también como vecino, como amigo, como desconocido, como hombre o mujer, (...) y con otros papeles que él se haya forjado o que le haya conferido el grupo que anali- za y con el que convive” o en el espacio por el cual se desplaza. La observación participante y la deriva: dos ejemplos de técnicas móviles2 Mediante esta técnica se pueden observar las actividades comunes y corrientes de las personas; esto es, facilita que la observación se produzca en los contextos urbanos cotidianos, con interacciones, prácticas sociales y comportamientos igualmente naturales, justo tal y como ocurren. Al respecto, y según precisa Delgado (1995), “el [investigador] debe estar consciente de que la sociedad tiende a aprender reglas culturales que se convierten en hábitos” (p. 149). Esta técnica de recogida de datos supone un acercamiento al objeto de estudio y, a la vez, un proceso de socialización por medio de la técnica. Cuando se usa, el in- vestigador se convierte en un intérprete y un conocedor de aquello que analiza, por lo que es capaz de interiorizar y compartir la lógica radicante. Es entonces cuando se genera conocimiento: ocurre cuando se produce la transformación del investi- gador en un miembro competente del espacio que analiza, al participar y vivir, de una manera íntima, con él. En la sociedad “en tránsito”, la observación participante ofrece la oportunidad de formar parte de los desplazamientos diarios que están relacionados con los procesos y los fenómenos urbanos y sociales que son objeto de investigación. De esta manera, se le abre al investigador la posibilidad de compren- der los distintos recorridos y trayectos que realizan las personas y cómo mediante ellos se construyen los signifcados sociales de la ciudad, a la vez que se ve envuelto en las prácticas cotidianas relacionadas con su tema de estudio. La deriva se presenta en este artículo como una técnica de recogida de datos es- pecífca para el estudio de la ciudad. Su origen se asocia con la Internacional Situa- cionista (Debord, 1958). Esta técnica es un tipo de observación participante que permite captar el movimiento desde el movimiento. La deriva supone también una refexión sobre las formas de ver y de experimentar la vida urbana. Así, mediante la deriva, el investigador se escapa de la prisión de la rutina cotidiana y se deja llevar por las ciudades, siguiendo caminos y trayectos inciertos. Mediante la deriva, las situaciones urbanas se miran de una forma nueva y radical, por el paseo. Certeau (1999) consideraba que “el acto de caminar es al sistema urbano lo que el habla es al lenguaje”. Y lo que la deriva pretende es recoger la multiplicidad de los “actos de habla” (Searle, 1980) de la ciudad para su posterior análisis. 3 El recurso a la deriva, cuando se la realiza con rigor y sistematización, no implica, en ningún caso, que esta observación de lo urbano deba quedar en una mera descripción o deleite estético. Según Perinola (1972), “no se parece al deambular de los surrealistas, una experiencia meramente urbana” (p. 25). A partir de ella se obtiene información valiosa del espa- cio, siendo una técnica de estudio que no altera el espacio ni los fujos que en él se dan, ya que el investigador que la usa los sigue, se involucra y forma parte de ellos. La observación participante y la deriva: dos ejemplos de técnicas móviles2 La característica principal de la deriva es que no se acota el campo de observa- ción. Sólo se escoge el entorno urbano objeto de estudio, por el cual se va a derivar. Durante la caminada se recogen los datos, a modo de texto/discurso. La trayecto- ria/relato permite al investigador reconocer e interpretar los aspectos urbanos y ©EURE \| VOL 39 \| N O 116 \| ENERO 2013 \| pp. 119-139 130 sociales. Así pues, mediante la deriva y gracias al movimiento, al deambular, al tras- lado, se recopila información sobre la ciudad, que posteriormente será analizada. El investigador que usa esta técnica adopta el modo de actuar del fâneur (Baudelaire, 1994; Benjamín, 1927), transformándose de paseante en “observador más directo e investigador de los signifcantes de la ciudad” (Frisby, 2007, p. 49). Frisby resalta el carácter productor de esta fgura: “como observador urbano, el fâneur ‘va a hacer botánica al asfalto’, a recoger y registrar imágenes urbanas, interacciones y tipifca- ciones sociales” (p. 51). En la deriva, el investigador renuncia durante un tiempo más o menos largo a desplazarse o actuar por los motivos habituales vinculados a los quehaceres diarios o de ocio, con el objetivo de dejarse llevar por las solicitaciones de los espacios y los encuentros que a ellos corresponden (Debord, 1958). De alguna manera, el inves- tigador busca alienarse para poder observar con una mirada crítica. Mediante esta técnica se pretende captar la información que el espacio desprende, información que en una observación más focalizada podría pasar inadvertida. Lo fundamental es, como proponía Debord (1958), que mediante la deriva puedan observarse las situaciones urbanas de una forma nueva, radical, realizán- dose una lectura de la ciudad acorde con esa observación. La deriva, según Vivas, Pellicer y López (2008, pp. 132–133), “como técnica y en lo que implica a nivel epistemológico, [supone] la renuncia a una mirada totalizadora y genérica sobre el espacio urbano, para detenerse en la importancia que las prácticas sociales efímeras, invisibles e insignifcantes puedan tener para la comprensión [de este espacio]”. Esta técnica ha sido tildada como una práctica lúdico-festiva de escaso aporte científco, fruto de que en algunas ocasiones se ha perdido de vista su objetivo: la re- colección de datos para leer y acercarse con una mirada interrogadora y de carácter crítico a lo urbano3. La observación participante y la deriva: dos ejemplos de técnicas móviles2 Es decir, se ha dejado de lado su carácter consciente, lo que se pretende con ella, que es la recopilación de datos para luego analizarlos y responder a la pregunta o preguntas derivadas del objetivo de la investigación realizada. La técnica de la deriva es sustancialmente distinta a la fâneurie de Baudelaire, tanto en la forma como en su objetivo y en la actitud y propósito del ‘investigador’, aun- que comparte con ella el interés por lo urbano (el enigma que hay que descifrar), la producción de lecturas de la ciudad, y el dejarse abstraer por las solicitudes del territorio. La deriva va más allá del paseo, del deambular, diferenciándose cualitati- vamente de ellos porque apunta al reconocimiento de ciertos efectos del contexto urbano (Perinola, 1972). En resumen, con el uso de la observación participante se trata de ir buscando el movimiento y la transformación social. La técnica de la deriva nos propone ir encontrando el movimiento y la transformación social, fruto de observar con una mirada interrogadora, crítica y atenta a los detalles, lo que ocurre en dichos pro- 131 Pellicer et al. \| La observación participante y la deriva… \| ©EURE cesos. En defnitiva, se propone, con ambas técnicas, mirar de nuevo, o mirar de manera diferente, la ciudad y los espacios urbanos: transitarlos y reinterpretarlos, cuestionando los discursos que de ella y ellos aforan. Ambas técnicas generan datos descriptivos, y pueden resultar complementarias para el estudio de lo urbano. Con la deriva se capta lo que ocurre en los espacios urbanos por los que se deambula, capturando el movimiento estando en movimiento; y con la observación partici- pante se busca la comprensión de los signifcados y de las prácticas. Por lo que cada una aporta, es útil que, para el estudio de los espacios urbanos contemporáneos, se utilicen ambas técnicas de manera complementaria. Cuadro 2 Aspectos claves de la observación participante y de la deriva Cuadro 2 Aspectos claves de la observación participante y de la deriva Observación participante Deriva Elegir el “campo urbano” por observar. Elegir el punto de inicio. Fijar días y horarios de la estancia en el campo. Fijar el tiempo de “perdida” por la ciudad. Participar e interactuar con la situación y los protagonistas. Disfrazarse de fâneur y dejarse llevar por las oscilaciones de la ciudad. La observación participante y la deriva: dos ejemplos de técnicas móviles2 Usar herramientas de recogida de datos que permitan una observación efcaz (notas de campo, grabadora, cámara digital, etcétera). Usar herramientas de recogida de datos que provoquen una deriva efectiva (grabadora, cámara de video, etcétera). Intentar observar con detalle y con mirada interrogadora todo lo que acontece. Intentar observar en detalle y con mirada interrogadora todo lo que acontece. Traducir y “pasar a limpio” la información recopilada durante la jornada de observación. Traducir y “pasar a limpio” la información recopilada durante la deriva. fuente elaboración propia. Aplicando las técnicas móviles a la ciudad actual La urbe y, por ende, la sociedad, sufren una metamorfosis diaria ilimitada. Este aspecto condiciona que, en la actualidad, las instituciones y los conceptos socia- les no sean “fjos”. Las estructuras y las concepciones de lo social, y de la ciudad, son móviles, maleables y en proceso de constante transformación. Debido a que las ciudades, y las sociedades, se transforman permanentemente, se impone el uso de una metodología y, por ende, unas técnicas de investigación que nos permitan recoger “datos sociales” que a la vez que se desplazan, se mueven, se deslizan, se están transformando, transmutando, metamorfoseando. Así pues, en este apartado queremos ejemplifcar el uso de estas técnicas en una ciudad/sociedad móvil y que se transforma cotidianamente. A lo largo del trabajo se subraya que la observación ©EURE \| VOL 39 \| N O 116 \| ENERO 2013 \| pp. 119-139 132 participante y la deriva, dos técnicas móviles para el estudio de la ciudad actual, son sumamente aconsejables si se quiere investigar lo urbano, ya que ellas permiten que el investigador estudie los procesos urbanos formando parte del movimiento, dejándose llevar por los fujos que se producen y traspasan la ciudad. En el estudio que aquí se presenta, la puesta en práctica de estas dos técnicas se concretará en una ciudad (Barcelona) y en dos infraestructuras de la misma (el metro y el aeropuerto). Los criterios de elección de esta urbe y de estas dos infraestructuras están rela- cionados con su carácter transformador y móvil. Barcelona, a lo largo de las últimas décadas, ha experimentado un proceso urbanístico, social y económico muy im- portante, que la ha llevado a erigirse como una de las capitales de Europa. Según el informe del consistorio municipal, algunas cifras actuales (2009) ponen de ma- nifesto este carácter global de la urbe. Aplicando las técnicas móviles a la ciudad actual El área de Barcelona es la sede de 467.000 empresas; la ciudad localiza más de un millón de puestos de trabajo y su área de infuencia concentra cerca de 2,5 millones de trabajadores; el área de Barcelona concentra 113 centros tecnológicos y de investigación, 33 infraestructuras para la innovación y 9 instalaciones científcas y técnicas de referencia internacional; la ciudad recibe 6,7 millones de visitantes (2008), distribuidos en partes iguales entre el turismo vacacional y el de negocios; es la primera destinación de cruceros de los puertos del Mediterráneo, con más de dos millones de pasajeros; Barcelona ocupa la tercera posición en la escala mundial de organización de ferias y congresos, con un total de 280.000 metros cuadrados de superfcie destinada a exposiciones y cerca de 700.000 asistentes a congresos. Para poder sustentar estas cifras, este fujo de personas, de mercancías y bienes económicos, de ideas sociales, se están ampliando diferentes infraestructuras: aeropuerto, metro, redes de trenes de cercanía y de alta velocidad, puerto, entre otras. En este contexto, para aplicar ambas técnicas, hemos elegido: a) El aeropuerto de Barcelona, por la ampliación de sus instalaciones cuando en- tró en funcionamiento la nueva terminal, en junio de 2009; y porque es una infraestructura que, durante ese mismo año, registró un tráfco de 27.421.682 pasajeros, 278.981 operaciones y 89.815 toneladas de carga. b) La red del metro de Barcelona, por la constante ampliación de su red. Por ejemplo, durante 2009 entraron en funcionamiento las líneas 9 y 10 —que estarán entre las mayores de Europa— y en 2010 se fnalizó la ampliación de dos líneas (L2 y L5). También se la eligió porque registró un total de 361,65 millones de viajeros en 2009, y representa un emplazamiento vital que sostiene el entramado urbano y social del área metropolitana. Las cifras señaladas muestran cómo el aeropuerto y el metro se han convertido en dos de las principales infraestructuras estratégicas que soportan una parte muy impor- tante de Barcelona como ciudad global. Por tanto, ambas son espacios icónicos en el sentido apuntado por Gottdiener (2001), Adey y Bevan (2006) y Sheller y Urry (2006). En el caso del aeropuerto de Barcelona, se decidió que la técnica más adecuada 133 Pellicer et al. \| La observación participante y la deriva… \| ©EURE para observarla, con las terminales elegidas (T1 y T2), era la observación partici- pante. Aplicando las técnicas móviles a la ciudad actual Los criterios de selección de esta técnica estaban relacionados con el objetivo de observar las prácticas y uso del espacio por parte de los usuarios de dicha infraes- tructura. Lo que la observación participante permite, en este caso, es el acceso y acercamiento a los signifcados que los actores construyen y asignan a su mundo, permitiendo al investigador formar parte de lo que en el aeropuerto sucede y estar en contacto con ello. Para analizar la red del metro de Barcelona (L1, L2, L3, L4, L5) se consideró que la técnica apropiada era la deriva. Los motivos de selección de esta técnica es- taban relacionados con la voluntad de analizar qué ocurre y cómo se construye este emplazamiento urbano vital en la articulación de la red urbana y social actual, es decir, de este espacio de tránsito (Pellicer, Rojas y Vivas, 2010). La deriva permitió recopilar discursos y prácticas que tenían lugar en él. Observando la nueva terminal 1 y la antigua terminal 2 del aeropuerto de Barcelona Observando la nueva terminal 1 y la antigua terminal 2 del aeropuerto de Barcelona La construcción de la nueva terminal del aeropuerto (T1), y su reciente entrada en funcionamiento, signifcaron una excelente oportunidad para iniciar una ob- servación participante de un nuevo espacio urbano, y ello por diferentes motivos. En primer lugar, porque permitiría analizar el tipo de prácticas y movilidades que se llevan a término en un espacio que se acaba de inaugurar. Es decir, cómo se usa y se practica un espacio de nueva construcción con unas determinadas características arquitectónicas y unas específcas intenciones económicas y sociales; por ejemplo, convertirse en un hub europeo de referencia. Una segunda razón que llevó a elegir- lo fue la posibilidad de estudiar cómo la tecnología está integrada en el edifcio y cómo potencia y participa en la gestión de las prácticas y las movilidades sociales. En esa misma línea, se quería indagar de qué forma los usuarios que están en tránsi- to, y los que no, usan las tecnologías en sus trayectos o en sus momentos de espera. Por otro lado, la antigua terminal (T2) del aeropuerto posibilitó observar otras rea- lidades que quedaron al descubierto, tales como las personas “sin techo” que viven en el aeropuerto, poniendo de manifesto las carencias del sistema poscapitalista. Aplicando las técnicas móviles a la ciudad actual El trabajo de observación se inició en diciembre de 2009 en la T1 y en marzo de 2010 en la T2. El uso de una técnica como la observación participante implicó que los investigadores, cuando se encontraban en el “campo aeroportuario”, fueran, a la vez, usuarios de las terminales. Ello signifcó también que se aprovechó cualquier viaje para profundizar todavía más en alguno de los objetos de estudio. Se amplió la información recopilada conversando con cinco informadores. ©EURE \| VOL 39 \| N O 116 \| ENERO 2013 \| pp. 119-139 ©EURE \| VOL 39 \| N O 116 \| ENERO 2013 \| pp. 119-139 134 trayectos, viajes, transbordos, etcétera. Además, es un eje de movilidad ciudadana que está en permanente transformación y adecuación a las necesidades y los avances tecnológicos. Este hecho nos permitió observar la evolución y las nuevas maneras de pensar y vivir estos espacios. Al igual que en el caso del aeropuerto, se pretendía indagar sobre el tipo de prácticas y de movilidades que se llevan a término en la infraestructura del metro, y cómo la tecnología integrada en la estructura de las estaciones, trenes, intercambia- dores y otros de sus componentes, potencia y participa en la gestión de prácticas y movilidades. Se quería observar cómo los usuarios que están en tránsito en el metro, y los que no, usan las tecnologías en sus trayectos o en sus momentos de espera. El periodo de derivar por el metro de Barcelona se inició el año 2006 y se alarga hasta hoy. Cada vez que se realiza una deriva, se programa el lugar de inicio de la misma. Con ello aseguramos la variabilidad de líneas, de estaciones, de intercam- biadores por los cuales pasear y obtener una mayor versatilidad de prácticas y de fujos de usuarios. Derivando por las líneas de la red del metro de Barcelona El metro de Barcelona, como el de muchas otras ciudades —es decir, sus trenes, estaciones, intercambiadores, andenes y vestíbulos—, conforma una realidad ur- bana y social repleta de personas que provocan una multiplicidad e infnidad de Prefacio de Barcelona mediante sus desplazamientos Así pues, como seres sociales “interiorizamos” aquellas prácticas que podemos llevar a cabo en estos espacios de movilidad, y aquellas que no están permitidas. Se entra y se usa estos espacios realizando los rituales de paso esperados, sin que im- porte (aparentemente) que, por ejemplo, una persona de seguridad nos registre, una cámara de videovigilancia controle cuando se usa el wc, una máquina detecte ele- mentos metálicos y substancias prohibidas en el arco de control, y así por delante. Se hace evidente que las personas desean perder el menor tiempo posible cuan- do se mueven, anhelan aprovechar el tiempo de viaje. Esa búsqueda por ganar tiempo al tiempo empieza, por ejemplo, con la evolución del procedimiento de las compañías aéreas para la emisión del billete de avión. Tecnológicamente se está in- novando constantemente a fn de realizar los trayectos con mayor rapidez no sola- mente en el inicio de trayecto, sino también durante el tiempo del mismo (emisión del pase de abordar, lector del código de barras en el primer control, mensaje sms con código QR, etcétera). Asimismo, la arquitectura de los edifcios y de las infraes- tructuras incorpora elementos para procurar ganar tiempo a los periodos de viaje (cintas transportadoras, escaleras mecánicas, entre otros implementos). Otro aspecto destacable durante el tiempo del trayecto remite al uso de disposi- tivos móviles por parte de los usuarios de los medios de transporte, ya sea para inte- ractuar con otras personas (amistades, negocios, otros) o para buscar información durante el tiempo de viaje. El transumerismo (Muñoz, 2010) se hace visible en estos espacios. Los actos de consumo mediante el viaje que lo constituyen se practican durante el tiempo del trayecto, debido a que se pone al alcance del viajero una multitud de posibilidades para llevarlos a cabo. Tanto en las instalaciones de los aeropuertos como en las es- taciones del metro se han incrementado las posibilidades de consumo. Los espacios interiores de estas infraestructuras se han convertido en calles, plazas o centros co- merciales en los cuales se pueden realizar actividades de ocio, de consumo o cultu- rales. La presencia de franquicias comerciales hace que el paisaje comercial se mul- tiplique en ellos. La ubicación de restaurantes, bares o máquinas dispensadoras de bebidas y alimentos proporciona una variabilidad de posibilidades gastronómicas. Prefacio de Barcelona mediante sus desplazamientos La amalgama de fragmentos e informaciones sobre el aeropuerto y el metro refejan una realidad híbrida y compleja, teñida de un conjunto de prácticas sociales y ur- banas que están relacionadas con el movimiento, con el fujo de usuarios y con las nuevas tecnologías. Lo tecnológico se desdibuja y se imbrica en estos espacios y con sus usuarios. A continuación, se detallan brevemente algunos aspectos de una reali- dad urbana como la de Barcelona, que se encuentra en cualquier parte del mundo. La movilidad colectiva debe ser segura y, en todo momento, las prácticas y las trayectorias sociales están sujetas a la vigilancia. La disposición de elementos de control (cámaras de vídeo, policía, empresas de seguridad, personal del aeropuer- to, etcétera) provoca una serie de efectos concretos. El primero, asegurar que en el interior de estos espacios sólo se lleven a cabo las prácticas sociales que están permi- tidas en ellos. El segundo, que los usuarios tengamos la sensación de estar seguros cuando usamos estos espacios, cuando estamos viajando (aunque no sepamos, de forma cierta, si los dispositivos funcionan o están atentos). El tercero, que aunque tenemos la sensación de estar en un espacio privado (o semiprivado), nuestras tra- yectorias y nuestras prácticas son, al estar vigiladas y controladas en todo momento, cada vez más públicas y visibles. Con relación a este último efecto, se destaca que los espacios del aeropuerto y del metro entran en continuidad con el espacio público de la ciudad, no solamente por la vigilancia y el control, sino también por el tipo de prácticas (de consumo, de ocio, de relación, etcétera) que realizamos en tales estructuras de movilidad. En estos espacios existe una normativa específca sobre el comportamiento co- lectivo esperado. Se regulan todos los aspectos relacionados con lo que se puede y no se puede hacer durante el tiempo de uso de la instalación, y durante el tiempo de trayecto. Así, la normativa cívica que regula el uso del espacio público de la ciu- 135 135 Pellicer et al. \| La observación participante y la deriva… \| ©EURE dad se conecta directamente con las diferentes normativas que regulan el uso del espacio del aeropuerto y del metro. Con ello se asegura que las prácticas y movili- dades estén normativizadas desde el momento preciso en que cruzamos el umbral de nuestras casas. Refexiones fnales Por medio de este artículo manifestamos que lo urbano no se encuentra fjo ni es- tático; es decir, este trabajo habla de las ciudades que se transforman mediante su permanente movilidad: la que experimenta la propia ciudad y la que se visualiza en las prácticas sociales de las personas que la habitan y la transitan. En este sentido, asumimos que las prácticas que se producen en la ciudad construyen colectivamente el espacio urbano, el cual es caracterizado por el fujo constante de movimientos, desplazamientos, comunicaciones, etcétera. Hay en la ciudad movimiento de vehículos, de gente, de mercancías, de información, de imá- genes, de sonido y de conocimiento. Tal movilidad es orquestada, directa o indi- rectamente, por los propios usuarios de la ciudad, sus habitantes, quienes compran, se desplazan, aprenden, se comunican, negocian e incluso llegan a amarse. De esta manera, la movilidad que genera la ciudad, y que a su vez la genera, da lugar a que las prácticas sociales de las personas se encuentren caracterizadas por el perpetuo movimiento y el constante recorrido. ¿Cómo analizamos lo que se mueve o lo que se desplaza en las ciudades? A tenor de esto, la metodología que puede sincronizarse con el movimiento es la metodolo- gía móvil. De la mano de dos técnicas, la observación participante y la deriva, que sirven para ejemplifcar dicha metodología, se recoge información sobre algunas de las prácticas sociales y urbanas observadas y llevadas a cabo por los usuarios en dos casos típicamente urbanos y completamente caracterizados por el movimiento: el metro y el aeropuerto de una gran urbe, en este caso la ciudad de Barcelona. g La metodología móvil es una forma de investigación que capta la transformación de la ciudad en su proceso, que indaga sobre el movimiento cuando la técnica o el in- vestigador están en movimiento. Es por ello que a través de esta metodología se accede a la lógica del contexto actual. Ella posee una mayor capacidad para seguir y refejar, de forma fexible, trayectorias, procesos y cambios (a nivel temporal y/o espacial). De ella se debe enfatizar su carácter cualitativo intrínseco, ya que en tanto metodología cualitativa, permite estudiar los procesos mientras se producen, al mismo tiempo que presta atención a los fenómenos relevantes o signifcativos concomitantes. Prefacio de Barcelona mediante sus desplazamientos La presencia estratégica de exposiciones o la realización de actividades culturales en ambos espacios de tránsito, amplía la oferta cultural de la ciudad. Se busca, de este modo, el máximo rendimiento económico del tiempo de viaje, y también de estas infraestructuras en las que transcurre una gran parte de nuestro tiempo. Teniendo en cuenta estos aspectos generales, que resaltan la ubicuidad social, se puede considerar que la ciudad global, como en el caso de la ciudad barcelonesa —y esto también se puede extrapolar a otras ciudades del mundo—, está habitada por una colectividad transumer, radicante, viajera... que multiplica e incrementa sus trayectorias y desplazamientos diariamente y que aprovecha el tiempo de trayecto ©EURE \| VOL 39 \| N O 116 \| ENERO 2013 \| pp. 119-139 136 para usar los dispositivos móviles (u otras tecnologías) para leer, escuchar música, comunicarse, informarse, todo bajo la “atenta mirada” de los dispositivos de control y de seguridad. A la vez que se produce el desplazamiento, se consume, dada la variabilidad de posibilidades que ofrecen las estructuras de movilidad de la ciudad. Refexiones fnales En este artículo, por tanto, se reivindica el uso de técnicas de investigación mó- vil, aproximaciones que permiten estudiar los procesos urbanos actuales estando en sintonía con el movimiento. Se considera la observación participante y la de- riva adecuadas para el estudio de lo urbano porque permiten acercarse a la ciudad durante su proceso constitutivo y observarla de manera refexiva, fexible y que da cabida a lo inesperado, a lo sorprendente y a lo efímero, que en cierto modo es 137 Pellicer et al. \| La observación participante y la deriva… \| ©EURE aquello que le da forma y signifcado. Al mismo tiempo que se usan estas técnicas, el investigador vive un proceso de transformación, no sólo de la ciudad, sino también de su experiencia como investigador. Las observaciones y registros de la experiencia urbana por medio de las técnicas móviles permiten replantear las transformaciones que ocurren en la interacción so- cial, replanteamientos que constituyen, a su vez, nuevas posibilidades de crear sig- nifcado en torno a la ciudad y lo social. Del mismo modo, y estrechamente relacio- nado con la ubicuidad social, la lógica radicante, al favorecer la habitabilidad entre los lugares y las experiencias nuevas, da pie a una fractura en la supuesta estabilidad de lo social. Pero, sobre todo, la observación participante y la deriva han permitido constatar que la movilidad es una característica sine qua non de la sociedad, de las ciudades y de sus habitantes. Las prácticas sociales tienen como eje articulador el hecho de ser ejecutadas por los actores-territoriantes de la ciudad; es decir, se dan en y por el movimiento y el desplazamiento, evocando al personaje con que hemos empezado este artículo, Ryan Bingham. La ciudad y lo urbano son creados mediante las prácticas “en trán- sito”, las cuales ilustran la movilidad tanto de la ciudad como de sus habitantes. La consecuencia de dichas prácticas es la potenciación de la ubicuidad social y de la lógica radicante, entendiendo la ubicuidad social como la capacidad de interacción diacrónica. Las nuevas tecnologías han transformado las maneras de interacción y, en parte, han creado nuevas. Muchas de las formas de relación están mediatizadas por las nuevas tecnologías. La lógica radicante facilita comprender la transforma- ción de habitante a territoriante, a transumer, adaptándose a las características del radicante, usuario/a de varios lugares a tiempos parciales. Refexiones fnales En alguna de estas fgu- ras, o en todas a la vez, nos hemos ido transformando, al mismo tiempo que experi- mentamos la transformación de la propia ciudad. Por lo que respecta a la lógica radicante, hemos distinguido lo siguiente. Por un lado, la agilización del movimiento, tanto a nivel de la comunicación como del desplazamiento por la superfcie. Agilizar el movimiento es algo recurrente por sms, whatsapp, gps y google maps, o por medio del billete multiviaje o de la lectura del có- digo de barras en el móvil. La velocidad de los desplazamientos asegura el no arraigo, la permanente movilidad entre los lugares. Suelos móviles, escaleras eléctricas, trenes automáticos, venta automática, e incluso autochecking y autoadjudicación de asientos. Por otro lado, está el consumo en la movilidad: el transumerismo. Toda la oferta que se pueda imaginar se encuentra no sólo en los escaparates de la ciudad “en tránsito”, sino también en los dispositivos electrónicos con los que las personas se proveen. La oferta de consumo se multiplica mientras el desplazamiento se lleva a cabo. 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https://openalex.org/W4376469607	https://china.elgaronline.com/downloadpdf/edcoll/9781786434685/9781786434685.00018.pdf	English	null	Index	Edward Elgar Publishing eBooks	2,017	cc-by	7,022	Daniel McFadden and Kenneth Train - 9781786434692 Downloaded from https://www.elgaronline.com/ at 10/24/2024 03:59:17AM via Open Access. Open https://creativecommons.org/licenses/by/3.0/igo/ Index cognitive burden 64 cost 66 income effects 64–5 provision mechanism 65 for reliability of CV 88–90 results 73–5 study discussion and conclusions 78 and truthfulness 60–61, 69, 78 adequate sensitivity to scope 129–37, 140 use of term 88, 140 algae 60, 69, 100, 117, 257, 259 alum 60, 69–70, 72, 74, 100, 117 American Trader spill litigation 299 anchoring biases 178 cost 2, 5, 10, 84 effects 2, 5, 84, 114, 160, 163, 215, 277 as issue surrounding CV 30, 36 in red knot study 55–6 response distortions in 165 ABERS approach 196 estimate for lower bound 200, 204, 206–7, 218 non-parametric estimator 74 adding-up test adaptation for 70–73 “adding-up condition” applying only to goods obtained incrementally 60 consumer preferences not adhering to 67–8 definition 59 description 61–3 deviations from 68 findings 61, 78 implementation of 59 in previous studies 60 testing 60–61 and adequate response to differences in scope 59, 66, 78 applied to restoration program for river system and lake 60–61, 69–75 elicitation method 63, 67–8, 78 explanation 61–3 findings 61, 78 income effects 64–5, 75–7 incremental parts for accelerated restoration 71 and cognitive burden 64 findings 61, 78 lack of studies on 59–60 meaning 59 in original study’s design 70 past studies review 66–8 use to test adding-up condition 60–61 limitations of scope tests 58–9 original study 68–70 potential difficulties in implementing applied to restoration program for river system and lake 60–61, 69–75 Daniel McFadden and Kenneth Train - 9781786434692 Downloaded from https://www.elgaronline.com/ at 10/24/2024 03:59:17AM via Open Access. Open https://creativecommons.org/licenses/by/3.0/igo/ behavior behavior choice 14, 176–9, 237–8 deterministic heuristic rule predicting 239–40 ideal, for validity of WTP estimates 224–5 irrational 48 market 153–4, 156–7 protest 232 purchase 172, 277 randomizer 234 surveys of consumer, in court cases 299–300 use of conjoint methods for predicting 162, 168–70, 173, 176 of utility maximizing 231, 255 behavioral anomalies 36, 112, 154 behavioral economics 155, 157, 159 elicitation method 63, 67–8, 78 elicitation method 63, 67–8, 78 explanation 61–3 findings 61, 78 income effects 64 5 75 7 incremental parts for accelerated restoration 71 and cognitive burden 64 findings 61, 78 lack of studies on 59–60 meaning 59 in original study’s design 70 past studies review 66–8 use to test adding-up condition 60–61 limitations of scope tests 58–9 original study 68–70 potential difficulties in implementing irrational 48 market 153–4, 156–7 307 307 Contingent valuation of environmental goods 308 single-focus approach bid levels 194–6 and WTP for meal vouchers 67–8 “bounded substitution” 84–5, 88–9, 131 brain activity 178–9 budget awareness adoption in survey 192 in CVM studies 188 findings 219 three related enhancements 193 “wording additions” intended to enhance 216–19 budget constraint affecting responses to valuation questions 189 findings 219 in single-focus valuation method 190 in WTP survey methods 189 single-focus approach bid levels 194–6 and WTP for meal vouchers 67–8 “bounded substitution” 84–5, 88–9, 131 brain activity 178–9 budget awareness adoption in survey 192 in CVM studies 188 findings 219 three related enhancements 193 “wording additions” intended to enhance 216–19 budget constraint affecting responses to valuation questions 189 findings 219 in single-focus valuation method 190 in WTP survey methods 189 behavioral response to high bids 17–18, 36 questions to explore 34–5 true 23 behavioral theories 78, 154–5, 157, 159, 252 belief in bid values 32–5 bias ratio (BR) 271–4, 277–85 bids “composite good” approach bid levels cognition difficulties with part- whole relationships 214–15 comparison between composite good and V1 COS 207 non-monotonicity 200 propensity to shift votes 204–6 marine species preservation case 3 percentage of respondents voting for alum treatments 73–4 red knot studies belief in bid values 32–5 bid amount non-parametric estimates by 27, 32, 35 percentage of respondents who believed they would pay more or pay less than offered 34 percentage of yes responses at highest 19–21 percentage of yes responses by 27 yes response irrespective of 30 yes responses v. responses adjusted for “believed” 33 yes responses v. Daniel McFadden and Kenneth Train - 9781786434692 Downloaded from https://www.elgaronline.com/ at 10/24/2024 03:59:17AM via Open Access. Open https://creativecommons.org/licenses/by/3.0/igo/ behavior 17, 19, 58, 60–61, 69, 78, 85–8, 91–8, 100, 112–14, 117, 123, 132, 135, 140, 155, 159, 179, 192, 196, 209, 252–3, 255, 257, 271 percentage of respondents who believed they would pay more or pay less than offered 34 , , 257, 271 CBC see choice-based conjoint (CBC) census weights 54–5 CBC see choice-based conjoint (CBC) census weights 54–5 percentage of yes responses at highest 19–21 CBC see choice-based conjoint (CBC) census weights 54–5 certainty adjustment 31–2 correction 138–40, 273–6, 279–81, 283–5 census weights 54–5 percentage of yes responses by 27 certainty adjustment 31–2 correction 138–40, 273–6, 279–81, 283–5 yes response irrespective of 30 elimination of uncertainty 54–5 levels 30–31 questions 30–31 of response 255–6, 262, 266 weights 54–5 cervical screening study 3–4 “cheap talk” in “composite good” approach 193, 215–16, 218–20 in hypothetical bias study 272, 274, 277, 279–81 in marine species preservation study 6, 228 in scope studies 137 8 elimination of uncertainty 54–5 levels 30–31 q of response 255–6, 262, 266 p weights 54–5 cervical screening study 3–4 share of “yes” votes 50, 52 in “composite good” approach 193, 215–16, 218–20 bid range 18 in hypothetical bias study 272, 274, 277, 279–81 non-monotonicity 26 in hypothetical bias study 272, 274, 277, 279–81 in marine species preservation study 6, 228 in scope studies 137–8 and yes-response function 26–7 309 Index hoke price 18, 28 ognition difficulties with part-whole relationships 214–16 sizeable proportion in responses 209–13 ognitive burden 64, 258 composite good” approach “cheap talk” 193, 215–16, 218–20 composite good version priority allocations for components 201–4 standalone COS reactions to scenario 204–5 values for program 205–6 valuing Version 2 199–200 context and rationale foci 188–9 and single-focus approach 189–91 study design 192–4 not-for votes 196–7, 200, 206–8, 214, 218, 220 and single-focus approach 189–91, 199, 205, 208–9, 211, 213–15 single-good version 1995 test-bed study comparisons 196–8 similarities and differences 194–6 study findings budget exercise and COS 213–14 cognition difficulties with part-whole relationships 214–16 sizeable proportion in responses 209–13 lack of sensitivity to huge scope difference 206–8 plausible rationalization 219–21 within-questionnaire “wording additions” 216–19 single-good v. behavior responses adjusted for certainty 31 bid design 22, 24–5 bid levels highest 17–23, 25, 28–36 low-end 22–3, 30 share of “yes” votes 50, 52 bid range 18 non-monotonicity 26 truncated 17–18, 23, 25, 27–9, 36 willingness to pay estimates 28–30, 36 and yes-response function 26–7 in WTP survey methods 189 California Environmental Improvement Program 193–4, 199–202, 205–8, 213, 215 California oil spill (COS) study see “composite good” approach California oil spill (COS) study see “composite good” approach Carson, R.T. 17, 19, 58, 60–61, 69, 78, 85–8, 91–8, 100, 112–14, 117, 123, 132, 135, 140, 155, 159, 179, 192, 196, 209, 252–3, 255, 257, 271 Carson, R.T. Daniel McFadden and Kenneth Train - 9781786434692 Downloaded from https://www.elgaronline.com/ at 10/24/2024 03:59:17AM via Open Access. Open https://creativecommons.org/licenses/by/3.0/igo/ behavior composite good WTP estimates 208–9 Comprehensive Environmental Response, Compensation, and Liability Act (CERCLA) 293–4, 296, 299, 301–3, 305 consequentiality 18, 23, 35–6, 189, 191, 219 consequentiality 18, 23, 35–6, 189, 191, 219 see also choice-based conjoint (CBC); dichotomous choice surveys consumer choice behavior 14, 176–9, 237–8 310 Contingent valuation of environmental goods incentive compatibility as issue for 171 consumer utility theory 83–4, 140, 157, 177 incentive compatibility as issue for 171 consumer utility theory 83–4, 140, 157, 177 as leading SP method 153 as leading SP method 153 consumer well-being 154, 156, 160, 168, 177–8, 180 168, 177–8, 180 contingent valuation (CV) concepts examined difficulty answering questions xiii–xv inadequate response to cost x–xi inadequate response to number of payments xii inadequate response to scope xii–xiii, xvii legal issues xvi restoration programs xvi–xvii restricted samples xv consequentiality as important issue in 35–6 definition 58 estimates of WTP for non-use environmental goods see scope tests hypothetical bias presence in 29–30, 36 prominent concern about 58 reliability adding-up test 88–90 examination of criteria 86–8 gradient 154, 179 as ignored issue 91 question of x, 58 recognizing unreliability xvii survey problems 300 use of term x use values 82–3 “warm glow” that can arise in 68 see also “composite good” approach; referendum-style CV contingent valuation method (CVM) applicability 153 assumptions of 191 comparison to CBC 154, 160 in “composite good” approach 188–9 as controversial in economics community 160 criteria of reliability of 86–7 development of 159–60 hypothetical bias observed with 220 approaches to estimating non-use value for NRD 295 as highly disfavoured by NRD regulations 301–3 for non-use damages 292–3 as not relied on in non-NRD contexts 299–301 as not relied upon in NRD cases 296–9 296 9 trustees abandoning as matter of policy 305–6 trustees avoiding 303–5 as mimicking direct democracy by referendum 170 possible reasons for lack of development 154–5 and “protest no” votes 212 recreational use value of birdwatching 179–80 as sensitive to context and survey design considerations 199 tendency to pass scope tests 112–13, 128 typical referendum elicitations 159 valuation of lost use 162, 179 value of experience of SP methods 154 as widely used to value environmental goods and services 1 COS (California oil spill) see “composite good” approach cost adding up v. scope test 66, 90 anchoring 2, 5, 10, 84 annual v. behavior composite good WTP estimates 208–9 Comprehensive Environmental Response, Compensation, and Liability Act (CERCLA) 293–4, 296, 299, 301–3, 305 onsequentiality 18, 23, 35–6, 189, 191, 219 onsumer choice behavior 14 176–9 choice based conjoint (CBC) behavioral effects 154 comparison to CVM 154, 160 elicitations directly mimicking market choice tasks 158 example of typical menu 158 as leading SP method 153 lessons for 179–80 method and applicability 160–62 study design data analysis 176–7 experimental design 166–8 failures 177–9 important issues 162–5 incentive alignment 170–71 making menus realistic 173–6 reconciliation and validation 171–3 sampling and recruitment 165–6 subject training 168–70 as variable in hypothetical bias study 278, 280–81, 283, 285 choice experiment (CE) choice based on absolute costs 5, 11, 14 based on relative costs 5, 11, 14 behavior 14 corner 10–11 in higher cost scale 8–11, 14 in lower cost scale 8–11 pattern for do-something 2, 5, 8, 10 pattern for status quo 8, 10–11, 14 proportions 4–5 tasks, performing 2 choice questions answering 6 sample 7, 229 components 1 definition 82 fat tails equivalent for 36 in hypothetical bias study 272, 278 implications for choice behavior 14 method 1–2 NOAA sponsored online study 202, 216 popular use of 1 in previous scope test studies 105–6 see also choice-based conjoint (CBC); dichotomous choice surveys study design data analysis 176–7 experimental design 166–8 failures 177–9 important issues 162–5 incentive alignment 170–71 making menus realistic 173–6 reconciliation and validation 171–3 context and rationale foci 188–9 and single-focus approach 189–91 study design 192–4 context and rationale foci 188–9 and single-focus approach 189–91 study design 192–4 sampling and recruitment 165–6 subject training 168–70 not-for votes 196–7, 200, 206–8, 214, 218, 220 not-for votes 196–7, 200, 206–8, 214, 218, 220 as variable in hypothetical bias study 278, 280–81, 283, 285 and single-focus approach 189–91, 199, 205, 208–9, 211, 213–15 and single-focus approach 189–91, 199, 205, 208–9, 211, 213–15 choice single-good version 1995 test-bed study comparisons 196–8 similarities and differences 194–6 single-good v. Daniel McFadden and Kenneth Train - 9781786434692 Downloaded from https://www.elgaronline.com/ at 10/24/2024 03:59:17AM via Open Access. Open https://creativecommons.org/licenses/by/3.0/igo/ fat tails effect on consumer goods 14 effect on estimated WTPs 108–12 effect on use and non-use amenities 5, 14 effects on “T&E” species status 11, 14, 207 , estimation results for conditional logit model by 12 in four survey versions 6, 8–11 frequency of choosing improvement option by 9 as having strong positive correlation with WTP 108 impact on utility function 11, 14 influence on WTP 2–5 and scope effects 109–12 status quo and corner choices by 10–11, 14 summary of studies 4 cumulative distribution function (CDF) 30 CV see contingent valuation (CV) CVM see contingent valuation method (CVM) as manifestation of yea-saying 30, 36 as manifestation of yea-saying 30, 36 estimation results for conditional logit model by 12 in four survey versions 6, 8–11 frequency of choosing improvement option by 9 sensitivity of WTP to response data with 23, 36 sensitivity of WTP to response data with 23, 36 as having strong positive correlation with WTP 108 truncating high-end bids in response to 36 truncating high-end bids in response to 36 follow-up questions follow up questions to assess validity of SP data earliest examples 253 in fourth section of survey 258 invalid responses 267 lack of consistency in use of 253 literature review 255–7 method and results 259–66 as used to identify problem responses 254 in hypothetical bias study 276 and identification of protestors 227, 232, 243 as inducing “bargaining” mind-set 162 in red knot study 34–6 follow up questions to assess validity of SP data earliest examples 253 in fourth section of survey 258 invalid responses 267 lack of consistency in use of 253 literature review 255–7 method and results 259–66 as used to identify problem responses 254 in hypothetical bias study 276 and identification of protestors 227, 232, 243 as inducing “bargaining” mind-set 162 in red knot study 34–6 DDT and PCBs case 296–8, 305 debriefing questions see follow-up questions Deepwater Horizon oil spill xvii, 121, 303–5 as inducing “bargaining” mind-set 162 in red knot study 34–6 yea-saying as result of 124–5 Desvousges, W., Mathews, K. and Train, K. (DMT) 17, 19, 22, 58, 69, 87, 90–92, 98–104, 112–13, 117, 126, 140 GfK Custom Research 25, 194 Groves, T. 60–61, 69, 78, 171, 252 GfK Custom Research 25, 194 Groves, T. behavior one-time payments xii, 43–56 application to endangered shorebird species xi, 17–36 in CBC studies 167, 177 in choice experiments 1–2 household 24–5, 30, 32, 212, 216–17 in logit model of yes/no vote 76–7 in SP valuation of environmental goods x–xi, 1–14 trustees abandoning as matter of policy 305–6 COS (California oil spill) see “composite good” approach cost adding up v. scope test 66, 90 anchoring 2, 5, 10, 84 annual v. one-time payments xii, 43–56 application to endangered shorebird species xi, 17–36 in CBC studies 167, 177 in choice experiments 1–2 household 24–5, 30, 32, 212, 216 1 application to endangered shorebird species xi, 17–36 in CBC studies 167, 177 in choice experiments 1–2 household 24–5, 30, 32, 212, in logit model of yes/no vote 76–7 in SP valuation of environmental goods x–xi, 1–14 311 Index cost-benefit trade offs see trade-offs cost prompts see bids cost scales effect of quadrupling costs on mean WTP estimates 8–11, 13–14, 108, 207 effect on consumer goods 14 effect on estimated WTPs 108–12 effect on use and non-use amenities 5, 14 effects on “T&E” species status 11, 14, 207 estimation results for conditional logit model by 12 in four survey versions 6, 8–11 frequency of choosing improvement option by 9 as having strong positive correlation with WTP 108 impact on utility function 11, 14 influence on WTP 2–5 and scope effects 109–12 status quo and corner choices by 10–11, 14 summary of studies 4 cumulative distribution function (CDF) 30 CV see contingent valuation (CV) CVM see contingent valuation method (CVM) cost-benefit trade offs see trade-offs cost prompts see bids cost scales Endangered Species Act (ESA) 5–7, 13, 228–9 Endangered Species Act (ESA) 5–7, 13, 228–9 environmental litigation see legal issues Exxon Valdez oil spill 86, 305 effect of quadrupling costs on mean WTP estimates 8–11, 13–14, 108, 207 familiarity 162 3, 168 9, 279 fat tails and consequentiality 35–6 extent in response data 18 in follow-up question responses 35 as manifestation of hypothetical bias 29–30, 36 as manifestation of yea-saying 30, 36 for non-parametric estimators 17, 27 for parametric estimators 17 paths for future research 36 phenomenon xi, 17 i i i f WTP d y , , fat tails and consequentiality 35–6 extent in response data 18 in follow-up question responses 35 as manifestation of hypothetical bias 29–30, 36 as manifestation of yea-saying 30, 36 for non-parametric estimators 17, 27 for parametric estimators 17 paths for future research 36 phenomenon xi, 17 i i i f WTP d fat tails endangered shorebird see red knot studies Daniel McFadden and Kenneth Train - 9781786434692 Downloaded from https://www.elgaronline.com/ at 10/24/2024 03:59:17AM via Open Access. Open https://creativecommons.org/licenses/by/3.0/igo/ fat tails 60–61, 69, 78, 171, 252 dichotomous choice surveys 2, 21–3, 82, 124, 126, 137, 192, 218, 271 disbelief of cost, elimination 54–5 DMT see Desvousges, W., Mathews, K. and Train, K. (DMT) dichotomous choice surveys 2, 21–3, 82, 124, 126, 137, 192, 218, 271 Hanemann, W.M. 58, 65, 101, 112, 118, 123, 126, 133, 136, 162 Hanemann, W.M. 58, 65, 101, 112, 118, 123, 126, 133, 136, 162 disbelief of cost, elimination 54–5 DMT see Desvousges, W., Mathews, K. and Train, K. (DMT) Hicksian welfare measures 252 Hicksian welfare measures 252 hypothetical bias adjusting for 30–32 effect of “wording additions” 189 fat tails as manifestation of 29–30, hypothetical bias adjusting for 30–32 effect of “wording additions” 189 fat tails as manifestation of 29–30, economic valuation approach 294–5 emotional intensity scales 85 economic valuation approach 294–5 emotional intensity scales 85 endangered shorebird see red knot studies economic valuation approach 294–5 emotional intensity scales 85 y endangered shorebird see red knot studies 36 312 Contingent valuation of environmental goods for non-use damages 292–3 as not relied upon in non-NRD contexts 299–301 as not relied upon in NRD cases 296–9 trustees abandoning as matter of policy 305–6 trustees avoiding 303–5 court cases American Trader spill litigation 299 Idaho v. Southern Refrigerated Transport Inc. 298 People v. Attransco, Inc., 299 Price v. Philip Morris, Inc 299–300 United States v. Montrose 296–8 NRD regulatory framework and basis for non-use damages 293–4 logit model binary 266 estimates for low-cost and high- cost data samples 11–13 mixed form v. model with heuristics 235–7, 244–9 multinomial 176–7, 226 of trade-off respondents 238, 240–45 traditional 225–6, 231, 242–6, 248 of yes/no vote 76 parametric estimator 17 of random utility model 1–2 regression 264–6 Loureiro, M.L. 98, 105–9, 112 Louviere, J. 6, 157–8, 164, 172, 252–3, 257 meta-analysis current v. prior studies on 270 data bias ratio 271–4 certainty correction 273–6 cheap talk 277 conjoint/choice experiment 278 familiar good 279 lab experiment 278 non-use 279 private good/public good 278–9 same respondents v. different respondents 277 student 278 regression analysis base model 279–81 fixed effects regression 284–6 functional form 281–2 time trend 282–4 study conclusions 286 method of detecting 29 observed with CVM surveys 220 meta-analysis as not relied upon in NRD cases 296–9 trustees abandoning as matter of policy 305–6 trustees avoiding 303–5 court cases American Trader spill litigation 299 Idaho v. Southern Refrigerated Transport Inc. 298 People v. Daniel McFadden and Kenneth Train - 9781786434692 Downloaded from https://www.elgaronline.com/ at 10/24/2024 03:59:17AM via Open Access. Open https://creativecommons.org/licenses/by/3.0/igo/ fat tails Attransco, Inc., 299 Price v. Philip Morris, Inc 299–300 United States v. Montrose 296–8 model Idaho v. Southern Refrigerated Transport Inc. 298 “inadequate”, use of term 88 incentive alignment 170–71, 175–6, 179 incentive compatibility 163–4, 170–71 income effects 64–5, 75–7 Kahneman, D. 44–5, 86–7, 92–4, 101–2, 119, 154, 157, 160, 178, 191 Knetsch, J.L. 44–5, 86–7, 93, 102, 119, 191 K l d N t k 6 7 25 6 194 , , , , , , Knetsch, J.L. 44–5, 86–7, 93, 102, 119, 191 Knowledge Networks 6–7, 25–6, 194, 228–9, 299 Knowledge Networks 6–7, 25–6, 194, 228–9, 299 lab experiment variable 273–4, 278–85 latent class models 226–7, 230–34 legal issues approaches to estimating non-use value for NRD contingent valuation methods 295 restoration-based approach 294–5 contingent valuation methods approach to estimating non-use value for NRD 295 as highly disfavoured by NRD regulations 301–3 mandatory payment mechanism 191 survey mode 138 marginal utility diminishing 60, 62, 83–5, 88–9, 112, 129–31 of income 231 levels for species improvement 231 313 Index marine species preservation case inferences from stated preference surveys 225–49 response to cost prompts xi, 5–14 Mathews, K. see Desvousges, W., Mathews, K. and Train, K. (DMT) menus see choice-based conjoint (CBC) migratory bird studies see red knot studies minced pork study 3–4, 14 Mitchell, R.C. 85–6, 94, 117, 155, 252 marine species preservation case inferences from stated preference surveys 225–49 response to cost prompts xi, 5–14 Mathews, K. see Desvousges, W., Mathews, K. and Train, K. (DMT) menus see choice-based conjoint (CBC) migratory bird studies see red knot studies minced pork study 3–4, 14 Mitchell, R.C. Daniel McFadden and Kenneth Train - 9781786434692 Downloaded from https://www.elgaronline.com/ at 10/24/2024 03:59:17AM via Open Access. Open https://creativecommons.org/licenses/by/3.0/igo/ NRD see natural resource damage (NRD) y Price v. Philip Morris, Inc 299–300 private good/public good variable 273–4, 278–81, 283–5 probit estimates 54 model of yes/no vote 53 multivariate regressions 138–40 parametric estimator 17, 43 protestors p , private good/public good variable 273–4, 278–81, 283–5 Oil Pollution Act (OPA) 192, 293–4, 296, 299, 301–4 Ojea, E. 98, 105–9, 112 one-time tax 24, 199, 216–17 ordering problem xiv, 164 Oil Pollution Act (OPA) 192, 293–4, 296, 299, 301–4 probit Ojea, E. 98, 105–9, 112 one-time tax 24, 199, 216–17 ordering problem xiv, 164 estimates 54 model of yes/no vote 53 multivariate regressions 138–40 parametric estimators 17, 22–3, 43–4, 46, 53, 55, 126–7 fat tails 85–6, 94, 117, 155, 252 recommendation of “Yes/No” follow-up questions 253 reliability of CV 86–8, 91, 140 scope studies 91 non-parametric estimators in red knot study estimates by bid amount 27 adjusted for believed bid 35 adjusted for hypothetical bias 32 estimates of WTP 28–9 WTP and truncation of bids 23, 36 relation to fat tails 17 in river and lake quality study 73–5 non-use amenities 3, 5, 14, 83, 107, 141 see also scope tests: and CV estimates of WTP for non-use environmental goods non-use damages in court case 298 NRD regulatory framework and basis for 293–4 role of trustees 302 survey valuation methods legal obstacles to use 305 non-use by trustees 303–4 non-use, in hypothetical bias study considerations 270, 273, 275, 279–80 variable 279–81, 283–5 non-use values approaches to estimating for NRD CV methods 82–3, 253, 292, 295, 302, 305 restoration-based approach 294–5, 303–4 as at bad end of reliability gradient 154 and brain activity 179–80 in California 192–3 compensation for 294 in court cases 296, 303–4 CV and CE as designed to infer WTP for 108 CVM eliciting from consumers 154–5 effects of training and context 170 greatest need for SP data in application to environmental 180 minced pork study 3–4, 14 natural resource damage (NRD) ability of survey methods to measure non-use values in 292 approaches to estimating non-use value for contingent valuation methods 295 restoration-based approach 294–5 assessment (NRDA) 253, 305 better approach for valuation of 306 contingent valuation methods as disfavoured by regulations of 301–3 court decisions associated with 296–9 Deepwater Horizon oil spill xvii, 303 5 NRD regulatory framework and basis for 293–4 role of trustees 302 survey valuation methods legal obstacles to use 305 non-use by trustees 303–4 survey valuation methods legal obstacles to use 305 non-use by trustees 303–4 court decisions associated with 296–9 Deepwater Horizon oil spill xvii, 303–5 non-use, in hypothetical bias study considerations 270, 273, 275, 279–80 variable 279–81, 283–5 non-use, in hypothetical bias study considerations 270, 273, 275, 279–80 variable 279–81, 283–5 regulatory framework and basis for non-use damages 293–4 “no purchase” option 163, 176 NOAA (National Oceanic and Atmospheric Administration) adding up test addressing concerns of 59, 63, 78 clarification of term “inadequate” 88 commissioning study of NRD 296 concern over scope 129, 140 conclusion on respondents’ WTP 114 and consequentiality 189 marine species survey application of 225–7 data 5–11, 228–9, 243 mixed logit analysis of 248 model 230–34, 237 purpose of 58, 86 recommendation of referendum regulatory framework and basis for non-use damages 293–4 “no purchase” option 163, 176 NOAA (National Oceanic and Atmospheric Administration) adding up test addressing concerns of 59, 63, 78 non-use values approaches to estimating for NRD CV methods 82–3, 253, 292, 295, 302, 305 approaches to estimating for NRD CV methods 82–3, 253, 292, 295, 302, 305 restoration-based approach 294–5, 303–4 as at bad end of reliability gradient 154 and brain activity 179–80 in California 192–3 compensation for 294 in court cases 296, 303–4 CV and CE as designed to infer WTP for 108 CVM eliciting from consumers 154–5 effects of training and context 170 greatest need for SP data in purpose of 58, 86 application to environmental 180 recommendation of referendum format question 195 314 Contingent valuation of environmental goods People v. fat tails Attransco, Inc., 299 power outages study 3–4 present value willingness to pay (PV WTP) analysis 50–55 conclusion 55 literature review 44–9 survey 49–50 People v. Attransco, Inc., 299 power outages study 3–4 present value willingness to pay (PV WTP) analysis 50–55 conclusion 55 literature review 44–9 survey 49–50 h l 299 300 lack of data to support WTP estimates 252 lack of success stories 180 relation to market benchmarks 162 SP discrete choice experiment for estimating 1 SP methods applicable to 180 NRD see natural resource damage (NRD) lack of data to support WTP estimates 252 lack of success stories 180 relation to market benchmarks 162 SP discrete choice experiment for estimating 1 SP methods applicable to 180 NRD see natural resource damage (NRD) NRD see natural resource damage (NRD) parametric estimators 17, 22–3, 43–4, 46, 53, 55, 126–7 protestors common practice of excluding 7, 9, 124, 225, 240 definition 7, 9 and sensitivity of lower-bound mean WTP 211–12 in stated preference assessing validity study 253, 255–6, 260 in stated preference inferences study and estimated population share 239–40 model with three heuristics to identify 225 in sensitivity analysis 242–5 status quo only respondents 232 study conclusion on 249 use of latent class models to payments, frequency of, inadequate response to analysis 50–55 annual payments comparison of split samples 52 implicit discount rates 44–9 lower-bound estimates 53 probit estimates 54 referendum question 50–51 relation to WTP 55–6 sensitivity results 55 share of “yes” vote by bid amount 52 in split-sample survey 43–4, 50 version B of survey instrument 50 literature review 44–9 one-time payments comparison of split samples 52 implicit discount rates 44–9 lower-bound estimates 53 probit estimates 54 referendum question 50–51 relation to WTP 55–6 sensitivity results 55 share of “yes” vote by bid amount 52 in split-sample survey 43–4, 50 version A of survey instrument 50 purpose and method 43–4 relation to WTP 55–6 study conclusions 55–6 survey 49–50 annual payments comparison of split samples 52 implicit discount rates 44–9 lower-bound estimates 53 probit estimates 54 referendum question 50–51 relation to WTP 55–6 sensitivity results 55 share of “yes” vote by bid amount 52 in split sample survey 43 4 50 annual payments comparison of split samples 52 implicit discount rates 44–9 lower-bound estimates 53 probit estimates 54 referendum question 50–51 relation to WTP 55–6 sensitivity results 55 share of “yes” vote by bid amount 52 in split-sample survey 43–4, 50 version B of survey instrument 50 literature review 44–9 one-time payments comparison of split samples 52 implicit discount rates 44–9 lower-bound estimates 53 probit estimates 54 referendum question 50–51 relation to WTP 55–6 sensitivity results 55 share of “yes” vote by bid amount 52 in split-sample survey 43–4, 50 version A of survey instrument 50 purpose and method 43–4 relation to WTP 55–6 study conclusions 55 6 version B of survey instrument 50 literature review 44–9 PV WTP see present value willingness to pay (PV WTP) Daniel McFadden and Kenneth Train - 9781786434692 Downloaded from https://www.elgaronline.com/ at 10/24/2024 03:59:17AM via Open Access. Open https://creativecommons.org/licenses/by/3.0/igo/ Daniel McFadden and Kenneth Train - 9781786434692 Daniel McFadden and Kenneth Train - 9781786434692 Downloaded from https://www.elgaronline.com/ at 10/24/2024 03:59:17AM via Open Access. Open https://creativecommons.org/licenses/by/3.0/igo/ Daniel McFadden and Kenneth Train - 9781786434692 Downloaded from https://www.elgaronline.com/ at 10/24/2024 03:59:17AM via Open Access. Open https://creativecommons.org/licenses/by/3.0/igo/ Qualtrics sample 25–6 random utility model (RUM) 1–2, 8 rational choice model (RCM) 252 red knot studies fat tails and truncated bids purpose and method 17–18 related literature 19–23 results adjusting for hypothetical bias 30–32 belief in bid values 32–4 follow up questions 34–5 random utility model (RUM) 1–2, 8 rational choice model (RCM) 252 red knot studies random utility model (RUM) 1–2, 8 rational choice model (RCM) 252 red knot studies fat tails and truncated bids purpose and method 17–18 related literature 19–23 results results adjusting for hypothetical bias 30–32 belief in bid values 32–4 follow up questions 34–5 315 Index willingness to pay estimates 28–30 yes-response function 26–7 study discussion 35–6 survey 24–6 response to frequency of payments analysis 50–55 literature review 44–9 purpose and method 43–4 study conclusions 55–6 survey 49–50 willingness to pay estimates 28–30 yes-response function 26–7 study discussion 35–6 survey 24–6 response to frequency of payments analysis 50–55 literature review 44–9 purpose and method 43–4 study conclusions 55–6 survey 49–50 regressions examining factors affecting 139–40 rejection of hypothesis 91, 97 “warm glow” as explanation for 86 for wolves 83 scope tests and adding-up test study adding-up test extending 62 cost 66 examining estimated WTP increases 58–9, 63 issue of adequate response 59, 66, 78 non-negative scope effects 63 restoration program discussion 78 results 73–4 of river and lake 69–70 response to frequency of payments analysis 50–55 literature review 44–9 purpose and method 43–4 study conclusions 55–6 survey 49–50 referendum-style CV in hypothetical bias study 271 red knot studies 18–21, 24–5, 43, 50–51, 55–6 restoration program discussion 78 results 73–4 of river and lake 69–70 restoration program discussion 78 results 73–4 of river and lake 69–70 in river and lake quality study 69, 76 in river and lake quality study 69, 76 in single-focus and “composite good” approaches 190, 192–7, 199–206, 208, 210–13, 218, 220 i d f h d d in single-focus and “composite good” approaches 190, 192–7, 199–206, 208, 210–13, 218, 220 and CV estimates of WTP for non- use environmental goods diminishing marginal utility 83–5, 88–9, 112, 129–31 in stated preference methods study 159–60, 163, 170–71 responsible parties (RPs) 303–4 restoration-based approach 294–5, 301–3, 305 inconsistent statistical significance results 127–9 pass and fail results affected by measureable survey characteristics 137–40 restoration programs and contingent valuation xvi–xvii oyster reef 44, 46 pros and cons of voting for 258 for river and lake 60–61, 69–75, 78 wetland 254–67 pass and fail results affected by measureable survey characteristics 137–40 reliability of CVM 86–90 scope effects 84, 88, 91, 98, 108, 112–13, 123, 128 reliability of CVM 86–90 scope effects 84, 88, 91, 98, 108, 112–13, 123, 128 revealed preference (RP) 82–3, 153–4, 156, 172, 178–9, 219 scope elasticities 112, 129–37, 141 river and lake quality study see adding- up test river and lake quality study see adding- up test scope insensitivity 83–6, 91, 97, 126, 139–40 scope literature Carson’s review 91–7, 112–14 DMT’s review 91, 98–104 effect of cost scale on estimated WTPs 108–12 flaws in 107–8, 140–41 frequency of CV studies passing scope test 128–9 Ojea and Loureiro 98, 105–9 review of scope results reported in 113–24 scope sensitivity in 83, 87, 96–8, 107–8, 112, 123, 126 f t t f 112 13 river health improvements study 3–5 scope literature Carson’s review 91–7, 112–14 DMT’s review 91, 98–104 effect of cost scale on estimate sample allocation 3 scope effects 63, 84, 88, 91, 98, 108, 112–13, 123, 128 scope elasticities 112, 129–37, 141 scope insensitivity categorical 84 demand for environmental amenity 83 evidence for 126 factors leading to 85–6 as often attributed to diminishing marginal utility and satiation 84–5 quantitative 84 sample allocation 3 scope effects 63, 84, 88, 91, 98, 108, p 112–13, 123, 128 WTPs 108–12 flaws in 107–8, 140–41 scope elasticities 112, 129–37, 141 frequency of CV studies passing scope test 128–9 scope insensitivity categorical 84 Ojea and Loureiro 98, 105–9 review of scope results reported in 113–24 evidence for 126 factors leading to 85–6 scope sensitivity in 83, 87, 96–8, 107–8, 112, 123, 126 as often attributed to diminishing marginal utility and satiation summary of state of 112–13 on use amenities 107 summary of state of 112–13 on use amenities 107 quantitative 84 316 Contingent valuation of environmental goods scope sensitivity demonstration of adequate 129–37 in scope literature 83, 87, 96–8, 107–8, 112, 123, 126 study conclusions 140–41 use of external 84, 91–104, 112–15, 123–4, 129 use values 82–3 variations in analytical models and statistical procedures 126–7 in data included in analysis 124–5 in survey design 125–6 sensitivity analysis 209–13, 242–8 single-focus approach 189–91, 199, 205, 208–9, 211, 213–15 SP see stated preference (SP) stated preference (SP) evaluation of environmental goods assessing validity of data using follow-up questions consistency of questions 253 criteria for 252–3, 266–7 literature review 255–7 methods and results cumulative assessment of validity of responses 261–4 descriptive statistics 260–61 regression analysis 264–6 three basic components 259–60 “problematic responses” 253–4 study design and data 257–9 study discussion and conclusions 266–7 survey subject 254 inferences from surveys when costs and benefits are not compared application of methodology to data 225 estimation 235–8 heuristic decision rules attentive to environmental costs only 233–4 attentive to environmental improvements only 232–3 environmental improvements ignoring costs and benefits 234 randomizers ignoring costs and benefits 234 status quo only 232 model alternative to benefit-cost trade- offs 231–4 trade-off respondents 230–31 related literature 226–7 results estimated population shares of choice rules 238–40 estimated WTP for species status improvement 240–42 sensitivity analysis 242–8 study conclusions 248–9 survey data 228–9 WTP estimate features determining 224 ideal behavior for validity 224–5 for species status improvement 240–42 th d d li bilit methods and applicability choice-based conjoint study design 160–79 history of 155–61 importance of 153–4 lessons for 179–80 possible reasons for lack of development 154–5 response to cost prompts choice experiment method 1–2 cost scale studies 2–5 study conclusion 11–14 survey data 5–11 methods and applicability choice-based conjoint study design 160–79 history of 155–61 importance of 153–4 lessons for 179–80 possible reasons for lack of d l 154 5 response to cost prompts choice experiment method 1–2 cost scale studies 2–5 study conclusion 11–14 survey data 5–11 status quo in stated preference validity study 258, 264, 266 steelhead fish population damage case 298 student variable 273–4, 278–81, 283–5 student variable 273–4, 278–81, subject training 168–70 “subjective well-being” methods 160 317 Index implications 25 intentional 23 as tempting response to fat tails 36 truthfulness 60–61, 69, 78, 161, 163–4, 169, 171, 176, 195 Turnbull, B.W. Qualtrics sample 25–6 one-time payments 46–7 cost scale study 3–4 measuring yes-response rates 19–21, 23 scope tests Carson 91–8 DMT 98–104 summary 117–24 use of debriefing questions 256 wetland restoration project study see stated preference (SP) assessing validity study willingness to accept (WTA) 63, 67, 190 willingness to pay (WTP) in adding-up test addressing adequate response 59–60 description 62–3 findings 61, 78 environmental improvements ignoring costs and benefits 234 randomizers ignoring costs and benefits 234 randomizers ignoring costs and benefits 234 status quo only 232 estimation 235–8 need for heuristics 225 and respondent behavior 224–5 respondents 230–31 results 238–49 status quo only 232 estimation 235–8 need for heuristics 225 and respondent behavior 224–5 respondents 230–31 results 238–49 use of debriefing questions 256 wetland restoration project study see stated preference (SP) assessing validity study between status quo and do- something options 8–11, 14 willingness to accept (WTA) 63, 67, 190 Qualtrics sample 25–6 17, 43, 50, 53, 109, 196, 219 implications 25 intentional 23 as tempting response to fat tails 36 truthfulness 60–61, 69, 78, 161, 163–4, 169, 171, 176, 195 threatened and endangered (“T&E”) species marine choice experiment effects of cost scales on status 11, 14, 207 NOAA survey data 5–11 study conclusions 11–14 stated preference survey estimation 235–8 model 230–34 threatened and endangered (“T&E”) species marine choice experiment effects of cost scales on status 11, 14, 207 NOAA survey data 5–11 study conclusions 11–14 stated preference survey estimation 235–8 model 230–34 NOAA survey data 228–9 related literature 226–7 results 238–48 study conclusions 248–9 scope tests 105, 130 total value equivalency method (TVE) Turnbull, B.W. 17, 43, 50, 53, 109, 196, 219 United States v. Montrose 296–8, 305 use amenities 3, 5, 14, 83, 107 use values applications of SP methods to environmental 179 and brain activity 179 CBC elicitations of 179–80 and contingent valuation 82–3 passive 1, 11, 86, 94, 121 from SP studies 154 utility function 1–2, 8, 11, 107, 155–6, 158, 233–4, 237 United States v. Montrose 296–8, 305 use amenities 3, 5, 14, 83, 107 use values NOAA survey data 228–9 related literature 226–7 results 238–48 applications of SP methods to environmental 179 and brain activity 179 CBC elicitations of 179–80 and contingent valuation 82–3 passive 1, 11, 86, 94, 121 from SP studies 154 tilit f ti 1 2 8 11 107 155 6 study conclusions 248–9 scope tests 105, 130 study conclusions 248–9 scope tests 105, 130 total value equivalency method (TVE) 295, 302 trade-offs utility function 1–2, 8, 11, 107, 155–6, 158, 233–4, 237 trade-offs in CBC studies 175, 178 criterion in assessment of SP 255 and familiarity 163 in red knot study 35–6 valuation of lost use 162, 179 “vignette analysis” 160 voluntary contributions 124, 191 voting question example 25, 51 valuation of lost use 162, 179 “vignette analysis” 160 valuation of lost use 162, 179 “vignette analysis” 160 voluntary contributions 124, 191 voting question example 25, 51 in stated preference inferences study alternatives to benefit-cost trade- offs “warm glow” concept 60, 68, 83–4, 86–7, 89–90, 115, 125, 131, 208, 216, 220 “warm glow” concept 60, 68, 83–4, 86–7, 89–90, 115, 125, 131, 208, 216, 220 attentive to environmental costs only 233–4 water quality studies annual v. Daniel McFadden and Kenneth Train - 9781786434692 Downloaded from https://www.elgaronline.com/ at 10/24/2024 03:59:17AM via Open Access. Open https://creativecommons.org/licenses/by/3.0/igo/ Train, K. see Desvousges, W., Mathews, K. and Train, K. (DMT) willingness to pay (WTP) truncated bids as common practice 18 effect on WTP 27–9 explanation 17 description 62–3 318 Contingent valuation of environmental goods income effects 64–5, 75–7 in original study 69 in previous studies 59–60 provision mechanism 65 questionnaire versions 71 relation to adding-up condition 59–60 in restoration program 60–61, 69, 71, 74–5 results 74–5 review of past studies 66–8 and willingness to accept (WTA) discrepancy 63 in CBC studies 165 in “composite good” approach in aggregate for large basket of alternative public goods 190 effect if valuation question delayed 199 effect of propensity to shift votes 204 embodying budget constraint 189 estimates effect of “wording additions” 216–19 sensitivity to cognitive difficulties 209–13 single-focus v. composite good marine species preservation case impact of fourfold increase in costs 8–11, 13–14, 108, 207 influence of cost scales on 2–5, 14 modeling consumer utility in “money metric” form 177 for non-use amenities 3, 5, 14 in red knot studies adjusting for hypothetical bias 31–2 belief in bid values 34 effect of bid truncation on 27–8 estimates 18, 28–30 fat tails of yes-response function 24–5, 30 i f i bid marine species preservation case impact of fourfold increase in costs 8–11, 13–14, 108, 207 influence of cost scales on 2–5, 14 modeling consumer utility in “money metric” form 177 for non-use amenities 3, 5, 14 in red knot studies adjusting for hypothetical bias 31–2 belief in bid values 34 effect of bid truncation on 27–8 estimates 18, 28–30 fat tails of yes-response function 24–5, 30 adjusting for hypothetical bias 31–2 belief in bid values 34 effect of bid truncation on 27–8 estimates 18, 28–30 fat tails of yes-response function 24–5, 30 fat tails of yes-response function 24–5, 30 importance of maximum bid selection 28–9 lower-bound mean 27–9, 31–2, 35–6, 50–51, 53, 55 present value (PV WTP) 44–55 problem of negative estimates 17 related literature 22–3 sensitivity to frequency of payments 43–4, 49, 55–6 of mean, to largest bid 36 results 55 size of mean bids 29 cope tests present value (PV WTP) 44–55 problem of negative estimates 17 related literature 22–3 iti it embodying budget constraint 189 estimates embodying budget constraint 189 estimates sensitivity to frequency of payments 43–4, 49, 55–6 effect of “wording additions” 216–19 effect of “wording additions” 216–19 sensitivity to cognitive difficulties 209–13 sensitivity to cognitive difficulties 209–13 single-focus v. composite good 208–9 single-focus v. Daniel McFadden and Kenneth Train - 9781786434692 Downloaded from https://www.elgaronline.com/ at 10/24/2024 03:59:17AM via Open Access. Open https://creativecommons.org/licenses/by/3.0/igo/ Daniel McFadden and Kenneth Train - 9781786434692 Downloaded from https://www.elgaronline.com/ at 10/24/2024 03:59:17AM via Open Access. Open https://creativecommons.org/licenses/by/3.0/igo/ Train, K. see Desvousges, W., Mathews, K. and Train, K. (DMT) composite good 208–9 scope tests exploration of, using split-sample design 188 and CV estimates of WTP for non-use environmental goods 82–141 impact of budget exercise 214 lower-bound mean 196–7, 200, 82–141 effect of cost scale on estimated WTPs 108–12 and increase in environmental benefits 58–9 and prominent concern of CV 58 in stated preference inferences study estimates features determining 224 fragility of 242–8 ideal behavior for validity 224–5 for maximization of utility 249 for species status improvement 240–42, 248 estimation 235–8 for landscape improvements 227 and trade-off respondents 231 for use amenities 3, 5, 14 203–4, 206–7, 210–11, 213, first environmental good 215–16 estimation 235–8 for landscape improvements 227 and trade-off respondents 231 for use amenities 3, 5, 14 319 Index in binary choice models 23 expectations in valid surveys 30 at higher bid prices effect of high 29 explanation for high 35 to highest bid in referendum-style CV studies 18–21 in non-parametric estimates by bid amount 27 to question about tax amount 33 relation to fat tails 17 share by bid amount 50, 52 in study of migratory birds 22 in WTP estimates 28 wine scent wheel 174 “wording additions” 216–19 wine scent wheel 174 “wording additions” 216–19 yea-saying 30, 36, 253, 255–6 yea-saying 30, 36, 253, 255–6 yea-saying 30, 36, 253, 255–6 y y g , , , yes-response function adjusting for hypothetical bias 30–31 fat tails of 17–18, 34–5 many studies with truncated 18–21, 35 pinning down tails of 24–5, 35 results in red knot study 26–7 WTP captured in high-end tail of 36 yes-response rates bid design for 25 y y g , , , yes-response function adjusting for hypothetical bias 30–31 fat tails of 17–18, 34–5 many studies with truncated 18–21, 35 pinning down tails of 24–5, 35 results in red knot study 26–7 WTP captured in high-end tail of 36 yes-response rates bid design for 25
https://openalex.org/W2530937961	https://lettersonmaterials.com/Upload/Journals/825/313-3181.pdf	English	null	Superplasticity in a fine-grained tin alloy processed by multi-directional forging	Pisʹma o materialah	2,015	cc-by	4,401	Superplasticity in a fine-grained tin alloy processed by multi-directional forging F. Akbaripanah1, Y. Moradi1, R.Mahmudi2† 1Department of Mechanical Engineering, Faculty of Engineering, Malayer University, Malayer, Iran 2School of Metallurgical and Materials Engineering, College of Engineering, University of Tehran, Tehran, Iran Superplastic deformation of engineering materials is often represented by high elongation values obtained in conventional tensile tests carried out in specific ranges of temperatures and strain rates. This behavior is characterized by high strain rate sen sitivity (SRS) indices obtained by a variety of techniques. The SRS of a fine-grained Sn–1 wt% Bi alloy, processed by multi-direc tional forging (MDF) was studied by indentation testing at room temperature (T > 0.6Tm). The microstructural homogeneity increased with increasing the number of MDF passes, and the grain size decreased from 3.2 to 2 μm, as the number of passes increased from 1 to 8. The SRS indices of 0.08, 0.24, 0.31, and 0.49 were obtained for the 2, 4, 6 and 8 passes of MDF, respectively. The high SRS index of 0.49, calculated from different analysis methods of the indentation tests are in good agreement with each other and with those of the other testing methods and severe plastic deformation processes on the same alloy reported in the literature. These SRS indices together with the uniform fine-grained equiaxed microstructure with an average grain size of 2 µm, observed after 8 MDF passes, are indicative of a superplastic deformation behavior dominated by grain boundary sliding. Keywords: multi-directional forging, strain rate sensitivity, superplasticity Сверхпластичность мелкозернистого сплава олова, подвергнутого всесторонней ковке Сверхпластическая деформация конструкционных материалов часто характеризуется высокими значениями удли нения, получаемыми в обычных испытаниях на растяжение, проводимых в определенных интервалах температуры и скорости деформации. Это поведение характеризуется высокими значениями показателя чувствительности к ско рости деформации (ЧСД), которые определяются различными методами. Путем испытаний на твердость при ком натной температуре (T > 0.6Tm) была исследована ЧСВ мелкозернистого сплава Sn–1 вес. % Bi, подвергнутого всесто ронней ковке (ВК). Однородность микроструктуры возрастает с увеличением числа переходов ВК, а размер зерен уменьшается с 3.2 до 2 мкм, когда число переходов увеличивается от 1 до 8. Получены показатели ЧСД 0.08, 0.24, 0.31 и 0.49 для 2, 4, 6 и 8 переходов ВК, соответственно. Высокие показатели ЧСД около 0.49, рассчитанные с примене нием различных методов анализа испытаний на микротвердость, находятся в хорошем согласии друг с другом и со значениями, полученными другими методами испытаний и для других процессов интенсивной пластической дефор мации, имеющимися в литературе. Эти показатели ЧСД, вкупе с однородной мелкозернистой равноосной микро структурой со средним размером зерен 2 мкм, наблюдаемой после 8 переходов ВК, свидетельствует о сверхпласти ческом характере деформации, доминирующим механизмом которой является зернограничное проскальзывание. Ключевые слова: всесторонняя ковка, параметр скоростной чувствительности, сверхпластичность Letters on materials 5 (3), 2015 pp. 313-318 Letters on materials 5 (3), 2015 pp. 313-318 www.lettersonmaterials.com PACS 62.20.fq, 81.40.Ef, 81.40.Lm Keywords: multi-directional forging, strain rate sensitivity, superplasticity 2.2. Indentation creep tests Most of the studies on the superplastic deformation of severely deformed materials have employed the conven tional tensile methods. Localized tests such as indentation creep test, however, can be very useful in this respect. This is because they can be particularly advantageous when the ma terial is only available as small test-pieces or there are some difficulties with the machining of samples made of very soft materials [6]. The indentation creep process can be defined as the time dependent penetration of a hard indenter into the material under constant load and temperature [7]. The variation in penetration depth, expressed as a change in di agonal length, is monitored with dwell time. Accordingly, the stress exponents and strain rate sensitivity (SRS) indi ces of materials can be determined by these simple hard ness tests. The superplasticity of the rolled Sn–Pb eutectic [8], Sn–40%Pb–2.5%Sb peritectic [9,10], and Sn–1%Bi alloy [11] has been investigated by indentation creep test. In all of these studies, it has shown that the parameters which are commonly considered as characteristics of superplasticity can be obtained by this simple test. One of these parameters is the SRS index (m), which can be used to assess the super plastic behavior. The samples were polished and then tested in a Vickers hard ness tester where the testing time was the only variable. The Vickers indenter was mounted in a holder that was positioned in the center of the vertical loading bar. The specimen was placed in an anvil below the loading bar. The Vickers hardness number is given by Hv =0.1854 F/d2, where F is the applied load in N and d is the average diagonal length in mm. Inden tation creep measurements were made on each sample using 10 N load for dwell times up to 120 min. After each loading schedule, the sample was unloaded and the indentation diag onal was measured under a traveling microscope. Each read ing was an average of at least three separate measurements taken at random places on the surface of the samples. It is the aim of this study to investigate the superplastic behavior of the fine-grained Sn–l%Bi alloy processed by MDF at room temperature (T >0.6Tm). This was achieved through indentation creep test by measuring the stress exponents and SRS indices, using different methods of analysis. Fig. 1. Schematic illustration of multi-directional forging. 1. Introduction for superplastic deformation of aluminum [1], magnesium [2], and titanium alloys [3]. In principle, MDF is a repeating compression process in which the axis of the applied strain changes in each step [4]. Considerable plastic strain is accumulated in the material as it is repeatedly deformed at ambient to elevated temperatures. It has been shown that the evolution of equiaxed fine grains relies mainly on the strain accumulation applied from various directions [5]. Severe plastic deformation (SPD) processes have received great attention for their capability in producing very fine grain sizes. Different processes have been developed and their potentials and limitations have been documented. Among these, multi-directional forging (MDF) has proved to be a possible means for attaining fine grain structures suitable 313 Akbaripanah et al. / Letters on materials 5 (3), 2015 pp. 313-318 3. Results and discussion length increases with the loading time and the number of MDF passes. In other words, the level of indentation curves and their slopes in the steady-state region are higher for the 8-pass condition with a grain size of 2 μm, while for the 2-pass material with a grain size of 3.5 μm there is not much increase in indentation diagonal length. Hence, indentation creep occurs more readily in the material with a finer grain size. The Optical micrographs of the material before the MDF process and after 8 passes of MDF are shown in Figs. 2 and 3, respectively. As can be observed in Fig. 2a, the unMDFed�� microstructure consist of some large grains to gether with many smaller grains which are believed to be formed as result of high cooling rates imparted by the cold mold and the possible static recrystallization at room tem perature. The corresponding grain size distribution data, collected from a number of samples, are shown in Fig. 2b. It is clear that a somewhat skewed distribution with an average grain size of about 4.2 µm and a standard deviation of 1.9 µm has been achieved. However, the microstructure of the mate rial after 8 passes of MDF, depicted in Fig 3a, is indicative of a more refined state, implying that dynamic recrystallization has occurred during the MDF process. The grain size data histogram in Fig. 3b exhibits a normal distribution with an average grain size of about 2 µm and a small standard de viation of 0.6 µm. In fine-grained materials, a uniform grain size distribution is important in achieving homogeneous mechanical properties.h i It is generally accepted that the mechanical behavior of metallic materials at homologous temperatures higher than 0.3 can be fairly expressed by the power-law creep in a wide range strain rates. 2.1. Materials and processing The material used was a Sn–1 wt% Bi alloy. It was prepared from high purity (99.9%) tin and bismuth, melted in an elec trical furnace under an inert argon atmosphere, and cast into 110×13×13 mm slabs. The slabs for MDF were cut into bil lets with dimensions of 20×13×13 mm. In order to ensure that the billets had similar initial microstructures, they were homogenized at 425 K for 8 h. The samples were multi-direc tionality forged at room temperature at a pressing speed of 2 mm min–1. A 0.05-mm thick PTFE film was wrapped around the samples as a lubricant during MDF. As shown in Fig. 1, the loading direction was changed by 90° se quentially along the three axes of the sample from pass to pass, in such a way that the dimension ratio of 2:1.3:1.3 of the billets was kept unchanged during MDF. The axial pass strain of ∆ε=0.43 was employed, which is defined as ∆ε=ln\|ho/hf\|, where ho and hf are the respective initial and fi nal heights of the billets, resulting in an equivalent strain of ε=(2/√3)ln\|ho/hf \|≈0.5 [12]. MDF was carried out up to a maximum cumulative strain of ∑∆ε=3.44, i.e., a total of eight passes.h The specimens were examined by optical microscopy to determine the grain size and distribution of the forged material. After mechanical polishing, samples were etched using a solution of 60 ml alcohol, 30 ml H2O, 5 ml HCl and 2 g FeCl3. The grain sizes and distributions were calculated using the Clemex professional image analysis program according to the ASTM E112 standard. Fig. 1. Schematic illustration of multi-directional forging. 314 Akbaripanah et al. / Letters on materials 5 (3), 2015 pp. 313-318 3. Results and discussion Thus, for steady-state creep, the relationship between the strain rate, ἐ, and the tensile stress, σ, at a constant temperature can be expressed by: 1/ = = n m A A ε σ σ (1) (1) where A is a constant, n is the steady-state stress exponent, and m is the SRS index defined as: ln ln ∂   =   ∂   m  ε σ ε (2) (2) (2) Assuming an analogy between strain and diagonal length from one side, and stress and hardness from the other side, the following relationship is obtained: Assuming an analogy between strain and diagonal length from one side, and stress and hardness from the other side, the following relationship is obtained: The results of indentation tests are shown in Fig. 4, where the indentation length is plotted against time. It can be inferred from this figure that the indentation diagonal Fig. 3. (a) Optical micrograph and (b) grain size distribution of the material after 8 passes of MDF. Fig. 2. (a) Optical micrograph and (b) grain size distribution of the unMDFed material. Fig. 3. (a) Optical micrograph and (b) grain size distribution of the material after 8 passes of MDF. Fig. 3. (a) Optical micrograph and (b) grain size distribution of the material after 8 passes of MDF. Fig. 2. (a) Optical micrograph and (b) grain size distribution of the unMDFed material. 315 Akbaripanah et al. / Letters on materials 5 (3), 2015 pp. 313-318 Fig. 4. Indentation creep curves for different conditions. Differentiating Eq. (5) with respect to time and substituting into Eq. (4) gives 1/ 1/ 4 0 0   =     m dA P C A dt A ε σ (6) (6) where C3 and C4 are constants. When P is held constant, Eq. (6) can be rewritten as 1/ 4 0 0 1      = −              m V V V dH H C H dt ε σ (7) (7) According to Eq. (7), a plot of ln(Hv) versus ln[(-1/Hv) (dHv/dt)] at a constant temperature has a slope of m. The results obtained by this approach are illustrated in Fig. 6 for all material conditions. Similar to the previous method of analysis, lines with different single slopes of m are obtained for each condition. 3. Results and discussion The obtained m-values are in the range 0.08–0.48 for the materials after 2 to 8 MDF passes. Fig. 4. Indentation creep curves for different conditions. ln ln ∂   =   ∂   v d H m d (3) (3) t Sargent and Ashby have also derived the following relationship between indentation hardness and dwell time: where Hv is the Vickers hardness number, d is the indentation diagonal length, and d is the rate of variation in indentation length. This implies that if Hv is plotted against d on a double logarithmic scale, a straight line would be obtained, the slope of which is the stress exponent, m. This is shown in Fig. 5, where m-values in the range 0.08–0.5 are obtained for the materials experiencing 2–8 MDF passes, respectively. ( ) 0 4 0 ( ) =  V m H t nC t σ ε (8) (8) where Hv(t) is the time-dependent hardness. Hence, from Eq. (8), the slope of a plot of ln(Hv) against ln(t) at a constant temperature is – m. The results obtained based on this approach are demonstrated in Fig. 7. This figure shows that the hardness of all tested conditions of the material can be represented as linear functions of time. Accordingly, m-values calculated from the slope of the fitted lines were found to be 0.08–0.48 for the materials after 2 to 8 MDF passes. Sargent and Ashby [13] carried out hot hardness tests on a wide range of materials and proposed a dimensional analy sis for indentation creep. According to their model, the dis placement rate of an indenter has been derived as: ( ) 1/ 0 1 2 0         =                   m C du P A dt C A ε σ (4) (4) t One interesting point on the hardness results in Figs. 4 through 7 is that the Hv values are reduced with increasing numbers of MDF passes whereas grain sizes are reduced. This may seem to be against the Hall–Petch relationship, from which one may expect higher hardness values at smaller grain sizes. The softening behavior of the HPT-processed materials is recently reviewed by Kawasaki [14]. 3. Results and discussion It is well accepted that at SRS index values close to 0.5, superplastic deformation mechanisms such as grain boundary sliding become prominent [19]. SRS indices of about 0.45 to 0.5 have been reported in the indentation creep of a superplastic lead-tin eutectic alloy [8]. More recently, SRS indices of about 0.4 have been reported for the Sn-1Bi alloy processed by four passes of ECAP [11]. It is therefore concluded that after 8 passes of MDF, the present Sn-1Bi alloy can develop a fine-grained microstructure with superplastic indentation behavior caused by grain boundary sliding. Fig. 7. Hardness-dwell time log–log plots giving straight lines with the slope of –m. HPT when the homologous temperature of processing is very high. This has been reported to be the case in ultrafine- grained Zn–22% Al eutectoid and ultrafine-grained Pb–62% Sn eutectic alloys after HPT at room temperature. Different microstructural reasons such as significant reduction in the distribution of the stable hexagonal close-packed Zn precipitates in Zn–Al [15] and similar loss in precipitation in the Pb–Sn alloy during deformation by HPT [16] have been put forward for the observed softening after severe plastic deformation of these materials. In a recent report, the plasticity and strain rate sensitivity of an ultrafine-grained Zn–22% Al alloy after HPT was examined by nanoindentation which demonstrated the clear softening and increasing m-values with increasing number of HPT turns [17]. t In all of the above studies, softening has been manifested by a detectable drop in either yield stress or hardness values measured after deformation. The nature of this softening behavior is different from that observed in the present study. Here, the short-term hardness values obtained at the dwell time of 0.5 min are very close for all conditions of the material, as shown in Fig. 4. On the other hand, the difference in the measured hardness values of the material after different MDF passes at longer dwell times is significantly high. This implies that creep resistance is strongly affected by the number of MDF passes, while the short-term hardness that is an indication of the yield stress is not much changed. In other words, smaller grain size has provided a higher density of grain boundaries, which can ease grain boundary sliding of the stressed material during dwell time, the effect which is not dominant in short-term hardness tests. 3. Results and discussion It is well accepted that at SRS index values close to 0.5, superplastic deformation mechanisms such as grain boundary sliding become prominent [19]. SRS indices of about 0.45 to 0.5 have been reported in the indentation creep of a superplastic lead-tin eutectic alloy [8]. More recently, SRS indices of about 0.4 have been reported for the Sn-1Bi alloy processed by four passes of ECAP [11]. It is therefore concluded that after 8 passes of MDF, the present Sn-1Bi alloy can develop a fine-grained microstructure with superplastic indentation behavior caused by grain boundary sliding. Table 1. The SRS indices of the materials after different passes of MDF derived from different methods. Condi tion SRS index, m Average m-value ln(Hv) vs. ln(do) ln(Hv) vs. ln[(-1/Hv) (dHv/dt)] ln(Hv) vs. ln(t) 2 Passes 4 Passes 6 Passes 8 Passes 0.08 0.25 0.32 0.50 0.08 0.23 0.30 0.48 0.08 0.24 0.30 0.48 0.08 0.24 0.31 0.49 Fig. 7. Hardness-dwell time log–log plots giving straight lines with the slope of –m. Table 1. The SRS indices of the materials after different passes of MDF derived from different methods. Table 1. The SRS indices of the materials after different passes of MDF derived from different methods. types. By increasing the number of MDF passes the grain size decreases and SRS index increases. This behavior is in agreement with the Gifkins' core-mantle theory suggesting that for a constant width of mantle, an increasingly fine grain size results in a domination of the mantle behavior and thus, the SRS index begins to increase [18]. It is worth noting that although the unMDFed material possesses a relatively small grain size of 4.2 µm, its SRS index of less than 0.08 is not as high as those of the material after MDF. This is believed to be due to the presence of some coarse grains, which have resulted in the non-uniformity of the grain structure with a relatively high standard deviation of 1.9 µm in the unMDFed condition. Moreover, the lack of high angle grain boundaries that are essential for the achievement of high SRS indices can be another possible reason for the observed behavior. On the contrary, it seems that processing by MDF not only refines the grain structure, but also increases the density of the high-angle grain boundaries. The high m-values of 0.49 obtained after 8 passes of MDF are in accord with this argument. 3. Results and discussion It has been stated that there is a unique softening behavior in metals and alloys after where A is the projected area of indentation, C2 is a constant, ἐ0 is the rate at a reference stress σ0, m is the SRS index, and P is the applied load. For a pyramid indenter the penetration is proportional to A, i.e., 3 = u C A (5) 3 = u C A (5) 3 u C A (5) Fig. 5. Plots of ln(HV) against ln(do) after different MDF passes to de termine the SRS index, m. Fig. 6. The ln(Hv) vs. ln[(-1/Hv)(dHv/dt)] curves with the slope of m. Fig. 6. The ln(Hv) vs. ln[(-1/Hv)(dHv/dt)] curves with the slope of m. Fig. 6. The ln(Hv) vs. ln[(-1/Hv)(dHv/dt)] curves with the slope of m. Fig. 6. The ln(Hv) vs. ln[(-1/Hv)(dHv/dt)] curves with the slope of m. Fig. 5. Plots of ln(HV) against ln(do) after different MDF passes to de termine the SRS index, m. Fig. 6. The ln(Hv) vs. ln[(-1/Hv)(dHv/dt)] curves with the slope of m. Fig. 5. Plots of ln(HV) against ln(do) after different MDF passes to de termine the SRS index, m. 316 Akbaripanah et al. / Letters on materials 5 (3), 2015 pp. 313-318 types. By increasing the number of MDF passes the grain size decreases and SRS index increases. This behavior is in agreement with the Gifkins' core-mantle theory suggesting that for a constant width of mantle, an increasingly fine grain size results in a domination of the mantle behavior and thus, the SRS index begins to increase [18]. It is worth noting that although the unMDFed material possesses a relatively small grain size of 4.2 µm, its SRS index of less than 0.08 is not as high as those of the material after MDF. This is believed to be due to the presence of some coarse grains, which have resulted in the non-uniformity of the grain structure with a relatively high standard deviation of 1.9 µm in the unMDFed condition. Moreover, the lack of high angle grain boundaries that are essential for the achievement of high SRS indices can be another possible reason for the observed behavior. On the contrary, it seems that processing by MDF not only refines the grain structure, but also increases the density of the high-angle grain boundaries. The high m-values of 0.49 obtained after 8 passes of MDF are in accord with this argument. References 9. R. Mahmudi, A. Rezaee-Bazzaz, Mater. Letts. 59, 1705 (2005). 1. M. Noda, M. Hirohashi, K. Funami, Mater. Trans. 44, 2288 (2003). 10. R. Mahmudi, A. Rezaee-Bazzaz, J. Mater. Sci. 42, 4051 (2007). unami, Mater. Trans. 44, 10. R. Mahmudi, A. Rezaee-Bazzaz, J. Mater. Sci. 42, 4051 (2007). 10. R. Mahmudi, A. Rezaee-Bazzaz, J. Mater. Sci. 42, 4051 (2007). 2. J. Y. Xing, X. Yang, H. Miura, T. Sakai, Mater. Trans. 48, 1406 (2007). 11. R. Mahmudi, H. Mhjoubi, P. Mehraram, Int. J. Modern Phys. B. 22, 2823 (2008). 2. J. Y. Xing, X. Yang, H. Miura, T. Sakai, Mater. Trans. 48, 1406 (2007). 1 11. R. Mahmudi, H. Mhjoubi, P. Mehraram, Int. J. Modern Phys. B. 22, 2823 (2008). 3. A. Gennady, E. A. K. Salishchev, V. Sergey, S. Zherebtsov, S L S i i M S i F 735 253 (2012) 12. R. Kapoor, A. Sarkar, R. Yogi, S. K. Shekhawat, I. Samajdar J. K. Chakravartty, Mater. Sci. Eng. A 560, 404 (2013). 3. A. Gennady, E. A. K. Salishchev, V. Sergey, S. Zherebtsov, S. L. Semiatin, Mater. Sci. Forum. 735, 253 (2012). 12. R. Kapoor, A. Sarkar, R. Yog J. K. Chakravartty, Mater. Sc 3. A. Gennady, E. A. K. Salishchev, V. Sergey, S. Zherebtsov, S. L. Semiatin, Mater. Sci. Forum. 735, 253 (2012). 1 g j J. K. Chakravartty, Mater. Sci. Eng. A 560, 404 (2013). 13. P. M. Sargent, M. F. Ashby, Mater. Sci. Tech. 8, 594 (1992). 4. O. Sitdikov, T. Sakai, A. Goloborodko, H. Miura, 4. O. Sitdikov, T. Sakai, A. Goloborodko, H. Miura, R. Kaibyshev, Mater.Trans. 45, 2232 (2004). 14. M. Kawasaki, J. Mater. Sci. 49, 18 (2014). R. Kaibyshev, Mater.Trans. 45, 2232 (2004). 5. O. Sitdikov, T. Sakai T, A. Goloborodko, H. Miura, Scr. Meter. 51, 175 (2004). 15. M. Furukawa, Z. Horita, M. Nemoto, R. Z. Valiev, T. G. Langdon, J. Mater. Res. 11, 2128 (1996). T. G. Langdon, J. Mater. Res. 11, 2128 (1996). 16. N. Zhang, M. Kawasaki, Y. Huang, T. G. Langdon, J. Mater. Sci. 48, 4582 (2013). 6. R. Mahmudi, R. Roumina, B. Raeisinia, Mater. Sci. Eng. A38, 2 15 (2004). 17. I. C. Choi, Y. J. Kim, B. Ahn, M. Kawasaki, T. G. Langdon, 17. I. C. Choi, Y. J. Kim, B. Ahn, M. Kaw 7. R. Mahmudi, A. R. Geranmayeh, S. R. Mahmoodi, A. Khalatbari, J. Mater. Sci.: Mater Electron. 18, 1071 (2007). J. 4. Conclusions 1. It is experimentally shown that an indentation creep test provides a convenient method for measuring strain rate sensitivity (SRS) and thereby assessing the ability of a material to undergo superplastic deformation.hf A summary of the SRS indices calculated via each of the above mentioned approaches are given in Table 1 for all tested conditions. As can be seen the m-values calculated by different approaches are in good agreement with each other, indicating the similarity of the derivation methods. It is also evident that the obtained m-values increase with increasing the number of MDF passes. The average m-values of 0.08, 0.24, 0.31 and 0.49 are obtained for the material after 2, 4, 6 and 8 passes of MDF, respectively.hf 2. The SRS indices calculated from different methods of analysis are in good agreement with each other. The average SRS values obtained from different approaches were found to be 0.08, 0.24, 0.31 and 0.49 for the 2, 4, 6 and 8 passes of MDF, respectively. This implies that with increasing numbers of MDF passes, the SRS indices increase and superplastic deformation is encouraged. 3. The highest m-value of 0.49, obtained for the alloy after 8 MDF passes, together with a very fine-grained microstructure with an average grain size of 2 µm are indicative of a grain boundary sliding mechanism at room temperature The observed difference in the indentation behavior, as indicated by different m-values of the material after several MDF passes, can be attributed to their grain sizes and grain 317 Akbaripanah et al. / Letters on materials 5 (3), 2015 pp. 313-318 Akbaripanah et al. / Letters on materials 5 (3), 2015 pp. 313-318 Akbaripanah et al. / Letters on materials 5 (3), 2015 pp. 313-318 8. A. Juhasz, P. Tasnadi, I. Kovacs, J. Mater. Sci. Lett. 5, 35 (1986).k 19. T. G. Langdon, Mater. Sci. Eng. A 2 References I. Jang, Scr. Mater. 75, 102 (2014).k 18. R. C. Gifkins, Metall Trans 7A, 225 (1976). 8. A. Juhasz, P. Tasnadi, I. Kovacs, J. Mater. Sci. Lett. 5, 35 (1986).k 19. T. G. Langdon, Mater. Sci. Eng. A 283, 266 (2000) . . A. Juhasz, P. Tasnadi, I. Kovacs, J. Mater. Sci. Lett. 5, 35 (1986)k 19. T. G. Langdon, Mater. Sci. Eng. A 283, 266 (2000) k 19. T. G. Langdon, Mater. Sci. Eng. A 283, 266 (2000) . 8. A. Juhasz, P. Tasnadi, I. Kovacs, J. Mater. Sci. Lett. 5, 35 (1986). 19. T. G. Langdon, Mater. Sci. Eng. A 283, 266 (2000) . 318
https://openalex.org/W1984877171	https://edit.elte.hu/xmlui/bitstream/10831/65989/1/2709627.pdf	English	null	Social Category Formation Is Induced by Cues of Sharing Knowledge in Young Children	PloS one	2,014	cc-by	6,359	Introduction expectations about a person’s behavior [16], however other studies suggest that some classifications have more limited power in guiding young children’s behavior towards people [17]. Human societies are unique among species in that our form of living entails a level of interdependence between conspecifics that cannot be found in any other animal species. Humans form alliances with other people for different purposes every day, during which they engage in a wide variety of joint actions and collaboration towards goals, such as creating artifacts, working towards scientific discoveries or – even – doing sports together [1,2]. Group-living constitutes a fundamental characteristic of the human race and group-affiliations can influence various aspects of our lives to a great extent. Therefore the ability to form cognitive representations of human groups can be seen as an evolutionary adaptive capacity of the human brain. More recently, in a new line of research, Kinzler and colleagues have shown that spoken language occupies a prominent role in young children’s representations of social categories [18–20]. Their findings also indicate that this distinction is more privileged in children’s eyes than some other, such as race [21], which suggests that language taps into some of the more fundamental processes underlying the propensity of category-based thinking. One of the most important findings of these experiments is that the accent with which a person speaks has a stronger effect on 5-year- olds attitude toward that person than their ethnicity. Despite the great and long interest and the social relevance of the question of social categorization, the cognitive basis and the function of this process is still somewhat under-explained. Theories in social psychology have provided numerous accounts of how affiliating with a group may benefit an individual in – for example – boosting their self-esteem [15,22]; justifying a bigger share of resources [23–24], etc. These accounts generally tend to empha- size the competition between different social groups and assume an antagonist relationship between them. Theorists from various fields of cognitive sciences tend to grasp a different aspect of the question and offer other explanations with respect to the function of group-based thinking. Katalin Ola´h1,2, Fruzsina Elekes1,2, Ga´bor Bro´ dy1, Ildiko´ Kira´ly1 1 Department of Cognitive Psychology, Eo¨tvo¨s Lora´nd University, Budapest, Hungary, 2 Institute of Cognitive Neuroscience and Psychology, Hungarian Academy of Sciences, Budapest, Hungary Abstract Funding: The project received financial funding from the National Development Agency (URL: www.nfu.hu/?lang = en, grant number: KMOP-4.2.1/B-10-2011- 0002) and the number: K-109352 and K-100695). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript ceived financial funding from the National Development Agency (URL: www.nfu.hu/?lang = en, grant number: KMOP-4.2.1/B-10-2011- 109352 and K-100695). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the Competing Interests: The authors have declared that no competing interests exist. Email: olah.katalin@ttk.mta.hu Abstract Previous research has shown that human infants and young children are sensitive to the boundaries of certain social groups, which supports the idea that the capacity to represent social categories constitutes a fundamental characteristic of the human cognitive system. However, the function this capacity serves is still debated. We propose that during social categorization the human mind aims at mapping out social groups defined by a certain set of shared knowledge. An eye- tracking paradigm was designed to test whether two-year-old children differentially associate conventional versus non- conventional tool use with language-use, reflecting an organization of information that is induced by cues of shared knowledge. Children first watched videos depicting a male model perform goal-directed actions either in a conventional or in a non-conventional way. In the test phase children were presented with photographs taken of the model and of a similarly aged unfamiliar person while listening to a foreign (Experiment 1) or a native language (Experiment 2) text. Upon hearing the foreign utterance children looked at the model first if he had been seen to act in an unconventional way during familiarization. In contrast, children looked at the other person if the model had performed conventional tool use actions. No such differences were found in case of the native language. The results suggest that children take the conventionality of behavior into account in forming representations about a person, and they generalize to other qualities of the person based on this information. ekes F, Bro´dy G, Kira´ly I (2014) Social Category Formation Is Induced by Cues of Sharing Knowledge in Young Children. PLoS ONE /journal.pone.0101680 Editor: Howard Nusbaum, The University of Chicago, United States of America Received July 24, 2013; Accepted June 10, 2014; Published July 11, 2014 Received July 24, 2013; Accepted June 10, 2014; Published July 11, 2014 Copyright: 2014 Ola´h 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. pyright: 2014 Ola´h et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, whic e, distribution, and reproduction in any medium, provided the original author and source are credited. July 2014 \| Volume 9 \| Issue 7 \| e101680 Method In all the videos the model first non-verbally, but explicitly demonstrated his goal by reaching for one of the goal-objects. Then he grabbed one of the tools, examined and then rejected it (shaking his head). After that he grabbed the other tool, nodded on examining it, and then used the tool to bring the goal about. After the goal had been attained, he expressed satisfaction by nodding at the outcome. Importantly, in both conditions all the goals and possible tools were familiar to the children, but in the Non- conventional condition the associations between tools and goals were unfamiliar (e.g. using a fork to brush his hair), while in the Conventional condition associations were always familiar (e.g. using a fork to eat food). The length of the videos varied between 16 and 20 seconds. For detailed description of the videos see the Appendix. Note that in both the Conventional and Non- conventional condition, the tool that was chosen by the model could efficiently bring about the outcome. Moreover, in both conditions, the model performed the action with equal confidence, and without communicating with the participant in any way (he did not look into camera, did not smile, wave, etc.). Thus, the familiarization events only differed in their level of conventionality. p To directly investigate the question whether children are indeed sensitive to the boundaries of culturally shared knowledge, we designed two experiments where we sought to test whether children form similar representations of people based on other cues of sharing knowledge (using tools in a conventional or a non- conventional way) as they do based on language. The conven- tionality of tool-using behavior is an adequate index of sharing cultural knowledge as artifact functions have an inherent cultural aspect. It has been proposed that the differentiation of tools was a result of recursive tool making practices. In consequence, function knowledge includes cognitively opaque properties that could only be understood through cultural practices and culturally evolved teaching situations. Csibra and Gergely argue that communication of generic knowledge was selected as a consequence of the learnability problem induced by cognitively opaque contents in tool making practices [27,28]. Our hypothesis was that distinct cues -potentially of shared knowledge- induce an organized representation of social categories. Children were first familiarized with videos depicting a model that performed either conventional or non-conventional tool-use actions. Method An additional seven children were tested, but later excluded from the sample due to experimenter error (2); the eye-tracker did not return any data for the given measure and behavior could not be coded by visual observation (2), children could not be calibrated properly (2) and child’s parents turned out to be of two different cultures (1). Ethic statement. The experiment was conducted with the approval of the Ethical Committee of the Faculty of Education and Psychology, Eo¨tvo¨s Lora´nd University. Parents of the children signed informed consent prior to participation. We propose that the relevance of representing social groups is that it enables us to find the boundaries of culturally shared knowledge. Spoken language is obviously a part of this and can serve as a perfect indication of cultural group membership from the earliest periods of our lives as even 2-day-old infants are sensitive to language [29]. However, conducting successful interactions with others not only requires a commonly spoken tongue, but also knowledge of other social norms and conventions. Therefore, we propose that the basis of social categorization may be culturally shared knowledge in general. This notion is plausible if we consider that this distinction is genuinely meaningful and helpful in guiding behavior, whereas other distinctions, such as one based on skin-color, are in themselves empty categories. That is not to say that in certain situations some features cannot correlate with each other (see [30] for how perceptual markers of ethnicity can become associated with fundamentally norm-based social groups). However, characteristics such as race are not determin- istic in this respect. Equipment. For video stimuli presentation and data collec- tion a Tobii T60XL eye-tracker was used with the TobiiStudio 3.2 software. The screen’s size was 52632 cm and 192061200 pixels. We used a five point calibration throughout the experiment. Children who were included in the final sample provided at least 80% valid eye-tracking data. Video stimuli. For the familiarization phase, three videos were created for each condition (Conventional and Non-conven- tional). In each of the videos, a male model performed a goal- directed action with a chosen tool. The three videos depicted three different tool-using actions. The setup in the video always included two visible goals (e.g. a plate of food and messy hair) and two possible tools to bring the goals about (e.g. a fork and a brush). Method An adaptive system that is responsible for representing social groups must correspond to the core characteristics and advantages of group living. Forming short or long term bonds with other people enables us to create the uniquely complex and developed cultural niche that we live in. These products of human cooperation are also unique in that they can be preserved over time by the help of a specially evolved communicational system that allows for the efficient transmission of knowledge from one generation to the other [27–28]. As a result, each human culture is characterized by a vast body of shared knowledge that contains generic information about the world (such as ‘‘snow is cold’’) as well as a set of mostly arbitrary cultural norms. The very nature of these cultural norms is that while they are in great part arbitrary, acquiring knowledge of the norms specific to a certain culture is crucial in managing everyday life and in interacting with others (imagine driving on the wrong side of the road!). Participants. Thirty (13 girls; mean age: 24.37 months, SD: 2.27 months) monolingual children between the age of 20 and 28 months participated in the study, of whom 15 were assigned in the Conventional condition and 15 in the Non-conventional condi- tion. Participants were selected from a database of volunteer families that had previously applied for participation. Children were excluded from participation if there was at least one person in their immediate family whose native tongue was not Hungarian. An additional seven children were tested, but later excluded from the sample due to experimenter error (2); the eye-tracker did not return any data for the given measure and behavior could not be coded by visual observation (2), children could not be calibrated properly (2) and child’s parents turned out to be of two different cultures (1). Participants. Thirty (13 girls; mean age: 24.37 months, SD: 2.27 months) monolingual children between the age of 20 and 28 months participated in the study, of whom 15 were assigned in the Conventional condition and 15 in the Non-conventional condi- tion. Participants were selected from a database of volunteer families that had previously applied for participation. Children were excluded from participation if there was at least one person in their immediate family whose native tongue was not Hungarian. Social Categorization by Cues of Sharing Knowledge Social Categorization by Cues of Sharing Knowledge Experiment 1, but not in Experiment 2 as we hypothesized that the strong familiarity effect of hearing their native language would overshadow other effects. argue that the human mind has evolved a special domain to reason about social kinds in order to make sense of the extremely complicated structure of human societies. Similarly, Cosmides, Tooby, and Kurzban [26] propose that the primary function of representing social categories is to map out potential coalitional partners. Introduction Sperber and Hirschfeld [25] for example The phenomenon that adults have a propensity to think of fellow humans as belonging to groups has been well documented in the social psychology literature (see [3] for a review) and there is also ample evidence suggesting that even infants are able to perceive the boundaries of certain social categories. Studies conducted in the field of developmental psychology as well as cultural anthropology have demonstrated that young children or even infants are sensitive to social categories such as age [4], sex [5–9], race [10–14]. It has also been demonstrated that children as young as 2.5 years of age – similarly to adults [15] – are willing to accept even arbitrary cues of group membership when forming 1 July 2014 \| Volume 9 \| Issue 7 \| e101680 PLOS ONE \| www.plosone.org July 2014 \| Volume 9 \| Issue 7 \| e101680 Experiment 2 Data analysis. Four groups of area of interest were created for analyses combining two factors: who was the target person (model or the other person) and time window (before the onset of the audio stimulus and after). The cut-off point was set at eight seconds, which marks the end of the first utterance; therefore by this point children already had the opportunity to judge the familiarity of the language. The length of the before time window was 8 seconds, while the after time window lasted for 12 seconds. Experiment 2 was designed in order to test whether similar results could be obtained using the children’s native language instead of a foreign language. Since first fixations can be regarded as part of an information seeking process after a certain stimulus, we expected it to be more indicative of children’s cognitive processes after events that violate their expectations. Due to the fact that hearing people speak in our native tongue is such a strong part of our every-day experiences, we hypothesized that it would not elicit any specific cognitive processing, leading to a random pattern of first fixations in the two conditions. However, this study provides important additional information for interpreting the above described results and exploring the validity of the hypothesis that cues of shared knowledge may play a part in forming representations of social groups. Social Categorization by Cues of Sharing Knowledge and the after time window in the test phase, which revealed a general preference towards the model (F(1,28) = 13.36, p = 0.001) before the onset of the audio stimulus (Mean looking times: Conventional condition/model: 2.84 sec; Conventional condi- tion/other: 2.051sec; Non conventional condition/model: 2.79 sec; Non-conventional condition/other: 2.23 sec). This pref- erence disappeared in the after time window (Mean looking times: Conventional condition/model: 2.59 sec; Conventional condi- tion/other: 1.99 sec; Non-conventional/model: 2.19 sec; Non- conventional/other: 2.36 sec). See also Figure 1. tracker (at a distance of approx. 60–70 cm) with the child sitting on the parent’s lap. The experimenter assisted with calibration, but left the room once it was finished. Before the stimuli began, the model entered the room and started manipulating the computer without looking at or talking to the children. This element of the procedure was added in order to avoid the possibility that in-group and out-group effects were weakened by the fact that it was the in- group experimenter that directed children’s attention to the stimuli. After the model had also left the room, children were presented with the three familiarization videos. Children always saw either three Conventional on three Non-conventional videos, depending on condition. Crucially, analyzing the directions of the first fixations we found a significant effect of condition in the after time window (x2 = 4.821, p = 0.028) showing that after the onset of the foreign language text (after time window), children were more likely to look at the model in the Non-conventional condition (10 out of 15 children), whereas the majority of children fixated on the other person first in the Conventional condition (11 out of 15 children). Results are depicted in Figure 2. No significant effect of condition was found before the audio stimulus (x2 = 2.4, p = 0.12). The familiarization phase was immediately followed by the test in which two photographs appeared side-by-side on the screen. The pictures were 21611 cm and were positioned on the left and the right side of the screen with a 10611 cm line in the middle, separating them. One picture depicted the model, while the other photo was taken of another young man matched in age. After 6 seconds had elapsed the voice of a man was heard from the speakers, who spoke in Swedish for 14 seconds. Method Then, we tested whether children differentially associated a foreign language (Experiment 1) or their mother tongue (Experiment 2) to the model or a stranger based on the kind of tool-use the model had previously performed. We expected to find a difference between conditions in Procedure. On arriving to the laboratory, children had some time to explore the room and get comfortable in the company of two experimenters, while parents were briefed about the experiment and signed informed consent. After this, one of the experimenters escorted the child and their parent into the testing room and seated them in a chair in front of the monitor of the eye- PLOS ONE \| www.plosone.org July 2014 \| Volume 9 \| Issue 7 \| e101680 2 Social Categorization by Cues of Sharing Knowledge Method Participants. Twenty-six (10 girls, mean age: 24.32 month, SD: 2.28) monolingual children participated in the study with equal number of children assigned in the two conditions (Conventional and Non-conventional). The criterion for partici- pation was the same as in Experiment 1. An additional three children were excluded from the sample due to inattentiveness. Materials and procedure. The applied stimuli and the procedure were identical to the ones used in Experiment 1 with the exception that during the test phase, the Swedish audio text was replaced by a Hungarian (native) text. The length of the audio stimulus and the duration of the first utterance were matched to those in Experiment 1. Materials and procedure. The applied stimuli and the procedure were identical to the ones used in Experiment 1 with the exception that during the test phase, the Swedish audio text was replaced by a Hungarian (native) text. The length of the audio stimulus and the duration of the first utterance were matched to those in Experiment 1. We also analyzed total visit duration in both time windows to explore any possible general preference towards either of the models. In addition we analyzed total visit durations for the familiarization videos in order to exclude the possibility that children were more attentive in the Non-conventional condition due to the surprising behavior exhibited by the model. Results. An alpha level of .05 was used for all statistical tests. Age and sex were first entered into all of the models used in the analyses, but were later removed as they were not significant in any of the cases. Results. An alpha level of .05 was used for all statistical tests. Sex and age were first always entered into the analyses, but were not significant in any case and were therefore removed from all models. An independent samples T-test was performed on the percent- age of looking at the three familiarization videos with condition as a between subject variable. The analyses revealed a marginal effect of condition (t(29) = 1.99, p = 0.057), showing that children attended to the videos slightly more in the Conventional condition (Mean percentage of looking in the Conventional and the Non- conventional condition: 98 and 94 percent, respectively). Discussion window. Children spent more time looking at the model than at the other person and they looked longer in the Non-conventional than the Conventional condition (Conventional/model: 2.82 sec; Conventional/other: 1.99 sec; Non-conventional/model: 3.55 sec; Non-conventional/other: 2.53 sec). No effects were found in the after time-window (Conventional condition/model: 2.69 sec; Con- ventional condition/other: 2.21 sec; Non-conventional condition/ model2.65 sec; Non-conventional condition/other: 2.47 sec, see also Figure 3). window. Children spent more time looking at the model than at the other person and they looked longer in the Non-conventional than the Conventional condition (Conventional/model: 2.82 sec; Conventional/other: 1.99 sec; Non-conventional/model: 3.55 sec; Non-conventional/other: 2.53 sec). No effects were found in the after time-window (Conventional condition/model: 2.69 sec; Con- ventional condition/other: 2.21 sec; Non-conventional condition/ model2.65 sec; Non-conventional condition/other: 2.47 sec, see also Figure 3). In this experiment we investigated whether 2-year-old children form similar representations of a person based on the observed level of conventionality in their tool using habits as they do based on the language they speak. This design allowed us to test whether children would associate a foreign and a native language differentially to people based on the conventionality of his behavior. We found that children associated a foreign language to the model if he had previously performed goal-directed actions in a non-conventional way, but formed an association between the foreign language and the other person if previously the model had been seen to act in a conventional way, making it unlikely that he was the source of the foreign language utterance. On the other hand, we found no evidence of children differentially associating a native language to the two men. The latter result indicates that different characteristics along which we form judgments about a person (or possibly about social group membership) may be organized hierarchically, and language represents a stronger cue than certain other qualities (in this case tool-use, but see [21]). Children meet a vast number of people every day that differ along countless traits but with a few exceptions they share a commonly spoken tongue. Method Pair-wise analysis of the percentages of the aggregated looking times during the three familiarization videos revealed a significant effect of condition, with children looking overall longer in the Conventional condition (d(25) = 3.08, p = 0.009). However, the difference between looking times was relatively small with a mean of 99 percent in the Conventional condition and 95 percent in the Non-conventional condition. A GLM analysis on the total visit durations in the test phase yielded a significant effect of condition (F(1, 24) = 24.34, p,0.001) and target person (F(1, 24) = 4.34, p = 0.048) in the before time Repeated measures GLM analyses on the total visit durations (factors: target person [model vs. other], condition [Conventional vs. Non-conventional]) were performed separately for the before PLOS ONE \| www.plosone.org July 2014 \| Volume 9 \| Issue 7 \| e101680 July 2014 \| Volume 9 \| Issue 7 \| e101680 3 Figure 1. Total looking times in the test phase of Experiment 1. The duration of overall looking times at the two photographs (depicting the model and the other person) in the Conventional and Non-conventional condition, presented separately for the periods before and after the onset of the foreign language stimulus. doi:10.1371/journal.pone.0101680.g001 Figure 1. Total looking times in the test phase of Experiment 1. The duration of overall looking times at the two photographs (depicting the model and the other person) in the Conventional and Non-conventional condition, presented separately for the periods before and after the onset of the foreign language stimulus. doi:10.1371/journal.pone.0101680.g001 Discussion Therefore hearing a text in their native language will come as no surprise to them and will probably not elicit such a Analyzing the direction of the first fixations we found an effect of the experimental condition in the before (x2 = 3.85, p = 0.05) time window showing that most of the children in the Conventional condition fixated on the model first (9 out of 13 participants) while the majority of children in the Non-conventional condition fixated on the other person first (9 out of 13 participants). However, after the onset of the stimulus there was no difference between conditions (x2 = 0.16, p = 0.69) with approximately the same number of children looking at the model first in both conditions (7 and 8 in the Conventional and Non-conventional condition, respectively). Results are depicted in Figure 4. Figure 2. Distribution of first fixations after the onset of the audio stimulus in Experiment 1. Number of children looking first at the model and at the other person in the Conventional and the Non-conventional condition after hearing the foreign language text. doi:10.1371/journal.pone.0101680.g002 Figure 2. Distribution of first fixations after the onset of the audio stimulus in Experiment 1. Number of children looking first at the model and at the other person in the Conventional and the Non-conventional condition after hearing the foreign language text. doi:10.1371/journal.pone.0101680.g002 July 2014 \| Volume 9 \| Issue 7 \| e101680 PLOS ONE \| www.plosone.org 4 Figure 3. Looking times in the test phase of Experiment 2. The duration of overall looking times at the two photographs (depicting the model and the other person) in the Conventional and Non-conventional condition, presented separately for the periods before and after the onset of the native language stimulus. doi:10.1371/journal.pone.0101680.g003 Figure 3. Looking times in the test phase of Experiment 2. The duration of overall looking times at the two photographs (depicting the model and the other person) in the Conventional and Non-conventional condition, presented separately for the periods before and after the onset of the native language stimulus. doi:10.1371/journal.pone.0101680.g003 strong response from children. Supposedly this is reflected in the fact that we found no differences in Experiment 2. qualities (in this case, language) of the person based on this information. The phenomenon that even young children organize information about people systematically has been recently shown in a study where 6-month-old infants matched a non-native language to an other-race face [31]. Discussion This study, similarly to ours analyzed the looking-time patterns with photographs and spoken texts as stimuli. First fixations were chosen as the subject of analyses as an indication of participants’ expectation about who is more likely to be the source of the foreign language. Since children were presented with photographs, the question of to whom the voice belongs is rather ambiguous, which becomes evident to partici- pants once they start to visually explore the static stimuli. However, the direction of the first fixation provides information about children’s expectations before the realization that the visual stimuli will not help clear the ambiguity. Spoken language has been shown to occupy a prominent role in children’s representations of humans. Children not only prefer people belonging to the same linguistic group as themselves [18], but they extend this preference to objects associated with a linguistic in-group [20], and they selectively learn from people speaking their own language [19,32]. This study has demonstrated that these representations are not constrained to the domain of language but are possibly part of a wider module designated to reason about humans in terms of familiarity and conventionality of their behavior. These characteristics are ultimately indications of whether a person is in possession of the same cultural knowledge as oneself. Analyzing the total visit durations in the test phase, we found a preference towards the model before the onset of the audio stimulus. This suggests that familiarity did have an effect in the test phase, but preferences based on mere familiarity faded away by the time the audio stimulus started and were also suppressed by the conflicting information that was provided about the model’s behavioral habits (this is reflected in the between-conditions difference in first fixations in Experiment 1 and the even distribution of first fixations in Experiment 2). Differentiating between people based on whether they share the same cultural knowledge has great adaptive value in managing every day life. In conducting interactions with others we unconsciously rely on an immense amount of shared knowledge Our results suggest that children take the familiarity and the conventionality of performed actions into account in forming representations about a person, and they generalize to other References Quinn PC, Yahr J, Kuhn A, Slater AM, Pascalis O (2002) Representation of the gender of human faces by infants: A preference for female. Perception 31: 1109– 1121. 27. Csibra G, Gergely G (2006) Social learning and social cognition: The case for pedagogy. In: Munakata Y, Johnson HM, editors. Processes of change in brain and cognitive development. Attention and performance XXI. Oxford: Oxford University Press. pp. 249–274. 9. Walker-Andrews AS, Bahrick LE, Raglioni SS, Diaz I (1991) Infants’ bimodal perception of gender. Ecol Psychol 3: 55–75. 10. Baron AS, Banaji MR (2006) The Development of Implicit Attitudes Evidence of Race Evaluations From Ages 6 and 10 and Adulthood. Psychol Sci 17(1): 53– 58. 28. Gergely G, Csibra G (2006) Sylvia’s recipe: The role of imitation and pedagogy in the transmission of cultural knowledge. In: Levenson S, Enfield N, editors. Roots of human sociality: Culture, cognition, and human interaction. Oxford: Berg Publishers. pp. 229–255. 11. Dunham Y, Baron AS, Banaji MR (2008) The development of implicit intergroup cognition. Trends Cogn Sci 12 (7): 248–253. 29. Moon C, Cooper RP, Fifer WP (1993) Two-day-olds prefer their native language. Infant Behav Dev 16(4): 495–500. 12. Hirschfeld LA. (1995) Do children have a theory of race? Cognition 54: 209– 252. 13. Hirschfeld LA (1996) Race in the Making. Cambridge: MIT Press. g g ( ) 30. McElreath R, Boyd R, Richerson P (2003) Shared norms and the evolution of ethnic markers. Curr Anthropol 44(1): 122–130. 14. Kelly DJ, Quinn PC, Slater AM, Lee K, Gibson A, et al. (2005) Three-month- olds, but not newborns, prefer own race faces. Dev Sci 8(6): 31–36. 14. Kelly DJ, Quinn PC, Slater AM, Lee K, Gibson A, et al. (2005) olds, but not newborns, prefer own race faces. Dev Sci 8(6): 31–36 31. Uttley L, de Boisferon H, Dupierrix E, Lee K, Quinn PC, et al. (2013) Six- month-old infants match other-race faces with a non-native language. Int J Behav Dev 1–6. 15. Tajfel H, Billig MG, Bundy RP, Flament C (1971) Social categorization and intergroup behavior. 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Fiske ST (2000) Stereotyping, prejudice, and discrimination at the seam between the 305 centuries: Evolution, culture, mind, and brain. Eur J Soc Psychol 30(3): 299–322. p J g ( ) 21. Kinzler KD, Shutts K, DeJesus J, Spelke ES (2009) Accent trumps race in guiding children’s social preferences. Soc Cogn 27(4): 623–634. g g p g ( ) 22. Turner J, Hogg MA, Oakes PJ, Reicher SD, Wetherell MS (1987) Rediscovering h i l A i l i i h O f d B Bl k ll 22. Turner J, Hogg MA, Oakes PJ, Reicher SD, Wetherell MS (1987) Rediscovering the social group: A social categorization theory. Oxford: B. Blackwell. 4. Bahrick LE, Netto D, Hernandez-Reif M (1998) Intermodal Perception of Adult and Child Faces and Voices by Infants. Child Dev 69(5): 1263–1275. 23. Sherif M, Harvey OJ, White BJ, Hood WR, Sherif CW (1961) Intergroup cooperation and competition: The Robbers Cave experiment. 5. Leinbach MD, Fagot BI (1993) Categorical habituation to male and female faces: Gender schematic processing in infancy. Infant Behav Dev 16(3): 317– 332. cooperation and competition: The Robbers Cave experiment. 24. Sidanius J, Pratto F (1993) The inevitability of oppression and the dynamics of social dominance. In: Sniderman PM, Tetlock PE, Carmines EG, editors. Prejudice, politics, and the American dilemma. Stanford: Stanford University Press. pp. 173–211. 6. Miller CL (1983) Developmental changes in male/female voice classification by infants. Infant Behav Dev 6: 313–330. pp 25. Sperber D, Hirschfeld LA (2004) The cognitive foundations of cultural stability and diversity. Trends Cogn Sci 8(1): 40–46. 7. Poulin-Dubois D, Serbin LA, Kenyon B, Derbyshire A (1994) Infants’ intermodal knowledge about gender. Dev Psychol 30: 436–442. intermodal knowledge about gender. Dev Psychol 30: 436–442 26. Cosmides L, Tooby J, Kurzban R (2003) Perceptions of race. Trends Cogn Sci 7(4): 173–179. 8. Detailed description of the video stimuli In the Conventional condition the model uses the scissors to cut the paper, while in the Non-conventional condition the model uses them to cut a banana. In sum, this study has demonstrated that young children rely on the familiarity of tool using actions in forming judgments about a person and these representations are convergent with the ones based on language use. We propose that these two characteristics are alike in that they both belong to the body of culturally accumulated and shared knowledge. This study opens the ground for further investigations aiming to test the potential role that cues of culturally shared knowledge play in representing social groups. Author Contributions Conceived and designed the experiments: KO FE GB IK. Performed the experiments: KO FE GB. Analyzed the data: KO FE GB IK. Wrote the paper: KO FE GB IK. Conceived and designed the experiments: KO FE GB IK. Performed the experiments: KO FE GB. Analyzed the data: KO FE GB IK. Wrote the paper: KO FE GB IK. Social Categorization by Cues of Sharing Knowledge without which our interactions would be infinitely more difficult, unsuccessful or even dangerous (think of the example of driving on the inappropriate side of the road). Appendix Detailed description of the video stimuli Detailed description of the video stimuli 1. Food vs. Hair. The setup includes a fork and a brush (tools), a plate of food and the model’s hair being messy (implication of goals). In the Conventional condition the model uses the fork to eat some potato from a plate, while in the Non- conventional condition the model uses the fork to brush his hair. 1. Food vs. Hair. The setup includes a fork and a brush (tools), a plate of food and the model’s hair being messy (implication of goals). In the Conventional condition the model uses the fork to eat some potato from a plate, while in the Non- conventional condition the model uses the fork to brush his hair. From a developmental point of view, this differentiation also gains great importance as children are in the process of acquiring the necessary cultural knowledge to become a competent member of a particular society. For this, they must be able to identify reliable sources information, whose knowledge is likely to prove useful to them as well (see [20]). It is also worth noticing that children already at the age of two are extremely sensitive to violations of norms [33], which means that they are equipped with the ability to form judgments of violating or adhering to social conventions. 2. Liquid vs. Locket. The setup includes a key and a spoon (tools), a glass of liquid and a locket (implications of goals). In the Conventional condition the model uses the key to open the locket, while in the Non-conventional condition the model uses the key to stir the liquid. 2. Liquid vs. Locket. The setup includes a key and a spoon (tools), a glass of liquid and a locket (implications of goals). In the Conventional condition the model uses the key to open the locket, while in the Non-conventional condition the model uses the key to stir the liquid. 3. Banana vs. Paper. The setup includes a knife and a pair of scissors (tools), a banana and some crepe paper (implications of goals). In the Conventional condition the model uses the scissors to cut the paper, while in the Non-conventional condition the model uses them to cut a banana. 3. Banana vs. Paper. The setup includes a knife and a pair of scissors (tools), a banana and some crepe paper (implications of goals). Figure 4. Distribution of first fixations after the onset of the audio stimulus in Experiment 2. Number of children looking first at the model and at the other person in the Conventional and the Non-conventional condition after hearing the native language text. doi:10.1371/journal.pone.0101680.g004 Figure 4. Distribution of first fixations after the onset of the audio stimulus in Experiment 2. Number of children looking first at the model and at the other person in the Conventional and the Non-conventional condition after hearing the native language text. doi:10.1371/journal.pone.0101680.g004 July 2014 \| Volume 9 \| Issue 7 \| e101680 PLOS ONE \| www.plosone.org 5 Social Categorization by Cues of Sharing Knowledge Social Categorization by Cues of Sharing Knowledge Social Categorization by Cues of Sharing Knowledge References Buttelmann D, Zmyj N, Daum M, Carpenter M (2012) Selective Imitation of In- Group Over Out-Group Members in 14-Month-Old Infants. Child Dev 84(2): 422–428. 17. Kinzler KD, Spelke ES (2011) Do infants show social preferences for people differing in race? Cogn 119(1), 1–9. 33. Rakoczy H, Warneken F, Tomasello M (2008) The sources of normativity: Young children’s awareness of the normative structure of games. Dev Psychol 44(3): 875. 18. Kinzler KD, Dupoux E, Spelke ES (2007) The native language of social cognition. Proc Natl Acad Sci U S A 104(30): 12577–12580. July 2014 \| Volume 9 \| Issue 7 \| e101680 PLOS ONE \| www.plosone.org 6
https://openalex.org/W2982703877	https://bmcpublichealth.biomedcentral.com/track/pdf/10.1186/s12889-019-7637-9	English	null	Impact of the village health center project on contraceptive behaviors in rural Jordan: a quasi-experimental difference-in-differences analysis	BMC public health	2,019	cc-by	8,406	Komasawa et al. BMC Public Health (2019) 19:1415 https://doi.org/10.1186/s12889-019-7637-9 Komasawa et al. BMC Public Health (2019) 19:1415 https://doi.org/10.1186/s12889-019-7637-9 Open Access Impact of the village health center project on contraceptive behaviors in rural Jordan: a quasi-experimental difference-in- differences analysis Makiko Komasawa1* , Motoyuki Yuasa1, Yoshihisa Shirayama2, Miho Sato3, Yutaka Komasawa4 and Malak Alouri5 Makiko Komasawa1* , Motoyuki Yuasa1, Yoshihisa Shirayama2, Miho Sato3, Yutaka Komasawa4 Abstract 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. * Correspondence: mkomasa@juntendo.ac.jp 1Department of Public Health, Faculty of Medicine, Juntendo University, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan Full list of author information is available at the end of the article * Correspondence: mkomasa@juntendo.ac.jp * Correspondence: mkomasa@juntendo.ac.jp Correspondence: mkomasa@juntendo.ac.jp 1Department of Public Health, Faculty of Medicine, Juntendo University, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan Full list of author information is available at the end of the article p @j jp 1Department of Public Health, Faculty of Medicine, Juntendo University, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan Full list of author information is available at the end of the article Abstract Background: Appropriate contraceptive use remains a major health challenge in rural Jordan. The Japan International Cooperation Agency implemented a project aimed at enhancing the capacity of village health centers (VHCs) to improve the quality and quantity of family planning (FP) services in rural Jordan in 2016–2018. Facility- and community-based approaches were integrated into the interventions. We evaluated the project’s impacts on contraceptive behaviors and the effectiveness of the two approaches. Methods: We used a difference-in-differences analysis based on the project baseline and endline surveys, and logistic regression analysis to assess associations between eight primary outcomes and three secondary outcomes (impacts). The unit of intervention was five target VHCs; the unit of analysis was currently married women of reproductive age (15–49 years) in five intervention and five control villages. Results: Overall, 2061 married women participated; 83.8% were in need of FP. Compared with the control villages, significant effects, ranging from + 0.4% points (pp) to + 11.5 pp., were observed in the intervention villages for six primary outcomes in these categories: increasing the use of FP services at VHCs, participation in health promotion activities, and changing the sources of reproductive health information. There was a trend toward improved secondary outcomes in the intervention villages, but no significant differences were observed between the intervention and control villages regarding modern contraceptive use (mCU; + 4.3 pp), traditional contraceptive use (tCU; −0.5 pp), and spousal agreement on contraception (+ 5.1 pp). mCU was positively associated with five primary outcomes: obtaining contraceptives at VHCs [adjusted odds ratio (AOR) 3.44, 95% confidence interval (CI) 1.26–9.40], education sessions at VHC (AOR 7.41, 95% CI 1.60– 34.39), health activities in communities (AOR 7.41, 95% CI 3.28–16.78), counseling by private doctor/clinic (AOR 0.62, 95% CI 0.40–0.97), and information gained through TV (AOR 0.50, 95% CI 0.32–0.76). Spousal agreement on contraception showed similar positive trends. tCU was associated only with TV. Conclusions: The project had impacts on increased mCU and husbands’ perception of contraception in rural Jordan. The integration of facility- and community-based approaches may be effective in shifting from tCU to mCU in other rural areas. Keywords: Evaluation, Impact, Difference-in-differences analysis, Family planning, Modern contraceptives, Community- based approach, Jordan, Japan International Cooperation Agency © The Author(s). © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. * Correspondence: mkomasa@juntendo.ac.jp 1Department of Public Health, Faculty of Medicine, Juntendo University, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan Full list of author information is available at the end of the article Project description j p The Jordanian local health system comprises VHCs at the bottom and primary health centers (PHCs) and com- prehensive health center (CHCs) as core primary health- care facilities in the health directorate. The level of primary healthcare services provided by PHCs and CHCs is almost the same as that provided by general practitioners and midwives, including IUD insertions and removals. Generally, VHCs are located in rural areas where CHCs/PHCs are not available. These primary health care facilities provide services free of charge with the exception of some medications and IUD services. In principle, one nurse (various cadres from registered nurse to aid nurse) or midwife is assigned to one VHC apart from a part-time outreach doctor and a midwife from the upper CHCs/PHCs for 2–3 days per week. The minimum VHC services include provision of basic medi- cations by the part-time doctor and more simple care by the nurse or midwife, such as vital sign monitoring, minor surgery, and provision of essential medication as per the doctor’s prescription. The current MOH regula- tion restricts the provision of FP services by trained mid- wives. However, there is a severe shortage of trained midwives and lack of essential equipment in rural areas that has led to the absence of FP services in rural communities. Due to the efforts of MOH and its partners, the contraceptive prevalence rate in Jordan increased from 40% in 1990 to 61% in 2012 [6]. However, this improve- ment was dependent on the use of traditional contracep- tive methods (e.g. rhythm, withdrawal, and breastfeeding as a non-lactational amenorrhea method), and disparity remains between urban and rural areas [6]. The use of modern contraceptive methods [e.g. intrauterine devices (IUDs), pills, and male condoms, the top three contra- ceptive methods used in Jordan] has plateaued at 46% for currently married women of reproductive age (15– 49 years) [6], which is lower than the world average of 58% and the average of other Muslim-majority countries in the middle eastern region, such as Egypt, Morocco, and Tunisia (59, 61, and 57%, respectively) [7]. These facts indicate that Jordan is still facing difficulties in transitioning from traditional to modern contraceptive methods. Using traditional methods or failure to use modern methods causes unintended pregnancies, leading to adverse health effects in both mothers and children [8–11]. Thus, appropriate FP practice remains a major health challenge in the rural areas of Jordan. Background in Jordan [13]. Therefore, the present study aimed to evaluate the project’s impacts on contraceptive behaviors and determine the effectiveness of the two approaches in rural Jordan. Over the past several decades, Jordan has faced issues of severe shortage of natural resources and rapid growth of population [1]. Furthermore, the massive influx of people from surrounding countries during this decade has become a major burden on the Jordanian health sys- tem [2]. The Ministry of Health (MOH) and the Higher Population Council, along with various partners in Jordan, have continuously implemented reproductive health projects, including family planning (FP) programs since the 1990s [2, 3]. Projects funded by the United States Agency for International Development (USAID) and the Japan International Cooperation Agency (JICA) have played major supporting roles in the field of repro- ductive health in Jordan [4, 5]. © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Komasawa et al. BMC Public Health (2019) 19:1415 Page 2 of 10 Project description The community-based interventions also comprised five components: (i) supporting the establishment of a com- munity health committee in each village; (ii) providing workshops to the committee members; (iii) encouraging committee members to make an action plan for the ac- tivities; (iv) monitoring their activities monthly; and (v) providing seed money for the first 4 months (a total of 100 Jordanian dinars, equivalent to approximately 140 US dollars as of October 2017). The actual period of the interventions was from October 2016 to January 2018 (13 months) for the facility-based interventions and from April 2017 to January 2018 (10 months) for the community-based interventions. [counseling by VHC health staff or by private doctor/ clinic, and information gained through TV (TV)]. Sec- ondary outcomes were impacts on contraception behav- iors and spousal’s perception of FP use: the percentage of modern contraceptive use (mCU), percentage of trad- itional contraceptive use (tCU), and percentage of spou- sal agreement on contraception. A questionnaire was designed based on the Jordan Population and Family Health Survey 2012 (JPFHS 2012) [6], with three questions added from the survey con- ducted by the Jordan Communication, Advocacy, and Policy project funded by USAID [17]. The draft ques- tionnaire was tested in Irbid villages, which were neither intervention nor control areas, and was modified for suitability to the local participants. Questions included participants’ basic characteristics, household conditions, contraceptive behaviors, VHC use, participation in health promotion activities, and sources of reproductive health information. According to the project monitoring documents [12], four training sessions for the VHC health staff and three workshops for doctor/midwife were conducted. Conse- quently, the number of FP service users at six target VHCs in Irbid increased from 0 to 742 after a 13-month intervention. Regarding community activities, all villages established the community health committees, made ac- tion plans, and implemented various activities. Conse- quently, 103 health promotion events were conducted in six villages and the number of participants reached 2051 within a 10-month period. Activities conducted in each village varied from group educational sessions (e.g. at VHCs, schools, community-based associations, or village leaders’ houses), home-visits by VHC staff to free med- ical campaigns with a mobile clinic in collaboration with private companies. A field survey was conducted from September to Oc- tober 2016 for baseline and from January to February 2018 for endline. Trained female interviewers visited households and interviewed the eligible women using the structured questionnaire. Project description To address these situations, the project team, together with MOH, decided to equip VHCs to enable them to provide FP services using the currently available staff. The project sites were Irbid, Mafraq, and Balqa health directorates. The 14 target VHCs (six in Irbid, six in Mafraq, and two in Balqa) were purposely selected by MOH and the project team on the basis of the location of these VHCs in rural areas that do not have access to advanced health facilities, as well as the availability of basic health personnel. Detailed selection criteria for the study sites are described in the study population subsection. To improve the situation, JICA had implemented a technical cooperation project, entitled “Project for im- provement of services at village health centers in rural host communities of Syrian refugees (the VHC project),” in collaboration with the Jordanian MOH from April 2016 to April 2018 [12]. This project primarily aimed at enhancing the capacity of village health centers (VHCs) to improve the quality and quantity of FP services and eventually accelerate the appropriate modern contracep- tive use to reduce unintended pregnancies in rural northern Jordan. Under this project, two approaches were implemented: facility-based interventions for expanding quality FP service provision and community- based interventions for generating demands for modern contraceptive use. The facility-based interventions comprised five com- ponents: (i) providing a series of FP training for nurses or midwives in VHCs; (ii) conducting workshops for doctors and midwives who were periodically serving at VHCs; (iii) providing basic medical equipment; furniture; and information, education, and communication (IEC) materials required in FP services (i.e. FP counseling and providing pills and male condoms); (iv) conducting supervisory visits by maternal and child health supervi- sors from a local health directorate as well as MOH; and Notably, few studies have evaluated the effectiveness of FP projects using a rigorous epidemiological method Komasawa et al. BMC Public Health (2019) 19:1415 Page 3 of 10 Page 3 of 10 (v) updating the FP service manual for VHC staff. Project description Written informed consents were obtained from all participants. For married adoles- cents aged 15–17 years, in addition to the written in- formed consents, verbal consents were obtained from their husbands, parents-in-law, or other legal guardians during initial contact with the participants. Study design A diff Primary outcomes were the direct benefits of the project, comprising three categories with eight indica- tors: (i) use of VHC with three indicators (FP counseling, obtaining contraceptives, and general counseling); (ii) participation in health promotion activities (education sessions at VHCs and health activities in communities); and (iii) sources of reproductive health information Page 4 of 10 Komasawa et al. BMC Public Health (2019) 19:1415 Fig. 1 Map of Irbid health directorate. Footnote: The bold lines indicate the boundary of four health districts (Irbid, Kura, Al-Aghwar Shamaleh, and Bani Kenanah), and the dotted lines indicate the boundary of five districts in the Irbid health district (Kasbeit Irbid, Al-Wastiyah, Al-Taebah, Al- Mazar ash-Shamail and Bani Obeid) Fig. 1 Map of Irbid health directorate. Footnote: The bold lines indicate the boundary of four health districts (Irbid, Kura, Al-Aghwar Shamaleh, and Bani Kenanah), and the dotted lines indicate the boundary of five districts in the Irbid health district (Kasbeit Irbid, Al-Wastiyah, Al-Taebah, Al- Mazar ash-Shamail and Bani Obeid) Zα = standard normal deviation at 95% confidence level (1.96) W = confidence interval W = confidence interval Considering the stratification effect, invalid question- naires, and prevalence of currently infecund women, the sample size was increased to 500 for both the interven- tion and control groups. Thus, we intended to recruit a total of 1000 participants for both baseline and endline surveys. Study design A diff Among the three project-target directorates, the study team selected Irbid health directorate as the study area be- cause the other two health directorates had previously re- ceived similar interventions by other programs. Irbid health directorate has a population of 1.8 million [18]; it is located 100 km north of Amman, sharing a border with Syria. Generally, the upper northern and western parts of the Irbid health directorate are rural and remote areas where people experience difficulties in accessing health fa- cilities, whereas the southern parts are urban. The project team selected one intervention VHC from each health dis- trict (Fig. 1). Regarding Irbid health district, there are five districts; the project team selected one VHC in the Taebah district and two in the northern part of the Kasbeit Irbid district because of the large area and population of the lat- ter. Among the six intervention VHCs, the study team ex- cluded one VHC in the Kasbeit Irbid because two VHCs were closely located. With respect to the control VHCs, the study team chose a VHC to match an intervention VHC from the same health district on the basis of similar demographic and economic characteristics. However, be- cause the Kura health district has only one VHC, its con- trol VHC was chosen from the Al-Wastiyah district. A difference-in-differences (DID) analysis was used to compare outcome changes overtime between the inter- vention and control groups to measure the impacts of interventions [14]. DID is one of the quasi-experimental designs that is frequently used for the estimation of causal relationships in settings where randomized con- trol trials are infeasible or unethical [13, 15, 16]. Assum- ing that the outcome of the intervention group would follow “parallel trends” with the outcome of the control group over time in the absence of intervention, DID can help estimate the causal effects of the treatment [14]. The unit of intervention in this study was VHCs, and the unit of analysis was currently married women of re- productive age (15–49 years) who lived in the catchment areas of both the intervention and nonintervention VHCs. Sampling design To determine the sample size of the catchment popula- tion in each VHC, the probability proportionate to the population size was applied, based on the household frame of the 2015 Jordan Census [18]. In each village, participating households were selected by systematic random sampling. If more than one eligible woman was available in one household, the interviewer randomly se- lected one participant. In case of absence or unavailabil- ity of eligible woman on the second visit, the household was replaced by a neighboring house. P = estimated mCU in a study area (40%) P = estimated mCU in a study area (40%) Data analysis We estimated an mCU of 40% in the intervention area at baseline on the basis of data of JPFHS 2012 [6], and the result obtained using the following formula indicated that the required minimum sample size was at least 369: First, the basic characteristics of participants were com- pared between the intervention and control groups at baseline and endline. Second, the DID analysis was con- ducted using the eight primary outcome indicators at both baseline and endline between the two groups. To obtain mCU and tCU in the impacts, the number of women in need of FP (currently fecund women) was identified. Third, the DID analysis was used to evaluate the impacts (mCU, tCU, and spousal agreement on N ¼ 4Za2 Pð1−PÞ W 2 N = minimum sample size required for systematic ran- dom sampling Komasawa et al. BMC Public Health (2019) 19:1415 Page 5 of 10 Page 5 of 10 contraception). Finally, logistic regression analysis (refer- ence was “no”) was performed to examine the associ- ation between the eight primary outcomes and the three impacts separately at endline point. P < 0.05 was consid- ered statistically significant. STATA version 15 (STATA Corporation, Texas, USA) was used for DID analysis and SPSS version 25 (IBM, Chicago, USA) for other statis- tical analyses. indicators of participation in health promotion activities showed substantial effects between baseline and endline: education session at VHCs and community health activ- ities had intervention effects of + 11.5 pp. and + 8.1 pp., re- spectively, with P < 0.001 for both. Regarding the sources of reproductive health information, counseling by VHC staff showed an upward trend, whereas counseling by pri- vate doctor/clinic and TV exhibited a downward trend in both the intervention and control groups. Considering changes in the control group as counterfeit counterfac- tuals, six of the eight primary indicators in the interven- tion group showed expected effects at significant levels. Participant characteristics In total, 510 and 509 women at baseline and 508 and 534 at endline were enrolled in the intervention and control groups, respectively. There were no significant differences in the sociodemographic characteristics be- tween the intervention and control groups at both base- line and endline (Tables 1 and 2). The number of women in need of FP (fecund women) in the interven- tion and control groups was 434 and 432 at baseline and 426 and 435 at endline, respectively. Effects of intervention on secondary outcomes y Table 4 shows the DID estimates of the secondary out- comes (impacts). mCU increased from 47.0% at baseline to 51.9% at endline in the intervention group and from 48.8% to 49.4% in the control group, resulting in + 4.3 pp. effect (P = 0.376). Similarly, the effects of tCU and spousal agreement on contraception were −0.5 pp. (P = 0.901) and + 5.1 pp. (P = 0.057), respectively. Expected ef- fects were observed after controlling, although there was no significant difference in the secondary outcomes be- tween the two groups. Effects of intervention on primary outcomes Table 3 presents the DID analysis results for primary out- comes. Regarding the use of services at VHCs, the inter- vention effects for the three indicators FP counseling, obtaining contraceptives, and general counseling increased by + 4.1 percentage points (pp.), + 7.6 pp., and + 4.9 pp., with P = 0.004, < 0.001, and 0.005, respectively. The two Association between primary and secondary outcomes Table 5 summarizes the results on the association be- tween the three secondary outcomes (mCU, tCU, and Association between primary and secondary outcomes Conversely, tCU was associated with only one primary outcome: TV (AOR 0.40, 95% CI 0.21– Table 2 Basic characteristics of participants of intervention and control groups at endline Variable Intervention group Control group P-value (N = 508) (N = 534) n (%) n (%) Age (mean [SD]) 34.5 [7.97] 34.4 [8.62] 0.79a Nationality Jordan 482 (94.9) 507 (94.9) 0.24b Syrian 25 (4.9) 22 (4.1) Others 1 (0.2) 5 (0.9) Schooling (years) No education 6 (1.2) 5 (0.9) 0.84b 1–10 139 (27.4) 159 (29.8) 11–12 210 (41.4) 215 (40.3) 13+ 152 (30.0) 155 (29.0) Age at first marriage (mean [SD]) 21.0 [4.35] 21.2 [4.46] 0.44a Number of children (mean [SD]) 3.5 [2.13] 3.3 [1.96] 0.08a Years at current residence (mean [SD]) 9.2 [7.89] 9.4 [8.05] 0.72a Monthly household incomec (mean [SD]) 391.8 [204.31] 374.8 [177.73] 0.16a (Medium) (350.0) (350.0) SD standard deviation at-test bChi-square test cJordan dinar Table 3 Effects of the intervention on primary outcomes from the DID analysis Variable Intervention group (%) Control group (%) Difference Assumptiona DID P-value Baseline Endline P-value Baseline Endline P-value e f (e – f) (N = 510) (N = 508) (N = 509) (N = 534) a b c d b −a d −c Use of VHCs’ services FP counseling 0.2 4.9 0.001 2.4 3.0 0.52 4.7 0.6 4.1 0.004 Obtaining contraceptive 1.4 8.9 < 0.001 2.2 2.1 0.91 7.5 −0.1 7.6 < 0.001 General counseling 1.4 6.3 < 0.001 4.7 4.7 0.98 4.9 0.0 4.9 0.005 Participation in health promotion activities Education sessions at VHCs 2.9 18.9 < 0.001 2.4 6.9 0.54 16.0 4.5 11.5 < 0.001 Health activities in communities 0.8 8.7 < 0.001 0.4 0.2 < 0.001 7.9 −0.2 8.1 < 0.001 Source of reproductive health information Counseling by VHC staff 5.5 14.2 < 0.001 3.1 6.2 0.02 8.7 3.1 5.6 0.012 Counseling by private doctor/clinic 42.0 35.6 0.04 43.0 34.5 0.01 −6.4 −8.5 2.1 0.639 TV 65.9 52.0 < 0.001 68.2 53.9 < 0.001 −13.9 −14.3 0.4 0.963 DID difference-in-differences, VHCs village health centers, FP family planning aCounterfactual assumption Table 2 Basic characteristics of participants of intervention and control groups at endline Variable Intervention group Control group P-value (N = 508) (N = 534) n (%) n (%) Age (mean [SD]) 34.5 [7.97] 34.4 [8.62] 0.79a Nationality Jordan 482 (94.9) 507 (94.9) 0.24b Syrian 25 (4.9) 22 (4.1) Others 1 (0.2) 5 (0.9) Schooling (years) No education 6 (1.2) 5 (0.9) 0.84b 1–10 139 (27.4) 159 (29.8) 11–12 210 (41.4) 215 (40.3) 13+ 152 (30.0) 155 (29.0) Age at first marriage (mean [SD]) 21.0 [4.35] 21.2 [4.46] 0.44a Number of children (mean [SD]) 3.5 [2.13] 3.3 [1.96] 0.08a Years at current residence (mean [SD]) 9.2 [7.89] 9.4 [8.05] 0.72a Monthly household incomec (mean [SD]) 391.8 [204.31] 374.8 [177.73] 0.16a (Medium) (350.0) (350.0) SD standard deviation at-test bChi-square test cJordan dinar Table 2 Basic characteristics of participants of intervention and control groups at endline spousal agreement on contraception) and the eight pri- mary outcomes at endline using the logistic regression analysis separately. Association between primary and secondary outcomes Table 1 Basic characteristics of participants of intervention and control groups at baseline Variable Intervention group Control group P-value (N = 510) (N = 509) n (%) n (%) Age (mean [SD]) 34.4 [7.86] 34.4 [8.18] 0.91a Nationality Jordan 483 (94.7) 490 (96.3) 0.32b Syrian 26 (5.1) 17 (3.3) Others 1 (0.2) 2 (0.4) Years of school attendance (years) No education 5 (1.2) 2 (0.9) 0.68b 1–10 141 (27.4) 135 (29.8) 11–12 219 (41.4) 225 (40.3) 13+ 145 (30.0) 146 (29.0) Age at first marriage (mean [SD]) 21.1 [4.32] 21.6 [4.80] 0.06a Number of children (mean [SD]) 3.5 [2.18] 3.4 [2.04] 0.28a Years at current residence (mean [SD]) 9.1 [7.89] 8.4 [7.70] 0.14a Monthly household incomec (mean [SD]) 380.3 [175.92] 382.2 [185.25] 0.87a (Medium) (350.0) (350.0) SD standard deviation Table 1 Basic characteristics of participants of intervention and control groups at baseline Komasawa et al. BMC Public Health (2019) 19:1415 Page 6 of 10 spousal agreement on contraception) and the eight pri- mary outcomes at endline using the logistic regression analysis separately. mCU showed positive association with five primary outcomes with a significant difference: obtaining contraceptives at VHCs [adjusted odds ratio (AOR) 3.44, 95% confidence interval (CI) 1.26–9.40], education sessions at VHC (AOR 7.41, 95% CI 1.60– 34.39), health activities in communities (AOR 7.41, 95% CI 3.28–16.78), counseling by private doctor/clinic (AOR 0.62, 95% CI 0.40–0.97), and TV (AOR 0.50, 95% CI 0.32–0.76). aCounterfactual assumption Association between primary and secondary outcomes mCU showed positive association with five primary outcomes with a significant difference: obtaining contraceptives at VHCs [adjusted odds ratio (AOR) 3.44, 95% confidence interval (CI) 1.26–9.40], education sessions at VHC (AOR 7.41, 95% CI 1.60– 34.39), health activities in communities (AOR 7.41, 95% CI 3.28–16.78), counseling by private doctor/clinic (AOR 0.62, 95% CI 0.40–0.97), and TV (AOR 0.50, 95% CI 0.32–0.76). Conversely, tCU was associated with only one primary outcome: TV (AOR 0.40, 95% CI 0.21– Table 3 Effects of the intervention on primary outcomes from the DID analysis Variable Intervention group (%) Control group (%) Difference Assumptiona DID P-value Baseline Endline P-value Baseline Endline P-value e f (e – f) (N = 510) (N = 508) (N = 509) (N = 534) a b c d b −a d −c Use of VHCs’ services FP counseling 0.2 4.9 0.001 2.4 3.0 0.52 4.7 0.6 4.1 0.004 Obtaining contraceptive 1.4 8.9 < 0.001 2.2 2.1 0.91 7.5 −0.1 7.6 < 0.001 General counseling 1.4 6.3 < 0.001 4.7 4.7 0.98 4.9 0.0 4.9 0.005 Participation in health promotion activities Education sessions at VHCs 2.9 18.9 < 0.001 2.4 6.9 0.54 16.0 4.5 11.5 < 0.001 Health activities in communities 0.8 8.7 < 0.001 0.4 0.2 < 0.001 7.9 −0.2 8.1 < 0.001 Source of reproductive health information Counseling by VHC staff 5.5 14.2 < 0.001 3.1 6.2 0.02 8.7 3.1 5.6 0.012 Counseling by private doctor/clinic 42.0 35.6 0.04 43.0 34.5 0.01 −6.4 −8.5 2.1 0.639 TV 65.9 52.0 < 0.001 68.2 53.9 < 0.001 −13.9 −14.3 0.4 0.963 DID difference-in-differences, VHCs village health centers, FP family planning aCounterfactual assumption Komasawa et al. BMC Public Health (2019) 19:1415 Page 7 of 10 Table 4 Effects of the intervention on secondary outcomes from the DID analysis Variable Intervention group (%) Control group (%) Difference Assumptiona DID P-value Baseline Endline P-value Baseline Endline P-value e f (e - f) (N = 434) (N = 426) (N = 432) (N = 435) a b c d b −a d −c Modern contraceptive use 47.0 51.9 0.15 48.8 49.4 0.86 4.9 0.6 4.3 0.376 Traditional contraceptive use 23.7 25.4 0.58 22.2 24.4 0.46 1.7 2.2 −0.5 0.901 Spousal agreement on contraception 86.5 92.5 0.001 88.6 89.5 0.07 6.0 0.9 5.1 0.057 DID difference-in-differences aCounterfactual assumption 0.75). Association between primary and secondary outcomes Spousal agreement on contraception was influenced by six primary outcomes: FP counseling at VHC (AOR 12.01, 95% CI 1.49–96.57), obtaining contraceptives at VHC (AOR 3.28, 95% CI 1.35–8.00), education sessions at VHC (AOR 6.72, 95% CI 2.28–19.84), health activities in communities (AOR 6.22, 95% CI 3.45–11.21), counseling by private doctor/clinic (AOR 0.71, 95% CI 0.53–0.95), and TV (AOR 0.48, 95% CI 0.36–0.65). The results obtained for the primary outcomes showed that the project interventions of expanding FP services at VHCs promoted the utilization of FP services at VHCs. This result may be explained by two reasons. First, the project began to provide free FP services (i.e. counseling and providing pills and male condoms) at VHCs, which were easily accessible for local women whose primary transportation means was walking: 85.1% of all women who used VHCs in the previous year walked to the facility according to the information col- lected in the endline survey. Second, the quality of ser- vices at VHCs may have visibly improved (e.g. better attitude of trained health personnel and availability of standardized counseling using updated IEC materials and other tools for FP counseling in VHCs). This was supported by the fact that 47.9% of study participants who used VHCs in the past year recognized improve- ment in the services within 1 year at endline: 82.7% par- ticipants reported improvements regarding increased variety of services, 35.1% reported improvement in hard- ware setting, 34.6% reported improvement in IEC mate- rials, and 51.4% reported a better attitude of nurses or Discussion The community-based inter- vention in this project was to start establishing commu- nity health committees comprising local leaders, both male and female, in each community; subsequently, ap- propriate FP messages would be delivered by health pro- fessionals and local networks to male decision makers in communities or at home. This is in line with a previous study in the Islamic world [24], which illustrated that a community-based approach is effective in addressing the social barriers and lack of access to information con- cerning FP and in increasing the adoption of modern contraception. Contrarily, tCU showed a weak associ- ation with the primary outcomes, which might be be- cause the project did not involve direct intervention of tCU. These findings imply that the positive impacts are attributable to facility- and community-based interven- tions, in line with the common findings of previous studies [8, 14, 25, 26]. midwives. Moreover, participation in health promotion activities substantially increased. Although some local women from the control areas may have attended the health promotion activities in intervention areas, it did not result in overvaluation of the project effect. These primary outcomes may induce impacts regarding FP be- havior and perception changes. p p g There have been changes in the trends in sources of reproductive health information: an increase in counsel- ing by VHC staff, whereas a decrease in counseling by private doctor/clinic and FP information through TV. These results indicate that the project interventions led to a shift from gaining information through private facil- ities or mass media to face-to-face counseling by local VHCs. This tendency was inconsistent with the findings of previous studies. First, a Jordanian study reported that Jordanian women in urban areas relied more on private doctor/clinic for using modern contraceptive services [11]. Second, it is believed that mass media has been ef- fectively delivering FP messages for several decades worldwide [8, 19]; however, the effect is observed only when mass media is used through programmatic ap- proaches, such as creating entertainment (e.g. soap opera), combining with social marketing, or developing interpersonal communication mechanisms [13]. Our re- sults indicated that in rural Jordan, free, interpersonal counseling at walking distance was more appreciated than that provided in private facilities or provided through mass media as a source of FP information. The expected impacts of the project were contracep- tive behavior changes and acceptance. Discussion To the best of our knowledge, this is the first study to evaluate the impacts of the project on FP behavior changes in Jordan using a rigorous method. The DID analysis revealed that the project interventions increased the utilization of FP services at VHCs and participation in health promotion activities. The estimated effects also indicated an increase in modern contraceptive use and spousal acceptance of contraception. The association be- tween the primary indicators and secondary indicators (impacts) shows that integrating the facility- and community-based approaches may contribute to the emergence of these impacts. 5 Factors associated with three secondary outcomes (impacts) by multivariate logistic regression analysis d d l l Table 5 Factors associated with three secondary outcomes (impacts) by multivariate logistic regression analysis Variable Modern contraceptive use Traditional contraceptive use Spousal agreement on contraception (N = 427) Adjusted odds ratio (95% CI) (N = 212) Adjusted odds ratio (95% CI) (N = 911) Adjusted odds ratio (95% CI) Use of VHCs’ services FP counseling 5.01 (0.55–45.82) – 12.01 (1.49–96.57)* Obtaining contraceptives 3.44 (1.26–9.40)* 2.33 (0.15–37.38) 3.28 (1.35–8.00) General counseling 2.26 (0.54–9.45) 1.99 (0.45–8.69) 2.43 (0.98–6.07) Participation in health promotion activities Education sessions at VHC 7.41 (1.60–34.39) – 6.72 (2.28–19.84) Health activities in communities 7.41 (3.28–16.78)* – 6.22 (3.45–11.21)*** Source of reproductive health information Counseling by VHC staff 1.25 (0.53–2.94) 1.70 (0.53–5.48) 1.45 (0.83–2.55) Counseling by private doctor/clinic 0.62 (0.40–0.97)* 0.53 (0.28–1.00) 0.71 (0.53–0.95)* TV 0.50 (0.32–0.76)** 0.40 (0.21–0.75)* 0.48 (0.36–0.65)*** CI confidence interval, VHCs village health centers, FP family planning; −, not presented due to the small numbers of prevalence at base line time P < 0.05, P < 0.01, **P < 0.001 with three secondary outcomes (impacts) by multivariate logistic regression analysis Komasawa et al. BMC Public Health (2019) 19:1415 Page 8 of 10 Page 8 of 10 association between the impacts and primary outcomes. mCU was positively associated with obtaining contracep- tives at VHCs and two kinds of health promotion activ- ities and negatively associated with counseling by private doctor/clinic and TV. The spousal agreement on contra- ception showed similar trends, with additional positive association with FP counseling at VHCs. These results suggest that in a facility-based intervention, free contra- ceptives at easily accessible delivery points, with suffi- cient information through qualified interpersonal counseling, could encourage rural wives to communicate with their husbands and easily convince them for a broad consensus [13, 21]. Discussion The DID esti- mates suggested that the project may have increased mCU (+ 4.3 pp.) and decreased tCU (−0.5 pp.) within the 13 months of its implementation; however, these im- pacts were not significantly different between the inter- vention and control groups. One possible reason behind the moderate increase in mCU was that VHCs did not provide IUD services, which was the most popular mod- ern method (used by 21.3% of all married women aged 15–49 years) in Jordan [6], due to the lack of equipment and skilled personnel. From a different viewpoint, Brown et al. [20] estimated that the annual modern contracep- tive prevalence growth rate was 2.8% for high growth levels between 2016 and 2020 worldwide. In comparison with this estimated rate, the growth rate in our interven- tion group in 13 months was 4.9%, whereas this rate in the control group was 0.6%. The estimated effect of spousal agreement on contraception was also positive, indicating that the project interventions may have raised the husbands’ awareness regarding the importance of FP and encouraged couples’ attitude and practice toward the use of modern methods [21–23]. Generally, to produce behavioral outcomes of FP, five es- sential elements are required: (i) adequate supply of safe and effective methods, (ii) quality of care, (iii) conscientious rights of both service providers and users, (iv) community engage- ment in introducing the FP concept, and (v) commitments to laws and policies [13, 27, 28]. The facility-based approach in this project covered the elements from (i) to (iii) and the community-based approach coped with (iv). In addition, MOH as a co-implementer committed to integrate the pro- ject approaches into the new governmental strategy [29]. Roudi-Fahimi F et al. [27] also insisted that a combination of supply-side and demand-side interventions was necessary to meet the FP demands in the Arab countries. The project in the present study considered these aspects and was designed to obtain synergistic effects of integrating the facility- and community-based approaches by strengthening VHCs. The integration of two approaches may contribute to produce visible outcomes within a relatively short duration of imple- mentation. However, the extent of each element’s contribu- tion, such as providing contraceptives alone, quality counseling, women’s convenience to visit VHCs, local leaders’ commitment, and local leaders’ influence on the hus- bands’ attitude, toward the impacts and degree of synergy effects among elements was not measured. AOR: Adjusted odds ratio; CHCs: Comprehensive health centers; CI: Confidence interval; DID: Difference-in-differences; FP: Family planning; IEC: Information, education and communication; IUD: Intrauterine device; JICA: Japan International Cooperation Agency; JPFHS: Jordan Population and Family Health Survey; mCU: percentage of modern contraceptive use; MOH: Ministry of Health; PHCs: Primary health centers; pp.: Percent point; Funding Japan International Cooperation Agency funded the data collection. The funder had no role in study design, data collection, preparation of the manuscript, and publication. Discussion To assess the effectiveness of integrating the facility- and community-based approaches, we investigated the Page 9 of 10 Komasawa et al. BMC Public Health (2019) 19:1415 Page 9 of 10 Page 9 of 10 Komasawa et al. BMC Public Health (2019) 19:1415 There are some limitations to our study. First, the main limitation of this study is that we used the quasi- experimental design instead of the randomized con- trolled design. The DID analysis does not perfectly elim- inate unobserved biases, which means that the estimated results may have been biased or invalid [13]. Despite this limitation, we believe that the DID analysis was best suited to measure the impact of government-led projects because performing evaluation of projects of this nature in a purely experimental manner is difficult due to both ethnical and practical issues [13, 14]. Second, seasonally mobile people were observed in the study areas between summer (July–September) and winter (November–Feb- ruary). Moreover, during the endline survey in January, several women were away from home during daytime because they were harvesting olives as their part-time job, which may have caused selection bias. However, we believe that the selection bias may not have affected the comparison much because such women were present in both the intervention and control populations. Third, our study did not include the factors related to the knowledge of contraceptives because the previous stud- ies have reported that the knowledge of contraceptives was universal and not a barrier to practice FP in Jordan [6, 17]. However, sufficient knowledge of modern methods, including fears of adverse health risks, side ef- fects, and future childbearing, called for increasing atten- tion as crucial factors related to the nonuse of modern methods [26, 30]. Therefore, further studies are neces- sary to identify the extent of knowledge of modern methods and how to shift from tCU to mCU. Fourth, it is important to assess the project sustainability after the intervention is completed. This study did not examine whether the impact will sustain after the end of the pro- ject and did not predict the future impacts. Thus, further studies are required to address these limitations. tCU: Percentage of traditional contraceptive use; USAID: United States Agency for International Development; VHC: Village health center tCU: Percentage of traditional contraceptive use; USAID: United States Agency for International Development; VHC: Village health center Ethics approval and consent to participate Ethical approvals were obtained from the Ethics Committee of the Faculty of Medicine, Juntendo University, Japan (reference no. 2015104, dated 20 January 2016) and the Jordan Department of Statistics (reference no. 3058/4/ 2/6, dated 7 September 2016). All women participating in the study were informed about the purpose and expected benefit of the study. Participants were also informed about their right not to participate or stop the interview at any time. All study participants gave written informed consent to participate in the study. For married adolescents aged 15–17 years, verbal consents were also obtained from their husbands, parents-in-law, or other legal guardians dur- ing initial contact with the participants. Confidentiality of the study participants was maintained via the use of identification numbers rather than names. Acknowledgments The authors would like to extend their appreciation to the Ministry of Health of Jordan and the Japan International Cooperation Agency for their entire supports. The authors also gratefully acknowledge the Department of Statistics of Jordan and the Center of Strategic Studies, Jordan University, who assisted in the conducting the field surveys. The authors particularly thank the women who participated in this study. Consent for publication Consent for publication Not applicable. Consent for publication Not applicable. Availability of data and materials The data that support the findings of this study are available from the Japan International Cooperation Agency, but restrictions apply to the availability of these data, which were used under license for the current study. However, data are available from the authors upon reasonable request and with permission from Japan International Cooperation Agency. Competing interests The authors declare that they have no competing interests. Competing interests The authors declare that they have no competing interests. Authors’ contributions MK h l MK wrote the proposal, analyzed the data, and drafted the manuscript. MK, MY, MS, and MA conceptualized the methodological design of the manuscript. MK, YK, and MA were responsible for field work, data collection, and analysis. MK wrote the proposal, analyzed the data, and drafted the manuscript. MK, MY, MS, and MA conceptualized the methodological design of the manuscript. MK, YK, and MA were responsible for field work, data collection, and analysis. MY, YS, and MS contributed in data analysis and manuscript preparation. All authors read and approved the final manuscript. MY, YS, and MS contributed in data analysis and manuscript preparation. All authors read and approved the final manuscript. authors read and approved the final manuscript. Author details 1 1Department of Public Health, Faculty of Medicine, Juntendo University, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan. 2Faculty of International Liberal Arts, Juntendo University, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan. 3School of Tropical Medicine and Global Health, Nagasaki University, 1-12-4 Sakamoto, Nagasaki-city 852-8102, Japan. 4Atelier 514, 5-1-18 Kinuta, Setagaya-ku, Tokyo 157-0073, Japan. 5Directorate of Woman and Child Health, Ministry of Health, Prince Hamzah Bin Al Hussein Street, P.O. Box 940370, Amman, Jordan. Authors’ information MK: Department of Public Health, Faculty of Medicine, Juntendo University, Tokyo, Japan, Research fellow, MUP. MK: Department of Public Health, Faculty of Medicine, Juntendo University, Tokyo, Japan, Research fellow, MUP. MY: Department of Public Health, Faculty of Medicine, Juntendo University, Tokyo, Japan, Professor, M.D., Ph.D. MY: Department of Public Health, Faculty of Medicine, Juntendo University, Tokyo, Japan, Professor, M.D., Ph.D. YS: Faculty of International Liberal Arts, Juntendo University, Tokyo, Japan, Assistant Professor, Ph.D. YS: Faculty of International Liberal Arts, Juntendo University, Tokyo, Japan, Assistant Professor, Ph.D. MS: School of Tropical Medicine and Global Health, Nagasaki University, Nagasaki, Japan, Assistant Professor, MA, MPH, Ph.D. MS: School of Tropical Medicine and Global Health, Nagasaki University, Nagasaki, Japan, Assistant Professor, MA, MPH, Ph.D. YK: Independent researcher, Tokyo, Japan, BSc. MA: Directorate of Woman and Child Health, Ministry of Health, Jordan, Director of Woman and Child Health Directorate, M.D. MA: Directorate of Woman and Child Health, Ministry of Health, Jordan, Director of Woman and Child Health Directorate, M.D. Conclusions The VHC project exerted the expected effects on the use of FP service at VHCs and participation in health promotion activities as well as the impacts on increasing mCU and hus- bands’ perception of contraception. The findings also indi- cated that the integration of the two approaches, facility- and community-based approaches, may have resulted in these impacts in the rural settings. This study will provide valuable insights for designing future FP projects not only in Jordan but also in rural areas of other countries that are still in the transitional phase from traditional to modern contraception. References 1. Higher Population Council (HPC). National Reproductive Health /Family Planning Strategy 2013–2017. 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https://openalex.org/W4210489056	https://www.frontiersin.org/articles/10.3389/frai.2022.806041/pdf	English	null	Artificial Interactionism: Avoiding Isolating Perception From Cognition in AI	Frontiers in artificial intelligence	2,022	cc-by	14,082	Edited by: Kristinn R. Thórisson, Reykjavík University, Iceland Edited by: Kristinn R. Thórisson, Reykjavík University, Iceland Reviewed by: Riccardo De Benedictis, National Research Council (CNR), Italy Antonio Chella, University of Palermo, Italy Correspondence: Mathieu Guillermin mguillermin@univ-catholyon.fr Reviewed by: Riccardo De Benedictis, National Research Council (CNR), Italy Antonio Chella, University of Palermo, Italy Correspondence: Mathieu Guillermin mguillermin@univ-catholyon.fr Artiﬁcial Interactionism: Avoiding Isolating Perception From Cognition in AI Mathieu Guillermin 1* and Olivier Georgeon 1,2 1 Sciences and Humanities Conﬂuence Research Center (EA 1598), Lyon Catholic University, Lyon, France, 2 UMR5205 Laboratoire d’Informatique en Image et Systèmes d’Information (LIRIS), Villeurbanne, France Mathieu Guillermin 1* and Olivier Georgeon 1,2 1 Sciences and Humanities Conﬂuence Research Center (EA 1598), Lyon Catholic University, Lyon, France, 2 UMR5205 Laboratoire d’Informatique en Image et Systèmes d’Information (LIRIS), Villeurbanne, France Mathieu Guillermin 1* and Olivier Georgeon 1,2 We discuss the inﬂuence upon the ﬁelds of robotics and AI of the manner one conceives the relationships between artiﬁcial agents’ perception, cognition, and action. We shed some light upon a widespread paradigm we call the isolated perception paradigm that addresses perception as isolated from cognition and action. By mobilizing the resources of philosophy (phenomenology and epistemology) and cognitive sciences, and by drawing on recent approaches in AI, we explore what it could mean for robotics and AI to take distance from the isolated perception paradigm. We argue that such a renouncement opens interesting ways to explore the possibilities for designing artiﬁcial agents with intrinsic motivations and constitutive autonomy. We then propose Artiﬁcial Interactionism, our approach that escapes the isolated perception paradigm by drawing on the inversion of the interaction cycle. When the interaction cycle is inverted, input data are not percepts directly received from the environment, but outcomes of control loops. Perception is not received from sensors in isolation from cognition but is actively constructed by the cognitive architecture through interaction. We give an example implementation of artiﬁcial interactionism that demonstrates basic intrinsically motivated learning behavior in a dynamic simulated environment. ORIGINAL RESEARCH published: 02 February 2022 doi: 10.3389/frai.2022.806041 INTRODUCTION Specialty section: This article was submitted to AI for Human Learning and Behavior Change, a section of the journal Frontiers in Artiﬁcial Intelligence Received: 31 October 2021 Accepted: 11 January 2022 Published: 02 February 2022 Citation: Guillermin M and Georgeon O (2022) Artiﬁcial Interactionism: Avoiding Isolating Perception From Cognition in Specialty section: This article was submitted to AI for Human Learning and Behavior Change, Specialty section: This article was submitted to AI for Human Learning and Behavior Change, a section of the journal Frontiers in Artiﬁcial Intelligence Received: 31 October 2021 Accepted: 11 January 2022 Published: 02 February 2022 From a superﬁcial phenomenological point of view, the subjective experience we have as humans tells us that perception, cognition, and action are three separate realms. Perceiving amounts to merely receiving information about the state of the world. Cognition, then, is about processing this newly acquired information in light of already possessed information, possibly to select an action suited to the current state of the world and to given goals. The appeal of this commonsensical view about perception as isolated and independent from cognition and possible consecutive actions may not come as a surprise. It relates to what Husserl calls the “natural attitude” of day-to-day life, according to which we do not question the existence of objects presented to us in perceptual experience (Husserl, 1982). This natural attitude is irreducible and drives our attention away from any consideration of processes possibly at play in the constitution of experience. Basic biological knowledge reinforces this naïve commonsense picture: sensory organs collect information about the world (perception), that is transmitted to the brain, which processes it (cognition) and drives motor organs (action). Received: 31 October 2021 Accepted: 11 January 2022 Published: 02 February 2022 Keywords: cognitive architecture, intrinsic motivation, constructivism, active perception, constitutive autonomy Citation: Guillermin M and Georgeon O (2022) Artiﬁcial Interactionism: Avoiding Isolating Perception From Cognition in AI. Front. Artif. Intell. 5:806041. doi: 10.3389/frai.2022.806041 February 2022 \| Volume 5 \| Article 806041 Frontiers in Artiﬁcial Intelligence \| www.frontiersin.org 1 Artiﬁcial Interactionism Guillermin and Georgeon been widely discussed and criticized for instance in philosophy and epistemology as well as from within cognitive sciences themselves. A few approaches in robotics and AI explicitly develop alternatives to this isolated perception paradigm. We will review them and describe our original proposition. This commonsense model of perception constitutes the ground for an extremely widespread analogy when describing the functioning of a robot or an AI program. It is very common to take a robot’s input data coming from its sensors as its perception. The processing of input percepts then stands for the robot’s cognition, possibly leading to setting output data traditionally labeled action. Output data drive the robot’s actuators. In resonance with the naïve picture sketched just above, sensors stand for the robot’s sensory organs, computation for its cognition, and actuators for its motor organs. Echoing our natural tendency to project our intentional life in artifacts we build (Searle, 1980, p. 419), it then becomes tempting to claim that the robot perceives, thinks, and acts as humans do. Through this paper, we intend to contribute to the study of what it could mean for AI and robotics to take distance with the isolated perception paradigm. We argue that abandoning this paradigm may lead to original ways of designing artiﬁcial agents. We thus propose Artiﬁcial Interactionism as a possible framework that rejects the paradigm of isolated perception. Before detailing this original framework, we ﬁrst ground our discussion of the possibility to take distance with isolated perception in the broader scope philosophy (phenomenology and epistemology), which provides interesting insights about the relationships between perception, cognition, and action. We then focus upon the manner these relationships are discussed in cognitive sciences and AI, which leads us to bring to the fore the question of extrinsic vs. intrinsic motivations. We ﬁnally expose underlying principles of Artiﬁcial Interactionism together with the ﬁrst implementations and experiments. This naïve view about perception and cognition does not only inﬂuence the general public, feeding fantasies about AI nature and future developments (according to which we would be on the verge of the emergence of strong AI, artiﬁcial systems that would reach and exceed the human level). Citation: It also inﬂuences researchers and engineers and pervades the ﬁelds of computer science, robotics, and AI. One of the most inﬂuential textbooks in AI deﬁnes the latter as the “study of agents that receive percepts from the environment and perform actions. Each such agent implements a function that maps percept sequences to actions (...)” (Russell et al., 2010, p. vii). Russell and Norvig go on: Philosophical Insights Upon Perception Among typical issues philosophical studies strive to address, one can mention the challenges of describing what are the contents of perception, of explaining perceptual intentionality—the fact that we have experiences about (what we hold to be) objects of the world and their properties—or of elucidating the diﬀerence between veridical and non-veridical perceptual experiences. We use the term percept to refer to the agent’s perceptual inputs at any given instant. (...) By specifying the agent’s choice of action for every possible percept sequence, we have said more or less everything there is to say about the agent” (Russell et al., 2010, p. 34–35). The ﬁrst philosophical stream deserving mentioning is Empiricism (Lock, Berkeley, Hume, Reid ...). Empiricists describe perception as the experience of “eﬀects in one produced by things in the world,” namely sensations or sense-data (Sarkar and Pfeifer, 2006, p. 545). The perceiver is directly aware of sensations or sense data. A strength of this empiricist approach is to provide a way of diﬀerentiating (conceptually and in an externalist fashion) veridical and non-veridical experiences. Veridical experiences are experiences of sensations caused by the right objects in the world while non-veridical ones are not. At ﬁrst sight, empiricists seem to present perceivers as passively experiencing sense data, thus resonating with the isolated perception paradigm. However, things may be less straightforward. To the extent that eﬀects that things in the world cause in perceivers can mobilize their cognitive processes, such approaches may well-depart from isolated perception. According to this explanation, the algorithm deﬁning actions an agent will perform in response to every possible sequence of perceptual inputs completely describes this agent. This leaves little room for escaping the idea that perception, cognition, and action constitute three separate realms. The agent’s cognition lies in the process of selecting actions in response to speciﬁc perceptual inputs. Thereby, perceptions and actions are what they are, independent of cognition so deﬁned1. In the rest of this text, we call this conception of perception as radically separated from cognition and action the isolated perception paradigm. Despite its pervasiveness in computer science, robotics, and AI, the isolated perception paradigm has 1The quotation from Russell et al. (2010) we reproduced just above comes with a ﬁgure that is particularly representative of this conception (therein Figure 2.1, p. PERCEPTION, COGNITION, AND ACTION IN PHILOSOPHY An agent is anything that can be viewed as perceiving its environment through sensors and acting upon that environment through actuators. (...) A human agent has eyes, ears, and other organs for sensors and hands, legs, vocal tract, and so on for actuators. A robotic agent might have cameras and infrared range ﬁnders for sensors and various motors for actuators. A software agent receives keystrokes, ﬁle contents, and network packets as sensory inputs and acts on the environment by displaying on the screen, writing ﬁles, and sending network packets. The topic of perception is widely discussed in philosophy, not only through eﬀorts to explain and account for perceptual experience, but also with the study of the role of perception in knowledge acquisition. Let’s ﬁrst focus on interesting insights from philosophical accounts of perception. 1The quotation from Russell et al. (2010) we reproduced just above comes with a ﬁgure that is particularly representative of this conception (therein Figure 2.1, p. 35): it includes the term percepts to denote a component impacting sensors (and thus pre-existing the sensors and any form of cognition on the agent’s behalf). Frontiers in Artiﬁcial Intelligence \| www.frontiersin.org Philosophical Insights Upon Perception 35): it includes the term percepts to denote a component impacting sensors (and thus pre-existing the sensors and any form of cognition on the agent’s behalf). This being said, many thinkers consider that empiricism fails at providing a satisfying account of perception as it does not February 2022 \| Volume 5 \| Article 806041 Frontiers in Artiﬁcial Intelligence \| www.frontiersin.org 2 Artiﬁcial Interactionism Guillermin and Georgeon explain how experiences of sensations (mental or bodily events) can present the world as being a certain way, can provide access to objects of the world and their properties (access to knowledge of the world and its objects). To the extent that perception is subjective contents, experiences of eﬀects internal to perceiver that are caused by interactions with objects of the world, why should we consider that certain sensations present properties of things of the world (like the redness of a fruit) while other do not (like pain when we hit our foot against a stone)? A way of phrasing this diﬃculty is to say that sensations or sense-data alone are not suﬃcient to account for the “intentional content of an experience—how it presents things as being” though this intentional content “is given immediately with the experience” and is core to perceptual consciousness (Sarkar and Pfeifer, 2006 p. 546–547). A possible answer (that is in fact as much an elimination of the problem) is phenomenalism, according to which “objects and states of aﬀairs are themselves just patterns of organization in subjective states of consciousness” (Sarkar and Pfeifer, 2006). “Physical objects are reducible to subjective sensory experiences, or sense-data” (Blaauw and Pritchard, 2005, p. 114). Thereby, perceptual access to, and knowledge of, physical objects are unproblematic. They are not external objects after all. (p. 193–194). Kant even describes acts of synthesis of recognition under concepts as judgments of apperceptions (p. 236). In sum, Kant grants to the mind and its cognitive processes an irreducible active role in perceptual experience. This does not necessarily mean that perception is entirely conceptualized. It is interesting to make the distinction between the intentional content of an experience as it presents the world being a certain way and the phenomenal character of experience, how it feels to have this experience (Blaauw and Pritchard, 2005, p. 52). Philosophical Insights Upon Perception While it is clear that intentional contents require cognition (acts of synthesis), one can appeal to non-conceptual contents such as qualia to approach the phenomenal dimension. pp p In a similar vein, Husserl also claims that sensations are not suﬃcient to account for perceptual experience (Husserl, 1982). In fact, an experience, in its intentional dimension (an experience of a given object), can be veridical or not, even though relying on the same sensations. Husserl’s phenomenological analysis put the focus on the natural attitude we mentioned earlier. Through this natural attitude, perceivers take the experience of the presence of objects for granted independently of the veridical or non-veridical nature of these experiences. This natural commitment to the existence of perceived objects is an irreducible component of perception. Husserl thus argues that phenomenology of perception must study intentional contents of perceptual experiences independently of any assumption about the existence of perceived objects. Such assumptions must be bracketed (Husserl’s famous epoché). The fact that an object really exists in the world or not when a perceiver has a perceptual experience of this object shall not enter into the phenomenological analysis of perception. Husserl calls noema this content of perceptual experience phenomenology shall study. As with Kant, Husserl’s noema includes, but does not reduce to, non-intentional and non-conceptual sense impressions (hýle). Husserl indicates that perceiving something as a given object requires the background of an intentional horizon composed of the many expectations one has about this object in terms of other possible (future) experiences. Again, the intentional content of perceptual experience irreducibly mobilizes cognition. Although with less intellectual coloration, similar connections between perception and cognition are brought to the fore by Heidegger and Merleau-Ponty (Dreyfus, 2007). Notably, Merleau-Ponty describes a “feedback loop between the embodied coper and the perceptual world” he calls “the intentional arc.” For him, what we learn in our past and what we see possible in our future is “sedimented” in how things look to us (Dreyfus, 2007, p. 1144– 1145). Husserl and Merleau-Ponty, like Kant, do not rely on the isolated perception paradigm. The intentional horizon or the intentional arc at play in perceptual experience clearly involves the knowledge and cognition of perceivers. j p y j This discussion directly leads us to the work of Kant, whose account of perception clearly opposes the isolated perception paradigm. Frontiers in Artiﬁcial Intelligence \| www.frontiersin.org Perception and Epistemology p p gy Let’s now turn to epistemological analyses of the role perception plays in knowledge acquisition and the insights they can provide to discuss the paradigm of isolated perception. In this respect, we can start from the “objectivist conception of science” (Baghramian, 2014) that promotes a universal and neutral scientiﬁc method permitting us to reliably infer knowledge about reality from empirical evidence and tools of logic. This conception relies on the famous dichotomy between analytic and synthetic statements, between statements “justiﬁable by a priori reasoning” and statements “justiﬁable only a posteriori” on the ground of empirical observation—“tertium non datur” (Uebel, 2014, p. 90–91). Arguably, the isolated perception paradigm is at play here. We reach analytic statements through cognition, with results warranted by the application of the laws of reasoning. By contrast, synthetic statements get their epistemic strength from their ground in neutral observation one can tie to isolated perception. Perceptions are reliable because they provide raw information about the world, information that is independent of what observers think or know. With his commonsense realism, Putnam also defends the possibility of genuine cognitive access to mind-independent reality despite the acknowledgment of the irreducible role of concepts and cognition (Putnam, 1999). Drawing on the work of American classical pragmatists such as James or Dewey, Putnam deploys an Aristotelian understanding of concepts (Aristotle’s direct realism) to which he subtracts essentialism: “when I think [or apperceive] that something is that way, and when the thing is that way, the ‘way’ in question is one and the same” (Putnam, 2002, p. 106). In sum, a correct conception is direct cognitive contact with mind-independent reality and allows direct cognitive contact with it in perception under the form of apperception. Apperceiving something involves this something. Apperceiving is a cognitive ability that functions “‘with long arms,’ arms that reach out to the environment” (Putnam, 2012, p. 352). We could reformulate this by saying that Putnam put upside down the discussion of issues induced by abandoning the isolated perception paradigm. There is no question about the way we could perceive and know mind-independent reality despite the involvement of cognition in perception. Perception and knowledge of mind-independent reality are possible thanks to (correct) cognition. Insights about the limits of the isolated perception paradigm we collected from philosophical accounts of perception are highly signiﬁcant with respect to the validity of this objectivist epistemological conception. Philosophical Insights Upon Perception Thus, notwithstanding their veridical or non-veridical nature, “some experiences are about things in the world external to the mind in virtue of representing possible interaction between the subject and the thing represented” (Siegel, 2021, Section 5.4). Notably, we can take diﬀerent perspectives on mind-independent objects, which are then expected to take diﬀerent looks. As we already evoked, such expectations about mind-independent objects enter what Husserl calls the intentional horizon in a quite conceptual approach. In slight contrast, Merleau-Ponty adopts a more action-oriented position, with mind-independence being connected to “‘readiness’ on the part of the subject to move her body relative to an object to get a better view, if she so wished” (Siegel, 2021). In addition, one could as well-mention indexical contents of perceptual experiences, “contents that must be speciﬁed by the use of indexical expressions, such as ‘over there’, ‘to the left’, ‘here’, ‘in front of/behind me’, ‘just a second ago’, ‘since a few second ago’, and so on” (Siegel, 2021, Section 3.4). These indexical contents seem necessary for experience to present spatially and temporally located events. To the extent they appeal to self-consciousness and situation awareness, these indexical contents would involve cognition and their irreducibility in perceptual experiences would mean opposition to the isolated perception paradigm. the occasion of scientiﬁc revolutions) can trigger changes in perceptual experience (at least in its intentional dimension; see Bird, 2000, p. 102–104). Through this view he calls the world- change thesis (Kuhn, 1996, chapter 10), Kuhn radically opposes the objectivist conception of science: experimental facts collected through perception cannot be considered as neutral grounds for (scientiﬁc) knowledge acquisition. g q As introduced with Kant’s transcendental idealism, the irreducible role of concepts and cognition in perception leads many authors to claim mind-independent reality either is unknowable or does not even exist. We can only know objects as they appear to us (phenomenal objects) depending on our concepts and cognitive processes. However, such conclusions are not inescapable. For instance, the theory-ladenness of empirical observation does not necessarily mean that empirical evidence cannot contribute to theory choice (Brewer and Lambert, 2001). In this respect, it may be fruitful to distinguish between two types of neutrality that one could require from observational statements (Sober, 2014). Philosophical Insights Upon Perception With his famous distinction between things in themselves we never perceive and phenomenal objects, Kant could, at ﬁrst sight, be seen as defending a form of phenomenalism. But, this would miss most of Kant’s transcendental idealism and its account of perceptual experience. Although Kant argues that we can only perceive the phenomenal world (the world as it appears to us), he nonetheless indicates that we experience objects and states of aﬀairs and that sensations alone are not enough to account for this intentional dimension of perceptual experience (Kant, 1998). According to Kant’s description, perceptual experience involves three faculties of the mind allowing acts of synthesis. An act of synthesis is an “act of putting diﬀerent representations together with each other and comprehending their manifoldness in one cognition” (p. 210). With the faculty of sensibility, the synthesis of apprehension organizes disparate perceptual inputs (intuitions) along with the a priori forms of space and time. We are never aware of intuitions independently of their organizations within these a priori forms of sensitivity. The faculty of understanding then allows the synthesis of recognition in a concept (with at minimum the Categories of the intellect such as the concepts of number, quality, and modality, together with the concept of the object that is recognized). The faculty of imagination permits the synthesis of reproduction for the mind to make the connection between intuitions and concepts. This Kantian account of perceptual experience also implies that only phenomenal objects are perceived while things in themselves remain irremediably out of cognitive reach. We only perceive objects resulting from the acts of synthesis of the mind. We will return to this epistemological question below. What is important for now is that, for Kant, perception is irremediably conceptualized and entangled with cognition. “Thoughts without content are empty, intuitions without concepts are blind” Remaining on the side of the phenomenological analysis of perception, one can evoke few other elements at odd with the isolated perception paradigm. First, Husserl and Merleau-Ponty can be understood as arguing that cognition is at play when we perceive ourselves as evolving in mind-independent reality. As long as we remain in the realm of phenomenology and Husserl’s epoché, whether we really are in contact with a mind- independent reality is irrelevant (it is rather an epistemological February 2022 \| Volume 5 \| Article 806041 3 Artiﬁcial Interactionism Guillermin and Georgeon question). Philosophical Insights Upon Perception One can (1) demand a set of observation statements that is independent of any theory and can be employed to test any theory (absolute theory neutrality) or, more modestly, (2) impose mobilizing observation statements that are independent of the theory to be tested (relative theory neutrality). With this second version, cognitive access to mind-independent reality may remain possible. Moreover, one can wonder whether the world-change thesis applies to any possible notion of world or whether one may be entitled to distinguish between scientiﬁc worlds that change with paradigm shifts and an empirical ordinary world that is left untouched during such modiﬁcations (Ghins, 2003). But, even in such a more optimistic epistemological framework, the detachment from the isolated perception paradigm remains. It is not the role of concepts and cognition that is at stake. It is rather the degree of stability of diﬀerent sets of concepts. Frontiers in Artiﬁcial Intelligence \| www.frontiersin.org Perception and Epistemology They resonate with many studies from epistemology and philosophy of science. One of the most famous among these is the work of Thomas Kuhn (1996 [1962]), who defends that scientiﬁc investigation relies on sets of methods and concepts or taxonomies (together with other elements forming paradigms) that are historically situated. In line with the criticism of isolated perception, Kuhn argues that this inﬂuence of paradigms reaches perceptual experience itself that relies on concepts available at a given place and time. In consequence, shifts in paradigms (for instance at February 2022 \| Volume 5 \| Article 806041 4 Artiﬁcial Interactionism Guillermin and Georgeon To conclude this brief review of questions associated with the isolated perception paradigm and its rejection, one can recall the epistemological shift operated in physics during the twentieth century. With the development of modern physics (Einstein’s relativity and quantum mechanics), the ideal of an observer looking at the world from the outside (as if she was not herself in the world) became more and more problematic (assuming that one could neglect speciﬁcities and actions of observers became less and less tenable). With Einstein’s relativity, spatial-temporal distances between events are relative to observers’ states of movement and situations in gravitational ﬁelds. With quantum mechanics, descriptions of particles include reference to observers’ choices and actions (to eﬀective experimental settings and devices experimenters settle to observe these particles). By contrast, classical physics was attempting to provide a picture of the world from the “God’s Eye point of view” (Putnam, 1981, p. 49) with no reference to observers. The increasing eﬃciency of experimental devices progressively unveiled that such an ideal was misled and that neglecting situations and actions of observers could be nothing but (at best) an acceptable provisory approximation. Even in the very controlled and reﬁned scientiﬁc practice of empirical observation, looking at the world mobilizes very complex networks of components that include observers’ situations, some of their cognitive processes, and actions (choices and judgments outcoming in particular experimental structures). and integrated in various ways, allowing for its later retrieval” (Hutto and Myin, 2018, p. 100). Perception is thereby separated from cognition. It provides information that becomes available for cognition through their encoding in mental representations. On the ground of this traditional conception, cognitive sciences and AI mutually inﬂuenced themselves. Perception and Epistemology We now propose to explore the manner these ﬁrst insights from philosophy resonate within the ﬁelds of cognitive sciences and AI. Perception and Epistemology The works of Shannon and Turing settled a bridge between the notion of cognition (as the processing of information in so far as it means something) and the work computers can do (the mechanistic processing of information qua physical object). One can also mention Newell and Simon’s physical symbol system hypothesis (Newell and Simon, 1976) according to which cognition amounts to the mechanical processing of physical symbols. Through such bridges, both traditional cognitive sciences and AI came to share the tendency to conceive cognition as syntactic-mechanical processing of representations. This idea is often associated with functionalism, “which claims that cognitive phenomena are fully determined by their functional role and therefore form an autonomous level of analysis” (Newen et al., 2018, p. 5). They could thus be adequately described by algorithms susceptible to be implemented in various information processing devices (biological as well as artiﬁcial). This widespread common ground contributed to instating isolated perception as an inﬂuential paradigm in AI. Again, if cognition is about processing information through operations upon representations (possibly to select actions to perform), then perception is easily conﬁned to the isolated role of providing contents to some of these representations. One can easily recognize the face of this isolated perception paradigm when Russell and Norvig themselves explain the important role for AI development of the premises of cognitive psychology in the middle of the twentieth century. They mention Craik who “speciﬁed the three key steps of a knowledge-based agent: (1) the stimulus must be translated into an internal representation, (2) the representation is manipulated by cognitive processes to derive new internal representations, and (3) these are in turn retranslated back into action” (Russell et al., 2010, p. 13). Cognition occurs only during the second step, in isolation from perception and action. A more recent and emblematic example is the development of AI algorithms capable of playing multiple games through Deep Reinforcement Learning (Mnih et al., 2013). A convolutional neural network is used to learn a control policy directly from sensory input (raw pixels of the Atari game scene). In this example, perception is isolated from the AI algorithm because a ready-made representation of the state of the game is provided as input to the AI algorithm. Moreover, the reward at the end of the game (win, draw, or lose) is also provided as though the agent could directly sense the world state value. PERCEPTION, COGNITION, AND ACTION IN COGNITIVE SCIENCES AND AI We begin this section by analyzing in more detail the manner the isolated perception paradigm is at play in cognitive sciences and AI. In a second time, we turn to the discussion of alternatives to this paradigm and of the consequences for AI. Frontiers in Artiﬁcial Intelligence \| www.frontiersin.org Isolated Perception that takes the form of more or less successful organism- environment couplings.” Similar to what Putnam proposes with his commonsense realism exposed in the previous section, with REC “perceiving is a matter of getting a grip on the world as opposed to representing it.” REC clearly opposes the isolated perception paradigm. REC’s cognition extends up to organism- environment couplings instead of operating upon the results of perception. Ecological approaches pioneered by Gibson (1979) and direct perception theories pertain to this stream. They oppose “conventional theories that suppose (at least tacitly) that nervous systems register impoverished, ambiguous, or otherwise inadequate variables of stimulation” (Michaels and Carello, 1981, p. 157). Gibson’s pioneering work is directed against Marr’s conception of visual perception as a computational process construing 3D representations on the ground of 2D information captured in the retinal images (Sarkar and Pfeifer, 2006, p. 547–550; Siegel, 2021, Section 8.1). According to ecological approaches, perception involves the perceiver as a whole (not just his brain) who sense “structured energy that invariantly speciﬁes properties of the environment of signiﬁcance to [her]” (Michaels and Carello, 1981, p. 156). For tenants of this line of thought, perception cannot be seen as isolated. The perceiver is “an active explorer of the environment—one who will make an eﬀort to obtain suﬃcient information” (Michaels and Carello, 1981, p.157–158). This is in line with the shift operated in quantum physics concerning the status of empirical observation whose active nature is fully acknowledged and accounted for. It also echoes theories of active perception according to which the brain should not be seen as passively receiving data from or during perception. On the contrary, perception is better conceived of along the analogy of an internet search, the brain playing the role of a query machine. “An agent is an active perceiver if it knows why it wishes to sense, and then chooses what to perceive, and determines how, when and where to achieve that perception” (Bajcsy et al., 2018, p. 178). Moreover, the depth of computation mobilized in an agent’s perception is irrelevant to assess whether the latter is isolated or not. One can for example mention Marr’s account of visual perception (Siegel, 2021, Section 8.1) that involves several layers of computation (from 2D levels of gray to reﬂectance information, then to isometric sketches, and ﬁnally up to 3D representations of objects). Isolated Perception Though quite complex, this perceptual computation remains in the scope of the isolated perception paradigm as long as it operates independently of a larger cognitive architecture. The same applies even when powerful algorithms, such as pre-trained neural networks, are integrated into sensors themselves (following the logic of Edge computing). In itself, this is not enough to escape the isolated perception paradigm. We could even have isolated perception in an agent equipped with life-long learning of perceptual module based on reinforcement learning (in case the rest of the cognitive architecture of the agent does not intervene in the reward deﬁnition). Now that the notion of isolated perception is clearer (perceptual processes independent from the rest of an agent’s cognitive processes, in particular, those in charge of selecting actions to perform), we can turn to what it could mean for AI to take distance with this paradigm. The role of action is also core to constructivist theories, such as Piaget’s, which propose to keep perception and action entangled in sensory-motor schemes (Piaget, 1954). According to constructivist epistemology, an agent learns through active interaction with its environment and constructs a dynamic data structure that characterizes its current situation (Riegler, 2002). For the same reasons, an agent does not passively receive percepts from the outside world, but actively constructs perception in his inner subjective realm, in compliance with his sensorimotor experience. This type of conception has an interesting resonance with Kant’s transcendental idealism and the active role of synthesis it grants to the mind. In this constructivist stream, we shall also mention O’Regan and Noë’s famous theory of perception based on implicit knowledge of regular patterns in sensory-motor schemes (O’Regan and Noë, 2001). This constructivist approach strongly resonates with Husserl and Merleau-Ponty’s accounts of perception. In granting an irreducible role in perception for active exploration based on available knowledge, it clearly departs from the isolated perception paradigm. Various studies in AI attempt at implementing these Piagetian and constructivist principles (see Isolated Perception To better understand the isolated perception paradigm and its pervasiveness in cognitive sciences and AI, it is interesting to evoke the “foundation of traditional cognitive science” constituted by “the representational and computational model of cognition” (Newen et al., 2018, p. 5). This model pictures cognition as the syntactic manipulation of mental representations. Somehow, the mind computes over a kind of representational “mental code” (Hutto and Myin, 2018, p. 101). This classical cognitivism takes its roots in philosophical views of thinkers such as Descartes—mental representations are the substrates of the mind’s activity—and Hobbes—thinking amounts to operating on representations (Hutto and Myin, 2018, p. 96). Though maybe not inescapably, this traditional conception in cognitive sciences naturally leads to the isolated perception paradigm. “According to the standard cognitivist account, information is supposed to be picked up via the senses through multiple channels, encoded and then further processed A clariﬁcation is in order here. Isolated perception as we intend it in this paper should not be understood as perception independent from computation or information processing. So deﬁned, isolated perception would be an empty notion. Any sensor comes with a certain dose of information processing (at minimum in the ﬁltering it operates by collecting only some of the information available in its environment). For instance, most cameras embed microcontrollers for low-level signal processing (noise ﬁltering, implementation of communication Frontiers in Artiﬁcial Intelligence \| www.frontiersin.org February 2022 \| Volume 5 \| Article 806041 5 Artiﬁcial Interactionism Guillermin and Georgeon protocols, etc.). Perception cannot be isolated in virtue of being independent of computation or data processing in general. Rather, and more precisely, there will be isolated perception in an (artiﬁcial) agent when the computational structures intervening in perception are independent of the rest of the agent’s computational architecture in charge of generating outputs (such as selection of actions to perform). This somehow echoes the discussion of the scope of Kuhn’s world-change thesis we evoked in the previous section. We saw in this respect that perceptual experience is irremediably mediated by available concepts. Nevertheless, the most radical form of the world-change thesis occurs when mediating concepts belong to, or depend on, sets of concepts empirical observation is meant to test. Frontiers in Artiﬁcial Intelligence \| www.frontiersin.org Theoretical Alternatives to Isolated Perception In line with a diﬀerent interpretation of constructivist epistemology, other authors have suggested following Wiener’s Cybernetics theory (Wiener, 1948) and the idea that an agent’s input data constitute feedback from action (Powers, 1973; Oyama, 1985; Pfeifer and Scheier, 1994; Laming, 2001; Georgeon and Cordier, 2014; Friston et al., 2017). These authors thus propose an inversion of the interaction cycle in which output data conceptually precedes input data. We will further develop this idea at the technical level in Section Towards an Interactionist Cognitive Architecture. to extrinsic motivation—like playing chess or Hanoi tower, by contrast with problems 4E-cognition tends to focus on such as perception, action, or emotional interaction (Newen et al., 2018, p. 5). In fact, extrinsic motivation is so widespread that intrinsic motivation has yet mostly been used in reinforcement learning as a means to better fulﬁll extrinsic goals. For example, Kulkarni et al. (2016) have demonstrated the utility of intrinsic motivation to help an agent to win games. p g g We believe there is a strong connection between this prominence of extrinsic approaches of agents’ goals in AI and the pervasiveness of the isolated perception paradigm. Extrinsic motivation problems request artiﬁcial agents to reach or produce speciﬁc states of the world through the selection of speciﬁc actions. It appears like a straightforward ﬁrst step to divide agents into separated (computational) modules who will (1) operate on representations (some of them being representations of states of the world) to select adequate actions to reach a goal itself deﬁnes in terms of states of the world (cognitive module), and (2) perceive the state of the world the agent is in (perception module) to provide inputs to the cognition module. Maybe the connection becomes clearer when we consider the rejection of the isolated perception paradigm. When an artiﬁcial agent’s perception involves the rest of its cognitive architecture, the designer loses control over the manner the agent represents its environment. Specifying directly goals the agent should reach in terms of states of the world becomes impossible. This makes designs principles that would reject the isolated perception paradigm highly unsuited for the achievement of extrinsic goals. Although it may not be impossible to specify extrinsic goals indirectly, no doubt it would prove extremely (and uselessly?) diﬃcult. By contrast, we think that the rejection of the isolated perception paradigm may open interesting perspectives to design artiﬁcial agents with intrinsic motivations. Theoretical Alternatives to Isolated Perception Deﬁning intrinsic goals does not require controlling the representations an artiﬁcial agent possesses of the external world. Rejecting the isolated perception paradigm acts as a kind of warrant that goals provided to artiﬁcial agents remain intrinsic. Diﬀerent forms of intrinsic motivation have been proposed, among which curiosity (Oudeyer et al., 2007), the autotelic principle (Steels, 2004), the principle of data compressibility (Schmidhuber, 2010), predictability-based exploration (Bugur et al., 2021), minimization of uncertainty through minimization of free energy (Friston et al., 2017). We have also proposed the notion of interactional motivation that seems to us highly compatible with a renunciation of isolated perception (Georgeon et al., 2012). Intrinsic motivation can be seen more like a drive or a value system than as a goal in the sense that it guides the behavior of the agent in an open-ended fashion. Beyond the idea of actively constructing knowledge through interaction, Piaget also theorized that agents construct themselves and autonomously develop their intelligence. This idea has been followed up in recent cognitive science with the concept of constitutive autonomy (e.g., Vernon et al., 2015). Froese and Ziemke (2009) argued that constitutive autonomy is necessary for sense-making, which makes it a desirable feature to achieve in artiﬁcial agents. Constitutive autonomy implies that the coupling between the agent’s cognitive processes and the environment evolves autonomously, meaning that the way the agent perceives the world should evolve through the agent’s development. Frontiers in Artiﬁcial Intelligence \| www.frontiersin.org Theoretical Alternatives to Isolated Perception p As classical cognitivism is tightly connected with the isolated perception paradigm, let’s have a look at approaches in cognitive sciences that reject this traditional conception. We can start by mentioning the diﬀerent views Albert Newen, Leon De Bruin, and Shaun Gallagher recently gathered under the umbrella term 4E-cognition (Newen et al., 2018). This stream departs from classical cognitivism by picturing cognition as Embodied (involving the body beyond the brain), Embedded (depending on extrabodily components), Extended (involving extrabodily components), and-or Enacted (with an active role for the agent in its environment). For instance, Hutto and Myin defend “Radically enactive and embodied accounts of cognition, REC” (Hutto and Myin, 2018, p. 96–97) according to which “cognitive processes are not, for example, conceived of as mechanisms that exist only inside individuals. Instead, they are identiﬁed with nothing short of bouts of extensive, embodied activity February 2022 \| Volume 5 \| Article 806041 Frontiers in Artiﬁcial Intelligence \| www.frontiersin.org 6 Guillermin and Georgeon Artiﬁcial Interactionism for instance: Thórisson, 2012; Miller, 2018). More speciﬁcally, Piagetian sensorimotor schemes have inspired a range of computer implementations called schema mechanisms (Arkin, 1989; Drescher, 1991; Stojanov et al., 1997; Holmes and Isbell, 2005; Guerin and McKenzie, 2008; Perotto, 2013). These implementations have however been criticized for their insuﬃcient account of the core ideas of constructivist epistemology (Bettoni, 1993), as we will examine in the technical discussion in Section Towards an Interactionist Cognitive Architecture. In line with a diﬀerent interpretation of constructivist epistemology, other authors have suggested following Wiener’s Cybernetics theory (Wiener, 1948) and the idea that an agent’s input data constitute feedback from action (Powers, 1973; Oyama, 1985; Pfeifer and Scheier, 1994; Laming, 2001; Georgeon and Cordier, 2014; Friston et al., 2017). These authors thus propose an inversion of the interaction cycle in which output data conceptually precedes input data. We will further develop this idea at the technical level in Section Towards an Interactionist Cognitive Architecture. for instance: Thórisson, 2012; Miller, 2018). More speciﬁcally, Piagetian sensorimotor schemes have inspired a range of computer implementations called schema mechanisms (Arkin, 1989; Drescher, 1991; Stojanov et al., 1997; Holmes and Isbell, 2005; Guerin and McKenzie, 2008; Perotto, 2013). These implementations have however been criticized for their insuﬃcient account of the core ideas of constructivist epistemology (Bettoni, 1993), as we will examine in the technical discussion in Section Towards an Interactionist Cognitive Architecture. Redeﬁning the Goals and Motivations of Artiﬁcial Agents g Theoretical alternatives to isolated perception have implications on the goals and motivations of artiﬁcial agents. When we delegate our goals to artiﬁcial agents we provide them with “criteria and policies for making decisions” instead of explicitly programming them with “every detail of what [they] should do in various circumstances” we give them motivation (Lieberman, 2020, p. 71–72). On this ground, one can distinguish between extrinsic and intrinsic motivation (Blank et al., 2005; Oudeyer et al., 2007; Lieberman, 2020). “Intrinsic motivation is like listening to music. You do it because you enjoy it for its own sake. Nobody has to force you or pay you to listen to music. Extrinsic motivation is when you are motivated for reasons other than the activity itself—you are paid a salary to work at a job, you get a reward, a gold star, social status, etc. (of course, some situations have both kinds of motivation)” (Lieberman, 2020, p. 73). Alternative Objectives for Research in AI Now, one could wonder why we, as human designers, should create intrinsically-motivated artiﬁcial agents. What could be our goals when attributing intrinsic goals to artiﬁcial agents? The answer to this question is manyfold. Intrinsic motivation may enter the design of an AI capable of autonomous mental development (Oudeyer et al., 2007; Nagai and Asada, 2015). Another goal could be to improve our understanding of natural cognition by trying to implement sensorimotor theories of The extrinsic approach of agents’ goals is extremely widespread in AI as it permits solving problems of interest for human designers (board games, self-driving cars ...). Most recent AI applications based on machine learning that produced impressive technological achievements belong to this category (e.g., Vinyals et al., 2019). Interestingly enough, typical issues traditional cognitive sciences study also seem to correspond February 2022 \| Volume 5 \| Article 806041 7 Artiﬁcial Interactionism Guillermin and Georgeon cognition such as the theory of sensorimotor contingencies (O’Regan and Noë, 2001). In the shorter term, implanting intrinsic motivation in artiﬁcial agents may allow designing robots who display increased levels of autonomy and with whom it would be interesting to interact and play. To measure progress in such directions we will need a measure of intelligence that does not involve the capacity to reach a predeﬁned goal. The measure should however reﬂect higher-level behavior than merely controlling a setpoint as it is done in control theory. ARTIFICIAL INTERACTIONISM In this section, we propose a technical approach we call Artiﬁcial Interactionism that rejects the isolated perception paradigm and implements intrinsic motivation. It relates to the frameworks of embodied cognition in that the agent learns through active interaction with its environment and constructs a dynamic data structure that characterizes its current situation (Riegler, 2002). We consider this approach to be constructivist and to contribute to the study of artiﬁcial developmental learning because the agent autonomously constructs its knowledge of the world and has some room for cognitive constitutive autonomy. We ﬁrst detail the proposed cognitive architecture before presenting an example of implementation. There are three major diﬀerences between this formalism and the reinforcement learning formalism as it is typically implemented in a Partially Observable Markov Decision Process (POMDP) (Kaelbling et al., 1998): (a) the cycle does not start from the environment but from the agent; (b) the agent’s input and output belong to the same set I rather than two diﬀerent sets (observations O and actions A); (c) there is no reward deﬁned as a function of the states (to avoid isolated perception). The agent’s policy π(et, Kt) → it+1 is the function that selects the intended interaction it+1 based on the enacted interaction et and the data structure Kt. The agent progressively constructs Kt from the experience of enacting interactions e0 to et. Kt includes a representation of long-term knowledge learned over the agent’s lifetime and a representation of the short-term situation of the agent. We implement π and the knowledge construction mechanism as a schema mechanism related to those introduced in Section Theoretical Alternatives to Isolated Perception. Our implementation, however, diﬀers from Drescher-style schema mechanisms which model Piagetian schemes as 3-tuples (pre-perception, action, post-perception). In our analysis, these implementations fall within the framework of isolated perception by directly including percepts in schemes, and, in so doing, they miss an important aspect of Piaget’s constructivist theory. Redeﬁning the Goals and Motivations of Artiﬁcial Agents Chollet’s measure of intelligence could be promising as it involves the capacity of the agent to acquire and reuse information on the ﬂow (Chollet, 2019). We could also use subjective evaluation of the agent’s behavior by human observers with questions such as “does the agent exhibit curiosity? Playfulness? Does it appear to pursue its own interests? etc..” This would constitute an adaptation of the Turing test to simple activities performed by robots. signal within a certain range. By contrast with most other cognitive architectures, the interactionist cognitive architecture does not manipulate isolated percepts and actions. Primitive interactions are the atomic items. The concepts of output data (written in output registers by the robot’s software) and input data (read from input register by the robot’s software) remain but they are not managed at the level of the cognitive architecture. Formally, we deﬁne the system as a tuple (S, I, q, v). S is the set of environment states. I is the set of primitive interactions oﬀered by the coupling between the agent and the environment. q is a probability distribution such that q(st+1\|st, it) gives the probability that the environment transitions to state st+1 ǫ S when the agent chooses interaction it ǫ I in state st at step t. v is a probability distribution such that v(et\|st, it) gives the probability that the agent receives the input et ǫ I after choosing it in state st. We call it the intended interaction because it represents the interaction that the agent intends to enact at the beginning of step t; and et the enacted interaction because it represents the interaction that the agent records as having been actually enacted at the end of step t. If the enacted interaction equals the intended interaction (et = it) then the attempted enaction of it is considered a success, otherwise, it is considered a failure. The series of enacted interaction e0 to et is the only source of information available to the agent about the environment. Frontiers in Artiﬁcial Intelligence \| www.frontiersin.org Toward an Interactionist Cognitive Architecture It is distinct from the software/world coupling deﬁned by the input and output registers. The enaction of primitive interactions takes place at the level of the software/world coupling. The behavior selection mechanism, however, takes place at a higher level called cognitive coupling. We deﬁne the higher-level policy 5(ET, KT) →IT+1 ǫ KT over a higher-level time scale, where ET is the previous enacted composite interaction at time T, and IT+1 is the next composite interaction to try to enact. As the agent learns new composite interactions, the cognitive coupling deﬁned by the policy 5 rises away from the software/world coupling, as if the agent saw the world in terms of increasingly sophisticated possibilities of interaction. (Georgeon and Ritter, 2012). This kind of bottom-up pairwise hierarchical chaining has also been studied by Martensen (2020) and Thórisson (2020). Hierarchical composite interactions are illustrated at the top of Figure 1 that pictures our agent’s interaction control mechanism. From the researcher-designer point of view, an interesting goal is to design agents and robots that have complex shapes and can interact with objects and environments of all kinds of conﬁgurations. The robot may need to coordinate diﬀerent body parts to achieve speciﬁc interactions in an environment where diﬀerent objects may be simultaneously moving. To this end, we built a cognitive architecture to extend the interaction control mechanism presented up to this point in adding speciﬁc features suitable to ensure the processing of spatial and temporal information. Like Rudrauf et al.’s (2017) Projective Consciousness Model, our cognitive architecture uses multiple points of view on a spatial representation of the world based on active inference principles. We draw inspiration both from philosophy (notably with the approach of Kant mentioned above) and from biological or cognitive sciences studies. We build upon the analyses of mammalian brains, which evolution has endowed with complex structures to handle space. In return, we expect our models to shed some light on the role those structures play in organizing behavior. For example (Grieves and Jeﬀery, 2017) list numerous spatially modulated structures in the brain beyond the well-known hippocampus. Such literature suggests that artiﬁcial cognitive architecture will need similar functionalities to generate well-adapted behaviors in complex environments. Moreover, authors like Buzsáki and Moser (2013) have emitted the hypothesis “that the neuronal At any given time, the agent stores a list of recently enacted interactions (primitive and composite) in short-term memory. Toward an Interactionist Cognitive Architecture Our approach integrates the inversion of the interaction cycle design principle introduced in Section Theoretical Alternatives to Isolated Perception. This contrasts with most cognitive architectures that are designed according to a perception-action cycle in which, conceptually, perception precedes action. The interaction cycle revolving indeﬁnitely, one may question why the order matters. This is because inverting the cycle already means taking distance with the isolated perception paradigm. The cognitive architecture’s input data do not constitute percepts representing some features of the environment but merely constitute feedbacks from motor controls. Other authors have implemented computer simulations following the inverted interaction cycle design principle (e.g., Porr et al., 2006; Franchi, 2013; Roesch et al., 2013) but, to our knowledge, we propose the ﬁrst cognitive architecture based on this principle. Instead of 3-tuples, our schema mechanism constructs hierarchies based on 2-tuples (pre-interaction, post-interaction) called composite interactions. Composite interactions are recursively recorded on top of primitive interactions in a bottom-up fashion. A ﬁrst-level composite interaction is a sequence of two enacted primitive interactions (et−1, et). Higher- level composite interactions are sequences of two lower-level composite interactions all the way up from primitive interactions. We presented the details of this algorithm in a previous paper More speciﬁcally, our implementation rests upon the notion of primitive interaction. A primitive interaction works as a reference to a predeﬁned subprogram that speciﬁes how a control loop can be enacted involving both motor control and expected feedback. An example of primitive interaction is moving a touch sensor during a predeﬁned time while receiving a tactile feedback February 2022 \| Volume 5 \| Article 806041 8 Guillermin and Georgeon Artiﬁcial Interactionism FIGURE 1 \| The interaction control mechanism. Cylinders and cubes with arrows represent predeﬁned primitive interactions enacted through control loops. The interaction learning mechanism (top) learns composite interactions (ﬂat arrows) made of hierarchies of interactions organized in time and space. The interaction selector (center) selects a previously learned interaction for enaction (right). The interaction enactor controls the sequential enaction of the selected interaction in time and space (bottom) and returns the enacted interaction (left), which may differ from that selected depending on the environment. The coupling between the interaction selector and the interaction enactor constitutes the cognitive coupling. It is distinct from the software/world coupling deﬁned by the input and output registers. interactions that are proposed in the current context. Toward an Interactionist Cognitive Architecture The criteria used for selecting interactions deﬁne the intrinsic motivation of the agent. For example, if the criteria consist of selecting interactions that have seldom been selected in this context, the robot will exhibit curiosity to try new things; if the criteria are based upon predeﬁned valences of interaction, the robot will appear motivated by hedonist pleasure because it seeks to enact interactions that have a positive valence and avoids interactions that have a negative valence. The selected interaction is processed by the interaction enactor that controls the enaction of the interaction through motor controls sent via the output registers and feedback received through input registers. The bottom of Figure 1 illustrates the enaction of primitive interactions. It is always possible that the attempt to enact an intended interaction fails due to the unexpected conﬁguration of the environment. For example, an interaction consisting of touching an object may fail due to the absence of the object in the expected position. The actually enacted interaction (in this example, moving without touching anything) is returned by the interaction enactor to the interaction selector. FIGURE 1 \| The interaction control mechanism. Cylinders and cubes with arrows represent predeﬁned primitive interactions enacted through control loops. The interaction learning mechanism (top) learns composite interactions (ﬂat arrows) made of hierarchies of interactions organized in time and space. The interaction selector (center) selects a previously learned interaction for enaction (right). The interaction enactor controls the sequential enaction of the selected interaction in time and space (bottom) and returns the enacted interaction (left), which may differ from that selected depending on the environment. The coupling between the interaction selector and the interaction enactor constitutes the cognitive coupling. It is distinct from the software/world coupling deﬁned by the input and output registers. FIGURE 1 \| The interaction control mechanism. Cylinders and cubes with arrows represent predeﬁned primitive interactions enacted through control loops. The interaction learning mechanism (top) learns composite interactions (ﬂat arrows) made of hierarchies of interactions organized in time and space. The interaction selector (center) selects a previously learned interaction for enaction (right). The interaction enactor controls the sequential enaction of the selected interaction in time and space (bottom) and returns the enacted interaction (left), which may differ from that selected depending on the environment. The coupling between the interaction selector and the interaction enactor constitutes the cognitive coupling. Frontiers in Artiﬁcial Intelligence \| www.frontiersin.org Toward an Interactionist Cognitive Architecture The interaction timeline (bottom) represents interactions as the robot experiences them over time. The cognitive architecture incorporates a hierarchical learning module (top right), different kinds of spatial memories (egocentric and allocentric) used to localize and track the position of interactions (center), and the phenomenal ontology (left) which stores categories of phenomena (objects as they appear through interaction) deﬁned by the interactions that they afford. Enacted interactions activate (1) representations of the context in spatial and temporal memory. Phenomenal representations of objects are progressively constructed (2). When an object is recognized, the interactions that it affords can be activated (3) in spatial memory. The spatial representation of the context affords (4) subsequent composite interactions. In return, afforded interactions can be simulated (5) in spatial memory. The policy 5 selects a new interaction, and the robot tries (6) to enact it (7). algorithms underlying navigation in real and mental space are fundamentally the same” (p. 130), suggesting that spatiotemporal functionalities could lay the ground toward abstract intelligence. To progress in this direction, we envision the interactionist cognitive architecture depicted in Figure 2. ontology (Figure 2, left). Among other types of phenomena, we study how the robot can learn a representation of its own body through interaction and store this representation in phenomenal ontology just as other types of objects. The relations between these diﬀerent modules are complex and open to research. In essence, the architecture constructs various data structures that characterize the agent’s current situation distributed across several modules: spatial memories, hierarchical temporal memory, phenomenal ontology. These dynamical data structures can be described as a kind of distributed perception that is not obtained in isolation from the other parts of the cognitive architecture. In line with phenomenological approaches of perception of external objects we recalled in Section Perception, Cognition, and Action in Philosophy (notably with Husserl and Merleau-Ponty), this kind of perception also relates to what Endsley (1995) called situation awareness in humans, in the sense that these data structures work as operational knowledge of the situation by informing the selection of future behavior based on anticipation of outcomes. With this interactionist cognitive architecture, we assume that the robot has some means to locate interactions in space, at least approximately, and to keep track of its own displacements in space (e.g., through an inertial measurement unit as an analogy to the vertebrate’s vestibular system). Toward an Interactionist Cognitive Architecture Interactions in short-term memory are organized in time and possibly in space if information about their approximate location relative to the agent is available. This implies that space and time are amongst the presuppositions hardcoded in the system. In this respect, our approach echoes Kant’s transcendental idealism that pictures space and time as indispensable a priori material of perception. In line with Husserl’s notion of intentional horizon or with what Gibson called aﬀordances (Gibson, 1979), the content of short-term memory constitutes a characterization of the agent’s current situation in terms of possibilities of interactions. Interactions in short-term memory may match the beginning of previously-learned higher-level interactions. When that happens, the continuation of these higher-level interactions is proposed for subsequent enaction. The agent then selects the next interaction to try to enact from among the proposed interactions. The center of Figure 1 illustrates this interaction cycle. The interaction selector selects a composite interaction to try to enact from amongst previously-learned composite February 2022 \| Volume 5 \| Article 806041 9 Guillermin and Georgeon Artiﬁcial Interactionism FIGURE 2 \| Interactionist cognitive architecture (adapted from Georgeon et al., 2020). As in Figure 1, interactions are represented as 3D blocks along a timeline to highlight the fact that they carry 3D spatial and temporal information. The interaction timeline (bottom) represents interactions as the robot experiences them over time. The cognitive architecture incorporates a hierarchical learning module (top right), different kinds of spatial memories (egocentric and allocentric) used to localize and track the position of interactions (center), and the phenomenal ontology (left) which stores categories of phenomena (objects as they appear through interaction) deﬁned by the interactions that they afford. Enacted interactions activate (1) representations of the context in spatial and temporal memory. Phenomenal representations of objects are progressively constructed (2). When an object is recognized, the interactions that it affords can be activated (3) in spatial memory. The spatial representation of the context affords (4) subsequent composite interactions. In return, afforded interactions can be simulated (5) in spatial memory. The policy 5 selects a new interaction, and the robot tries (6) to enact it (7). FIGURE 2 \| Interactionist cognitive architecture (adapted from Georgeon et al., 2020). As in Figure 1, interactions are represented as 3D blocks along a timeline to highlight the fact that they carry 3D spatial and temporal information. Toward an Interactionist Cognitive Architecture The interaction timeline (Figure 2, bottom) represents events of interaction as the robot experiences them, whether they are initiated by the robot (e.g., moving forward and hitting an obstacle) or not (e.g., the robot is pushed into an obstacle). Information related to the distal sensory system (vision, audition, olfaction) is also handled as interactions that are, as much as possible, located at the source of the interaction—the assumed phenomenon that causes the interaction. This allows the agent to update an internal representation (in terms of interactions) in spatial memory (Figure 2, center), and simultaneously construct temporal hierarchies of sequences of interactions (Figure 2, top). The robot must also construct representations of objects as it experiences them (i.e., phenomena) stored in phenomenal Frontiers in Artiﬁcial Intelligence \| www.frontiersin.org Example of Implementation We now present an experiment to demonstrate the behavior that can be obtained with the interactionist cognitive architecture. February 2022 \| Volume 5 \| Article 806041 10 Guillermin and Georgeon Artiﬁcial Interactionism FIGURE 3 \| An interactive experiment in a dynamic simulated environment (screenshot from Georgeon, 2021). (Top) two agents (sharks) interact with each other and with three kinds of objects: solid blocks (1 × 1 × 1 cubes), go-through tiles (1 × 1 × 0.5 tiles), food tiles (1 × 1 × 0.5 violet tile). Solid blocks and tiles are opaque to the agent’s visual system. The user can add and remove objects and agents in the environment through a mobile cursor (the wired cube). (Bottom) control panels of each agent: visual inputs (12 “pixels” spanning 180◦), tactile input (feeling solid blocks around the agent), outcome (bumping into a solid block, socializing by touching another agent, eating food). Egocentric spatial memory (head upward) recording phenomena in the surrounding of the agent. Agent 2’s spatial memory shows that Agent 2 remembers the presence of a violet tile at its rear right even though the tile exited the agent’s visual ﬁeld. FIGURE 3 \| An interactive experiment in a dynamic simulated environment (screenshot from Georgeon, 2021). (Top) two agents (sharks) interact with each other and with three kinds of objects: solid blocks (1 × 1 × 1 cubes), go-through tiles (1 × 1 × 0.5 tiles), food tiles (1 × 1 × 0.5 violet tile). Solid blocks and tiles are opaque to the agent’s visual system. The user can add and remove objects and agents in the environment through a mobile cursor (the wired cube). (Bottom) control panels of each agent: visual inputs (12 “pixels” spanning 180◦), tactile input (feeling solid blocks around the agent), outcome (bumping into a solid block, socializing by touching another agent, eating food). Egocentric spatial memory (head upward) recording phenomena in the surrounding of the agent. Agent 2’s spatial memory shows that Agent 2 remembers the presence of a violet tile at its rear right even though the tile exited the agent’s visual ﬁeld. the purpose of controlling a setpoint, which is not our goal. The outcome may be directly read from the input register or may be computed from input registers by a hard-coded subprogram. Primitive control loops are thus deﬁned by the tuple (action, outcome). 2The screenshot is from RI Interactive demo. Available online at: http:// oliviergeorgeon.com/demo/. CONCLUSION TABLE 1 \| Actions and outcome. In this paper, we have brought to the fore the pervasiveness of what we call the isolated perception paradigm in computer science, robotics, and AI. We studied the possibility, meaning, and consequences of taking distance with this paradigm, drawing on insights from philosophy (phenomenology and epistemology) and recent approaches in cognitive sciences and AI. We notably discussed relationships standing between the rejection of the isolated perception paradigm and the implementation of extrinsic or intrinsic motivations, arguing that this rejection opens an interesting path for designing artiﬁcial agents animated by intrinsic motivation. For example, behaviors could be reinforced based on intrinsic-motivation criteria rather than on their eﬃciency at maximizing an extrinsic reward. Such agents may not be able to win adversarial games but they may ﬁt other purposes such as becoming interesting companions. The community of reinforcement learning could contribute to this endeavor through the rich formalism and techniques that they have developed provided they freed themselves from the tyranny of the extrinsic reward, and imagined other applications than reaching predeﬁned goals. We may learn interesting lessons about cognition in the process. removed. Note that eating is not an action but the outcome of the action impulse forward when the agent reaches the food as if the agent had a reﬂex to ingest food. The social outcome is triggered when the agent touches another agent with its nose. The tactile outcome is a 3 × 3 array of Booleans (representing a somatotopic map) that are set to true by the presence of a solid block in the surrounding of the agent. The closer outcome is triggered when a color (diﬀerent from the background color) enlarges in the visual ﬁeld. removed. Note that eating is not an action but the outcome of the action impulse forward when the agent reaches the food as if the agent had a reﬂex to ingest food. The social outcome is triggered when the agent touches another agent with its nose. The tactile outcome is a 3 × 3 array of Booleans (representing a somatotopic map) that are set to true by the presence of a solid block in the surrounding of the agent. The closer outcome is triggered when a color (diﬀerent from the background color) enlarges in the visual ﬁeld. CONCLUSION We proposed a formalism devised from the Markov Decision Process formalism used in reinforcement learning, and a cognitive architecture escaping the scope of isolated perception through the approach we named artiﬁcial interactionism. Artiﬁcial interactionism goes beyond the idea of making perception active and controlled by cognition. It draws on the more fundamental assumption that perception and cognition should not be separated in the ﬁrst place. This is a more fundamental position that implies revisiting the very fundamental premises of artiﬁcial cognitive architectures. Nevertheless, we do not claim that the artiﬁcial interactionism paradigm is the only solution to avoid isolated perception. We rather defend it constitutes a proof of concept showing that the separation is not ineluctable. Artiﬁcial interactionism oﬀers a general blueprint for designing algorithms to control robots displaying natural behaviors and open-ended learning through interaction. In artiﬁcial interactionism, the agent knows its current situation through the enaction of control loops; and has no direct access to ready-made representations of the state of the environment. The lack of access to the environment’s state forbids the designer to encode extrinsic goal state recognition criteria. Since the agent is in control of the input data, and not trying to reach a goal state deﬁned within a pre-modeled problem, it is not facing information overload or problem-solving combinatorial explosion. The absence of extrinsic goal states makes room for studying how artiﬁcial agents could construct their own goals and also satisfy the objectives of their designers. The valences of outcomes deﬁne inborne behavioral preferences one can associate with intrinsic motivations. The positive valence associated with the closer outcome gives the agent an incentive to move toward salient objects and thus explore its environment. When the agent reaches an object, other outcomes are triggered, which the agent can then associate together when they overlap in spatial memory. The agent thus performs multimodal integration by learning which interactions are aﬀorded by which objects. The agent learns that solid blocks can be felt through touch, aﬀord bumping, and have speciﬁc colors. Violet tiles aﬀord eating and touching but not bumping. Other agents are gray, aﬀord socialization but not eating nor bumping. Once those associations are recorded (in phenomenal ontology in Figure 2) the agent will tend to move toward objects that aﬀord interactions that have a positive valence (food, other agents), and avoid objects that aﬀord a negative valence (sold blocks). Gay et al. Example of Implementation Finally, primitive interactions are made of such (action, outcome) tuples associated with spatial information and a scalar valence, as listed in Table 1. Agents in this experiment ﬁgure sharks evolving in a basin, as illustrated in Figure 3. The agent implementation has been described by Gay et al. (2012). It uses the early implementation of the cognitive architecture developed by Georgeon et al. (2013). The experiment was then re-implemented in a 3D dynamic environment originally developed by Voisin (2011) that allows interactions with the experimenter. The reader can interact with this experiment online (Georgeon, 2021). In our experiment, shark agents can perform three actions simulating ﬁn ﬂaps: impulse forward, impulse leftward, and impulse rightward. They however ignore the eﬀects of these actions before experiencing them. The agents’ displacements follow a ﬂuid friction law which makes linear and angular speed cumulate over impulses with a decay factor, resulting in a smooth continuous movement. Agents are not tied to the grid but an impulse forward would approximately propel them one unit forward before the next impulse. An impulse sideward would approximately turn them of π/4. In simulations such as this one, the control loops that specify primitive interactions can be reduced to a single round of output data followed by input data. In this case, we refer to output data with the term action. Although some authors in the cognitive science literature (e.g., Engel et al., 2013) have advocated reserving the term action to intentional acts, here we use it to designate mere motor control to comply with the vocabulary used in robotics literature. We refer to the signal received by the cognitive architecture by the term outcome. We prefer this term over the term feedback used in cybernetics control theory because the term feedback is connoted with Table 1 summarizes actions and outcomes shark agents can enact. The outcome bump is triggered when the agent bumps into a sold block. The outcome eat is triggered when the shark enters a violet patch (representing food). The violet patch is then February 2022 \| Volume 5 \| Article 806041 Frontiers in Artiﬁcial Intelligence \| www.frontiersin.org 11 Artiﬁcial Interactionism Guillermin and Georgeon TABLE 1 \| Actions and outcome. Example of Implementation Action Outcome Spatial properties Dimensionality Valence Impulse forward Closer Direction in [–π/2, π/2] (color index, direction) Positive Impulse leftward Eat x = 0, y = 0 Boolean Positive Impulse rightward Bump x = 1, y = 0 Boolean Negative Social x = 1, y = 0 Boolean Positive Tactile −1 < x < 1, −1 < y < 1 3*3 matrix of boolean Null Default None Null Frontiers in Artiﬁcial Intelligence \| www.frontiersin.org REFERENCES Friston, K., FitzGerald, T., Rigoli, F., Schwartenbeck, P., and Pezzulo, G. Friston, K., FitzGerald, T., Rigoli, F., Schwartenbeck, P., and Pezzulo, G. (2017). Active inference: a process theory. Neural Comput. 29, 1–49. doi: 10.1162/NECO_a_00912 (2017). Active inference: a process theory. Neural Comput. 29, 1–49. doi: 10.1162/NECO_a_00912 Arkin, R. C. (1989). Motor schema-based mobile robot navigation. Int. J. Robot. Res. 8, 92–112. doi: 10.1177/027836498900800406 Froese, T., and Ziemke, T. (2009). 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Made-up minds: a constructivist approach to artiﬁcial intelligence - a book review. AI Commun. 6, 234–240. doi: 10.3233/AIC-1993-63-413 Gay, S. L., Mille, A., Georgeon, O. L., and Dutech, A. (2017). Autonomous construction and exploitation of a spatial memory by a self- motivated agent. Cogn. Syst. Res. 41, 1–35. doi: 10.1016/j.cogsys.2016. 07.004 Bird, A. (2000). Thomas Kuhn. Princeton, NJ: Princeton University Press. Blaauw, M., and Pritchard, D. (2005). Epistemology A-Z. New York, NY: Palgrave Macmillan. Georgeon, O. L. (2014). The developmental sharks. Youtube. Available online at: https://youtu.be/vSUEoh-sjwU (accessed January 19, 2022). Blank, D., Kumar, D., Meeden, L., and Marshall, J. B. (2005). Bringing up robot: fundamental mechanisms for creating a self-motivated, self-organizing architecture. Cybernet. Syst. 36, 125–150. doi: 10.1080/019697205908 97107 Georgeon, O. L. (2021). RI Interactive Demo. Available online at: http:// oliviergeorgeon.com/demo/ (accessed January 19, 2022). Georgeon, O. L., and Cordier, A. (2014). Inverting the interaction cycle to model embodied agents. Proc. Comp. Sci. 41, 243–248. doi: 10.1016/j.procs.2014.11.109 Brewer, W. F., and Lambert, B. L. (2001). The theory-ladenness of observation and the theory-ladenness of the rest of the scientiﬁc process. Philos. Sci. 68, S176–S186. doi: 10.2307/3080944 Georgeon, O. L., Marshall, J. FUNDING MG and OG acknowledge ﬁnancial support from New Humanism at the time of Neurosciences and Artiﬁcial Intelligence project (NHNAI). CONCLUSION (2012) reported a detailed analysis of the agent’s behavior and learning process. A video with audio explanations can be consulted online (Georgeon, 2014). Note that by deﬁning the valences of interactions, the designer does not specify goal states for the agent, but indirectly deﬁnes what goals the agent will tend to reach. Goal-seeking behaviors result from the fact that the interaction selector preferentially selects interactions that have the highest positive valence. The user does not directly specify extrinsic goal states. At most, she can lure the agent to speciﬁc places by placing food in these places. Artiﬁcial interactionism raises many fascinating questions. An important one bears upon the assessment of the success of intrinsic motivation implementation (possibly by adapting the Turing test and involving human subjective judgments to evaluate the ability of artiﬁcial interactionist agents to interact and play with users). More theoretically, one could wonder: should the system be able to learn new primitives, February 2022 \| Volume 5 \| Article 806041 12 Guillermin and Georgeon Artiﬁcial Interactionism ACKNOWLEDGMENTS The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author. This paper beneﬁtted from discussions with Paul Robertson and Juan R. Vidal. AUTHOR CONTRIBUTIONS or is it possible to generate well-adaptable intelligence based on a ﬁnite predeﬁned set of primitives? How could the agent develop symbolic reasoning and problem solving grounded on interactionist experience? Is it possible to avoid presupposing space in the cognitive architecture (Gay et al., 2017)? In the same vein, an important feature of the artiﬁcial interactionism paradigm is that the cognitive coupling can evolve as the agent learns to represent the world in terms of new composite interactions. This opens the question of the possible implementation of cognitive constitutive autonomy (Georgeon and Riegler, 2019). OG designed the software implementation and experiment. MG wrote the ﬁrst draft of the manuscript. MG and OG wrote sections of the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version. 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https://openalex.org/W4229440826	https://www.frontiersin.org/articles/10.3389/fpsyt.2022.792460/pdf	English	null	Poor Sleep in Community-Dwelling Polysubstance Users: Association With Khat Dependence, Metacognition, and Socio-Demographic Factors	Frontiers in psychiatry	2,022	cc-by	8,093	ORIGINAL RESEARCH published: 10 May 2022 doi: 10.3389/fpsyt.2022.792460 ORIGINAL RESEARCH published: 10 May 2022 doi: 10.3389/fpsyt.2022.792460 Poor Sleep in Community-Dwelling Polysubstance Users: Association With Khat Dependence, Metacognition, and Socio-Demographic Factors Md Dilshad Manzar 1, Ahmad H. Alghadir 2, Masood Khan 2, Mohammed Salahuddin 3,4, Hamid Yimam Hassen 5, Ahmed M. Almansour 1, Dejen Nureye 3, Eyob Tekalign 6, Showkat Ahmad Shah 7, Seithikurippu R. Pandi-Perumal 8,9 and Ahmed S. Bahammam 10,11 1 Department of Nursing, College of Applied Medical Sciences, Majmaah University, Al Majmaah, Saudi Arabia, 2 Department of Rehabilitation Sciences, College of Applied Medical Sciences, King Saud University, Riyadh, Saudi Arabia, 3 Department of Pharmacy, College of Medicine and Health Sciences, Mizan-Tepi University (Mizan), Mizan-Aman, Ethiopia, 4 Pharmacology Division, Department of BioMolecular Sciences, University of Mississippi, Oxford, MS, United States, 5 Department of Public Health, College of Medicine and Health Sciences, Mizan-Tepi University, Mizan-Aman, Ethiopia, 6 Department of Medical Laboratory Sciences, College of Medicine and Health Sciences, Mizan-Tepi University, Mizan-Aman, Ethiopia, 7 Department of Economics, College of Business and Economics, Mizan-Tepi University (Mizan), Mizan-Aman, Ethiopia, 8 Somnogen Canada Inc., Toronto, ON, Canada, 9 Saveetha Medical College and Hospitals, Saveetha Institute of Medical and Technical Sciences, Saveetha University, Chennai, India, 10 The University Sleep Disorders Center, College of Medicine, King Saud University, Riyadh, Saudi Arabia, 11 National Plan for Science and Technology, College of Medicine, King Saud University, Riyadh, Saudi Arabia Keywords: polydrug use, khat, alcohol, nicotine, sleep problems, metacognition Edited by: Sairam Parthasarathy, University of Arizona, United States Reviewed by: Constance Fung, VA Greater Los Angeles Healthcare System, United States Axel Steiger, Ludwig Maximilian University of Munich, Germany Purpose: Poor sleep and cognitive deﬁcits are often associated with increased drug use. However, no study has addressed the relationship between poor sleep, substance dependence, and metacognitive deﬁcit in polysubstance users. Purpose: Poor sleep and cognitive deﬁcits are often associated with increased drug use. However, no study has addressed the relationship between poor sleep, substance dependence, and metacognitive deﬁcit in polysubstance users. Methods: This was a cross-sectional study with a simple random sampling involving community-dwelling polysubstance users (n = 326, age = 18–43 years) in Mizan, Ethiopia. Participants completed a brief sleep questionnaire, severity of dependence on khat (SDS-Khat), a brief meta-cognition questionnaire, and a socio-demographic survey. Correspondence: Masood Khan raomasood22@gmail.com; mkhan4.c@ksu.edu.sa Specialty section: This article was submitted to Sleep Disorders, a section of the journal Frontiers in Psychiatry Results: Majority (56.4%) of the polysubstance users had sleep disturbance. Chronic health conditions [adjusted odds ratio (AOR) = 2.52, 95% conﬁdence interval (CI) 1.31– 4.85], chronic conditions in the family (AOR = 2.69, 95% CI 1.40–5.20), illiterate-primary level of educational status (AOR = 2.40, 95% CI 1.30–4.04), higher SDS-Khat score (AOR = 1.39, 95% CI 1.13–1.72), and lower meta-cognition score (AOR = 0.90, 95% CI 0.84– 0.97) predicted poor sleep in the polysubstance users. Moreover, low metacognition score and high SDS score also predicted additional sleep disturbances like chronic sleep insufﬁciency, lethargy and restlessness after nighttime sleep, socio-occupational dysfunctions, and daytime disturbances in polysubstance users. Received: 10 October 2021 Accepted: 14 February 2022 Published: 10 May 2022 Abbreviations: SDS-Khat, severity of dependence on khat; PSU, polysubstance use; BSQ, brief sleep questionnaire. INTRODUCTION attempted to assess sleep, sleep-related symptoms, and their predictors in community-dwelling polysubstance users. There is a paucity of studies regarding these factors among polysubstance users. Therefore, this study explored the prevalence of poor sleep, poor sleep-related symptoms, severe dependence on khat, and level of metacognitive deﬁcits in polysubstance-using community adults. We hypothesized that history of chronic conditions in polysubstance users/family members, lower metacognitive ability, and higher level of dependence on substance use may predict poor sleep outcome. Polysubstance use (PSU) implies using more than one drug of abuse either simultaneously or sequentially within a deﬁned timeframe. PSU is common among illicit drug users with the desire to (1) obtain greater eﬀects compared to use of either drug alone, (2) acquire a notable increase in the subjective response to a drug, or (3) alleviate the adverse side eﬀects of one substance by the other (1). Most substance use research (including preclinical research) has not accounted for PSU as a variable. Globally, sleep disturbances have become one of the commonly prevalent mental health disorders, wherein one- third of general adult individuals suﬀer from sleep problems (2). Estimates vary, but a large proportion of community- dwelling adults show sleep disturbances ranging from ∼16 to 65.4% (3–5). Poor sleep and related sleep disturbances may lead to physiological, psychological, and social disturbances (6). For example, individuals with poor sleep have been associated with treatment-resistant hypertension (7), suicidal ideation (8), dysregulated circulating cholesterol and triglyceride levels (9), and diabetes mellitus (10). Moreover, individuals with poor sleep quality were more susceptible to neuropsychiatric complications, especially substance use and aﬀective and cognitive disorders (11–15). Intriguingly, the relationship between sleep disturbances and substance use disorders may be bidirectional, wherein sleep disturbances increase the risk of substance misuse (11, 12), and substance use may trigger sleep complications (16). Citation: Manzar MD, Alghadir AH, Khan M, Salahuddin M, Hassen HY, Almansour AM, Nureye D, Tekalign E, Shah SA, Pandi-Perumal SR and Bahammam AS (2022) Poor Sleep in Community-Dwelling Polysubstance Users: Association With Khat Dependence, Metacognition, and Socio-Demographic Factors. Front. Psychiatry 13:792460. doi: 10.3389/fpsyt.2022.792460 Conclusion: Poor sleep, severe khat dependence, and metacognitive deﬁcits are common in community polysubstance users. Moreover, poor sleep is associated with higher khat dependence, lower metacognitive ability, lower educational status, and the presence of chronic conditions in polysubstance users or their families. May 2022 \| Volume 13 \| Article 792460 Frontiers in Psychiatry \| www.frontiersin.org 1 Sleep Correlates in Polysubstance Users Manzar et al. Participants and Procedure A cross-sectional study was performed on community-dwelling habitual polysubstance users living in Mizan-Aman, Bench Maji Zone, Ethiopia. Houses were earmarked using simple random sampling (lottery method) from the list of houses provided by health post professionals. All households with a minimum of one adult member were the source population. Adults with habitual use of more than one substance for at least 6 months composed the study population. PSU was deﬁned as the habitual use of two or more of these substances: khat, alcohol, smoking, and caﬀeinated drinks. Those having memory problems or on neuro- psychotic medications based on self-report or on account of information given by the family members were excluded to avoid memory-related bias. A ﬁnal sample (n = 326) with certain age (range: 18–43 years; mean: 27.1 ± 3.7 years) completed this study involving a brief sleep questionnaire (BSQ), severity of dependence on khat (SDS-Khat), a brief metacognition questionnaire, and a socio-demographics tool (19, 28). A brief and precise summary of the objectives and methods to be followed in the study were given to the participants. Participation was voluntary and involved no risks or rewards. The participants gave informed written consent for participation and publication. Substance-using populations in Ethiopian demographics have pronounced sleep problems (16–20). Habitual khat (Catha edulis) is a plausible explanation for changes in sleep patterns. Khat has two important alkaloids, cathinone and cathine, that possess stimulant-like activity similar to amphetamines (17, 21, 22). Central nervous complications are associated with khat use, including deﬁcits in memory, concentration, sleep, headache, migraine, motor coordination, and stereotypical behavior (21, 22). Alcohol is often concurrently misused in the Ethiopian population (17, 18). The eﬀect of alcohol on sleep continuity is dose-dependent, with low dose increasing the sleep time and high dose leading to short-term withdrawal state, increased sympathetic activity, and sleep disruption, mainly in the second phase of the night (23). Smoking tobacco is associated with a constellation of sleep complications, including diﬃculty initiating sleep, staying asleep, daytime sleepiness, and aﬀective dysregulation, including anxiety and depression (24, 25). MATERIALS AND METHODS Participants and Procedure Meta-Cognition Questionnaire Metacognition is a person’s awareness about his own cognitive and emotive abilities (28). A brief measure of metacognition with nine items was developed and validated by Klusmann et al. (28). This structured questionnaire assesses two important aspects of metacognitive ability, namely, metamemory and metaconcentration (20, 28, 32, 33). An adapted version had been found to have adequate psychometric validity in collegiate young adults (32). Each of these nine items is scored on an ordinal scale of 1 (absolutely wrong) to 5 (absolutely true). Metacognition total score (range: 9–45) is obtained by adding scores for all the nine items. Lower scores indicate poor metacognitive ability in the respondent (28, 32). A similar and adapted meta-cognition questionnaire has been found to have robust psychometric validity measures in substance users, university students, and nurses (20, 32, 33). Brief Sleep Questionnaire A BSQ with four dichotomous items (yes/no) was used to assess the presence of poor sleep and poor sleep-related symptoms. The BSQ items recorded responses to determine these: (i) subjective report of sleep disturbances; (ii) duration of sleep complaints (3 or more months); (iii) daytime restlessness, irritability, and tiredness; and (iv) report of social and occupational disruptions related to sleep disturbances. The respondents were identiﬁed as having poor sleep if they had any one of the ﬁrst two symptoms, i.e., (i) or (ii) along with complaints of both (iii) and (iv) (20). Similarly, a clinical interview using slightly modiﬁed criteria based on the International Classiﬁcation of Sleep Disorders, Revised (ICSD-R) has been used in previous sleep research in similar settings (4, 29). The BSQ was found to have an excellent level of internal consistency, as shown by a McDonald’s Omega of 0.88 and the greatest lower bound to reliability of 0.92 in this study sample (30). All the four items loaded on a common factor, “poor sleep,” with a cumulative variance of 72. 30%. Further, a goodness of ﬁt index (GFI) = 0.994 and weighted root mean Metacognitive abilities do vary among insomnia patients in comparison to healthy people (26). Some of the identiﬁable features of circadian rhythm (a component in sleep regulation) are associated with dysfunctional metacognition and neuroticism (27). Sleep quality characteristics, and metacognition mediate between chorotype measures and poor well-being (27). These pieces of evidence do imply that PSU disorder is a comorbid disorder commonly associated with numerous inﬂuences such as poor sleep, stress, and other factors. However, no study has May 2022 \| Volume 13 \| Article 792460 Frontiers in Psychiatry \| www.frontiersin.org 2 Sleep Correlates in Polysubstance Users Manzar et al. square residual (WRMR) = 0.024 of the BSQ supported its unidimensional factor structure. univariate outlier in the age but was retained after verifying the correctness of the information. There was no issue of multicollinearity and singularity; all predictor variables were signiﬁcantly correlated with poor sleep except athletic activity: β = −1.71 to 8.7 (unadjusted odds ratio). All predictor variables had linear relation with their log odds; there were no signiﬁcant p-values for the interaction terms between continuous predictor variables and their natural logs in the model. Factor 10.10.03 for Windows was used to perform a factor analysis of the BSQ scores using categorical data assumptions (34, 35). Statistical Analysis All the statistical analysis was performed by SPSS-26.0 version and Factor 10.10.03 for Windows. Participants’ characteristics are presented using mean ± SD, range, frequency, and percentage. Binary logistics regression was employed to identify associated factors of poor sleep and related sleep disturbances after verifying the assumptions. Dichotomized measures—(i) presence or absence of poor sleep based on BSQ and (ii) presence or absence of related sleep disturbances—were outcome variables. There were no multivariate outliers as determined by the Mahalanobis criteria: X2(10) = 29.59, p < 0.001. There was one Participants’ Characteristics p More than 90% of the polysubstance users participating in this study were men (Table 1). The prevalence of poor sleep, chronic conditions in the participants, and chronic conditions in the participants’ family members was 56.4, 60.4, and 61.3%, respectively (Table 1). Most of the polysubstance users (69.9%) were found to have a more severe psychological dependence on khat. The majority of the study population (53.1%) were illiterate or primary-educated. More than half of the polysubstance users (60.4%) in this study were married or stayed with their partners (Table 1). The range of monthly income (in Birr), SDS-Khat total score, meta-cognition total score, and athletic activity every day (min) were 1,500–5,000, 2–12, 16–36, and 0–105, respectively (Table 1). Khat-chewing polysubstance users were the largest group among the polysubstance-using adults (Table 1). Socio-Demographic Information Information related to socio-demographic characteristics— age, gender, presence of chronic conditions, presence of chronic conditions in the family, educational status, marital status, monthly income (in Birr), and duration of athletic activity every day (min)—were collected. Self-reported accounts from respondents for the presence of medication of AIDS, cardiovascular complications, diabetes, epilepsy, hypertension, tuberculosis, and any other chronic diseases including mental health issues were recorded. The Severity of Dependence on Khat SDS-Khat has been found to have a moderate internal consistency, adequate internal homogeneity, convergent validity, and factorial validity in polysubstance-using adults (19). SDS- Khat is a brief measure to assess dependence on khat with ﬁve items each scored on the ordinal scale from 0 to 3. The least score of 0 is indicated for a frequency of never to almost never for khat use-related behavior, while a response of 3 indicates a frequency of always or nearly always for khat use-related behavior. Scores for all individual items are added to obtain SDS-Khat total score; a higher score indicates increasing severity of dependence (19). SDS-Khat is a valid and reliable tool for khat-chewing substance users (19). A cutoﬀscore of 6 and above has been used to indicate severe psychological dependence on khat (31). Associated Factors of Poor Sleep in Polysubstance Users Associated factors of poor sleep in the polysubstance users are shown in Table 2. A binary regression prediction model was adjusted for age (years) and gender. The prediction model explained 47.8% (Nagelkerke R2) of the variance in classifying poor sleepers among polysubstance users (36). This model was signiﬁcant compared to a model with only intercepts as indicated by χ2(10, N = 326) = 143.58, p < 0.001 with 79.4% accuracy in classifying those with poor sleep. The presence of chronic conditions [adjusted odds ratio (AOR = 2.52, 95% conﬁdence interval CI 1.31–4.85), the presence of chronic conditions in the family (AOR = 2.69, 95% CI 1.40–5.20), illiterate to primary level of educational status (AOR = 2.40, 95% CI 1.30–4.04), higher SDS total score (AOR = 1.39, 95% CI 1.13–1.72), and lower meta- cognition total score (AOR = 0.90, 95% CI 0.84–0.97) predicted poor sleep in the polysubstance users (Table 2). Frontiers in Psychiatry \| www.frontiersin.org Associated Factors of Poor Sleep-Related Symptoms in Polysubstance Users Characteristics Range; mean ± SD/ (N = 326) frequency (percentage) Age 18–43; 27.1 ± 3.7 Gender Male 294 (90.2) Female 32 (9.8) Poor sleep No 142 (43.6) Yes 184 (56.4) Presence of chronic conditions No 129 (39.6) Yes 197 (60.4) Presence of chronic conditions in the family No 126 (38.7) Yes 200 (61.3) Educational status Illiterate to primary level 173 (53.1) Secondary to higher level 153 (46.9) Marital status Unmarried/divorced/widowed 129 (39.6) Double/married 197 (60.4) Monthly income (In Birr)a 1,500–5,000; 3,647.8 ± 707.1 SDS-Khat total score 2–12; 6.5 ± 1.3 Dependence on khat Less psychological dependence 98 (30.1) More or severe psychological dependence 228 (69.9) Meta-cognition total score 16–36; 26.6 ± 4.1 Athletic activity everyday (min) 0–105; 15.0 ± 24.4 Polysubstance use with khat chewing 318 (97.5%)b Polysubstance use with alcohol 230 (70.6%)b Polysubstance use with smoking 221 (67.8%)b SD, standard deviation; SDS-Khat, severity of dependence on khat scale. The presence of chronic conditions was based on self-reported presence of diabetes, hypertension, epilepsy, tuberculosis, AIDS, other cardiovascular complications, and any other chronic diseases. Poor sleep was evaluated by a brief questionnaire; metacognition was assessed by a brief measure developed by Klusmann et al. aOne hundred Birr was approximately equal to 2.01 USD on January 23, 2022. bGroups were not exclusive. a model having only intercept χ2(10, N = 326) = 26.74, p < 0.01, which had an accuracy of 92.6% in classifying complaints of social and occupational disruptions related to sleep disturbances. p Low metacognition score predicted the presence of all the four sleep complaints: subjective report of sleep disturbances (AOR = 0.91, 95% CI 0.84–0.98), 3 or more months of complaints about sleep duration (AOR = 0.86, 95% CI 0.78–0.95), complaints of daytime restlessness, irritability, and tiredness (AOR = 0.85, 95% CI 0.78–0.92), and complaints of social and occupational disruptions related to sleep disturbances (AOR = 0.85, 95% CI 0.75–0.97; Table 3). Illiterate to primary level of educational status predicted the presence of all the four sleep complaints: subjective report of sleep disturbances (AOR = 3.30, 95% CI 1.71–6.36), 3 or more months of complaints about sleep duration (AOR = 2.57, 95% CI 1.22–5.40), complaints of daytime restlessness, irritability, and tiredness (AOR = 2.22, 95% CI 1.06– 4.64), and complaints of social and occupational disruptions related to sleep disturbances (AOR = 0.36, 95% CI 0.13–0.97; Table 3). and report of social and occupational disruptions related to sleep disturbances, respectively. Associated Factors of Poor Sleep-Related Symptoms in Polysubstance Users Predictors p-value AOR (95% CI of AOR) Presence of chronic conditions No 1 Yes <.01 2.52 (1.31–4.85) Presence of chronic conditions in the family No 1 Yes <0.01 2.69 (1.40–5.20) Educational status Illiterate to primary level <0.01 2.40 (1.30–4.44) Secondary to higher level 1 Marital status Single/divorced/widowed 1 Double/married 0.09 1.72 (0.93–3.20) Monthly income 0.10 1.06 (0.98–1.15) SDS-Khat total score <0.01 1.39 (1.13–1.72) Meta-cognition total score 0.01 0.90 (0.84–0.97) Athletic activity everyday (min) 0.10 1.01 (1.00–1.02) Poor sleep was evaluated by a brief structured questionnaire; the presence of chronic conditions was based on self-reported presence of diabetes, hypertension, epilepsy, tuberculosis, AIDS, other cardiovascular complications, and any other chronic diseases. Metacognition was assessed by a brief measure developed by Klusmann et al. Sample size (N = 326). SDS-Khat, severity of dependence on khat scale; CI, conﬁdence interval; AOR, adjusted odds ratio, adjusted for age (year) and gender. a model having only intercept χ2(10, N = 326) = 26.74, p < 0.01, which had an accuracy of 92.6% in classifying complaints of social and occupational disruptions related to sleep disturbances. Low metacognition score predicted the presence of all the four sleep complaints: subjective report of sleep disturbances (AOR = 0.91, 95% CI 0.84–0.98), 3 or more months of complaints about sleep duration (AOR = 0.86, 95% CI 0.78–0.95), complaints of daytime restlessness, irritability, and tiredness (AOR = 0.85, 95% CI 0.78–0.92), and complaints of social and occupational disruptions related to sleep disturbances (AOR = 0.85, 95% CI 0.75–0.97; Table 3). Illiterate to primary level of educational t t di t d th f ll th f l l i t TABLE 1 \| Participants’ characteristics. Associated Factors of Poor Sleep-Related Symptoms in Polysubstance Users Associated factors of poor sleep-related symptoms in the polysubstance users are shown in Table 3. All four models were adjusted for age and gender. Based on the criteria of Nagelkerke R2, the prediction models explained 54.9, 69.8, 58.0, and 19.3% of the variance in classifying polysubstance users with a subjective report of sleep disturbances, duration of sleep complaints (3 or more months), daytime restlessness, irritability, and tiredness, May 2022 \| Volume 13 \| Article 792460 Frontiers in Psychiatry \| www.frontiersin.org 3 Sleep Correlates in Polysubstance Users Manzar et al. TABLE 1 \| Participants’ characteristics. Characteristics Range; mean ± SD/ (N = 326) frequency (percentage) Age 18–43; 27.1 ± 3.7 Gender Male 294 (90.2) Female 32 (9.8) Poor sleep No 142 (43.6) Yes 184 (56.4) Presence of chronic conditions No 129 (39.6) Yes 197 (60.4) Presence of chronic conditions in the family No 126 (38.7) Yes 200 (61.3) Educational status Illiterate to primary level 173 (53.1) Secondary to higher level 153 (46.9) Marital status Unmarried/divorced/widowed 129 (39.6) Double/married 197 (60.4) Monthly income (In Birr)a 1,500–5,000; 3,647.8 ± 707.1 SDS-Khat total score 2–12; 6.5 ± 1.3 Dependence on khat Less psychological dependence 98 (30.1) More or severe psychological dependence 228 (69.9) Meta-cognition total score 16–36; 26.6 ± 4.1 Athletic activity everyday (min) 0–105; 15.0 ± 24.4 Polysubstance use with khat chewing 318 (97.5%)b Polysubstance use with alcohol 230 (70.6%)b Polysubstance use with smoking 221 (67.8%)b SD, standard deviation; SDS-Khat, severity of dependence on khat scale. The presence of chronic conditions was based on self-reported presence of diabetes, hypertension, epilepsy, tuberculosis, AIDS, other cardiovascular complications, and any other chronic diseases. Poor sleep was evaluated by a brief questionnaire; metacognition was assessed by a brief measure developed by Klusmann et al. aOne hundred Birr was approximately equal to 2.01 USD on January 23, 2022. bGroups were not exclusive. TABLE 2 \| Logistic regression predicting poor sleep in polysubstance users. Frontiers in Psychiatry \| www.frontiersin.org Associated Factors of Poor Sleep-Related Symptoms in Polysubstance Users The model with correlates was signiﬁcant compared with a model having only intercept χ2(10, N = 326) = 169.50, p < 0.001, which had an accuracy of 80.4% in classifying polysubstance users with a subjective report of sleep disturbances. The model with correlates was signiﬁcant compared with a model having only intercept χ2(10, N = 326) = 241.61, p < 0.001, which had an accuracy of 87.7% in classifying polysubstance users with 3 or more months of complaints about sleep duration. The model with correlates was signiﬁcant compared with a model having only intercept χ2(10, N = 326) = 173.13, p < 0.001, which had an accuracy of 83.4% in classifying complaints of daytime restlessness, irritability, and tiredness. The model with correlates was signiﬁcant when compared with A high SDS total score predicted the presence of the three sleep complaints: subjective report of sleep disturbances (AOR = 1.53, 95% CI 1.22–1.93), 3 or more months of complaints about sleep duration (AOR = 1.52, 95% CI 1.18–1.97), and complaints of daytime restlessness, irritability, and tiredness (AOR = 1.87, 95% CI 1.43–2.43; Table 3). The presence of chronic conditions in the family predicted the presence of the three sleep complaints: subjective report of sleep disturbances (AOR = 4.27, 95% CI May 2022 \| Volume 13 \| Article 792460 Frontiers in Psychiatry \| www.frontiersin.org 4 Sleep Correlates in Polysubstance Users Manzar et al. TABLE 3 \| Logistic regression predicting poor sleep-related symptoms in polysubstance users. Associated Factors of Poor Sleep-Related Symptoms in Polysubstance Users Predictors Subjective report of sleep disturbances Duration of sleep complaints Daytime restlessness, irritability, and tiredness Social and occupational disruptions related to sleep disturbances AOR (95% CI of AOR) AOR (95% CI of AOR) AOR (95% CI of AOR) AOR (95% CI of AOR) Presence of chronic conditions No 1 1 1 1 Yes 1.62 (0.80–3.30) 0.98 (0.41–2.38) 1.42 (0.66–3.02) 4.30 (1.47–12.54) Presence of chronic conditions in the family No 1 1 1 1 Yes 4.27 (2.10–8.68) 14.40 (5.73–36.20) 5.61 (2.59–12.15) 0.34 (0.11–1.09) Educational status Illiterate to primary level 3.30 (1.71–6.36) 2.57 (1.22–5.40) 2.22 (1.06–4.64)* 0.36 (0.13–0.97)* Secondary to higher level 1 1 1 1 Marital status Single/divorced/widowed 1 1 1 1 Double/married 1.17 (0.60–2.29) 3.65 (1.69–7.90)** 2.14 (1.05–4.36)* 2.20 (0.80–5.99) Monthly income 0.99 (0.98–1.00) 0.99 (0.98–1.00) 0.99 (0.98–1.00) 0.99 (0.98–1.00) SDS-Khat total score 1.53 (1.22–1.93) 1.52 (1.18–1.97) 1.87 (1.43–2.43)** 0.90 (0.63–1.29) Meta-cognition total score 0.91 (0.84–0.98)* 0.86 (0.78–0.95) 0.85 (0.78–0.92) 0.85 (0.75–0.97)* Athletic activity everyday (min) 1.02 (1.00–1.03)* 1.03 (1.02–1.05)** 1.00 (0.99–1.02) 0.99 (0.97–1.01) p < 0.05; *p < 0.01; sample size (N = 326). Poor sleep was evaluated by a brief sleep questionnaire; subjective report of sleep disturbances, duration of sleep complaints (3 or more months), daytime restlessness, irritability, and tiredness; and report of social and occupational disruptions related to sleep disturbances. The presence of chronic conditions was based on self-reported presence of diabetes, hypertension, epilepsy, tuberculosis, AIDS, other cardiovascular complications, and any other chronic diseases. SDS-Khat, severity of dependence on khat scale; CI, conﬁdence interval; AOR, adjusted odds ratio, adjusted for age (year) and gender. TABLE 3 \| Logistic regression predicting poor sleep-related symptoms in polysubstance users. p 0 05; p 0 0 ; sa p e s e ( 3 6) Poor sleep was evaluated by a brief sleep questionnaire; subjective report of sleep disturbances, duration of sleep complaints (3 or more months), daytime restlessness, irritability, and tiredness; and report of social and occupational disruptions related to sleep disturbances. The presence of chronic conditions was based on self-reported presence of diabetes, hypertension, epilepsy, tuberculosis, AIDS, other cardiovascular complications, and any other chronic diseases. SDS-Khat, severity of dependence on khat scale; CI, conﬁdence interval; AOR, adjusted odds ratio, adjusted for age (year) and gender. khat and nicotine-induced stimulants eﬀects like anxiety and impulsive behavior (37, 38). Frontiers in Psychiatry \| www.frontiersin.org Associated Factors of Poor Sleep-Related Symptoms in Polysubstance Users Given that the half-life of cathinone is 1.5 h (39), this combination may be worse, as the eﬀects of khat may wear oﬀquicker than alcohol (half-life is 4–5 h), triggering individuals to consume khat repeatedly. This may lead to a fatal inhibited state in these individuals when the eﬀects of alcohol are felt in isolation. Indeed, polysubstance users have poor health outcomes than single-drug users (38). As such, the present study revealed that PSU of khat, alcohol, and nicotine signiﬁcantly correlated with poor sleep, poor sleep-related disturbances, and metacognitive deﬁcits. 2.10–8.68), 3 or more months of complaints about sleep duration (AOR = 14.40, 95% CI 5.73–36.20), and complaints of daytime restlessness, irritability, and tiredness (AOR = 5.61, 95% CI 2.59– 12.15; Table 3). The presence of chronic conditions predicted the presence of social and occupational disruptions related to sleep disturbances (AOR = 4.30, 95% CI 1.47–12.54; Table 3). DISCUSSION In the present study, an association was observed between poor sleep and increasing severity of khat dependence, lower metacognition ability, the presence of chronic conditions, the presence of chronic conditions of a family member, and lower educational status. Moreover, a high proportion of polysubstance users had sleep disturbances and severe dependence on khat. Consistent with the crosstalk between substance use and poor sleep, this is the ﬁrst study to demonstrate further that polysubstance users have sleep complaints such as subjective sleep disturbances, short sleep duration, daytime complaints, and occupational problems related to sleep disturbances. This study showed that among polysubstance users, the majority of users chewed khat (97.5%), and ∼56% of the total population showed poor sleep. This PSU behavior among khat users is similar to the ﬁndings of prior studies, which also reported that khat users often use other substances (19, 40). Neurobiological mechanisms of khat which mimic amphetamine-like eﬀects on the sleep circadian system are just beginning to be understood. Dopamine is an important neurotransmitter that modulates the reward circuitry and regulates alertness, thus implicated in the sleep–wake cycle. It has been demonstrated that repeated use of amphetamine- like drugs led to increased sleep latency and a decrease in the total sleep time and slow-wave and rapid eye movement (REM) sleep (41). Sleep changes may further downregulate Mostly, psychostimulants like khat and nicotine are taken along with central nervous system (CNS) depressants like alcohol: either (a) to obtain greater subjective eﬀects when using these drugs together or (b) administration of khat or nicotine may oﬀset alcohol’s depressant eﬀects. Alcohol may temper May 2022 \| Volume 13 \| Article 792460 5 Sleep Correlates in Polysubstance Users Manzar et al. dopamine receptors, thereby increasing vulnerability to drug misuse (42). Indeed, PSU may elevate the risk of comorbid psychopathology and cognitive dysfunction (13). In support, we and others have previously demonstrated that concurrent use of khat and tobacco smoking (43) and concurrent use of alcohol, khat, and tobacco smoking were associated with poor sleep (18). In animal models of behavioral sensitization, which is a model of drug addiction, nicotine potentiated the amphetamine behavioral response and dopaminergic eﬄux in rats (44); in the drug reinforcement animal model, nicotine enhanced incentive motivational eﬀects of amphetamine (45). DISCUSSION In the light of the dynamic interaction eﬀects between nicotine, amphetamine, and alcohol on hypocretin/orexin, GABAergic, dopaminergic, and cholinergic neurotransmission, it is imperative to systematically study the neurological comorbidities in polydrug users to reveal new insights into the management of these addicts (41). alone did not impair the spatial memory, suggesting synergistic eﬀects when these drugs were combined (58). Furthermore, when rat hippocampus was assessed for markers of oxidative stress, the use of alcohol and METH showed a synergistic increase in reactive oxygen species, which are detrimental for the cell compared to the use of either drug alone. Hence, concurrent use of drugs may cause the drugs to interact with each other and may have additive or synergistic eﬀects on the physiological system. Consistent with the predisposition to sleep changes, cognitive deﬁcits may also interplay, albeit concurrently, following substance use (59, 60). In the present study lower metacognition score predicted the presence of all four sleep complaints in polydrug users. This is in line with previous preclinical and clinical neuroimaging studies, which showed that diﬀerential substance users have diﬀerential changes in the gray matter volume that are associated with psychomotor, aﬀective, and cognitive deﬁcits (22, 61–64). Furthermore, long-term use of drugs with abuse potential may produce excitotoxicity by over-activation of glutamatergic and dopaminergic synapses, thus leading to neuronal damage and associated behavioral changes (65–67). The present study also identiﬁed poor sleep-related disturbances in polysubstance users with the binary logistic regression models that were signiﬁcantly associated with a subjective report of sleep disturbances and three and more months of sleep complaints. These ﬁndings are consistent with a previous study, which demonstrated that adolescents’ substance use is signiﬁcantly associated with sleep disturbances, especially in maintaining sleep regularity, timing, eﬃciency, and duration (46). Recent evidence support changes in the gene expression level of Per2 with heavy drinking in alcoholics (47). These changes are modulated by the dopaminergic transmission between the ventral tegmental area and nucleus accumbens involving melatonin hormone, levels of which promote circadian rhythm, or via gene expression by the CLOCK protein (48, 49). Moreover, drugs of abuse mediate direct changes in the circadian rhythm independent of suprachiasmatic nucleus (SCN) or light/dark cycle (50, 51) and also changes the sensitivity of various drugs of abuse like opiates, nicotine, stimulants, and alcohol based on the diurnal cycle (51). DISCUSSION In the present study increasing severity of khat dependence was signiﬁcantly associated with sleep complaints, namely, subjective reports of sleep disturbances, sleep duration complaints (3 or more months), daytime restlessness, irritability, and tiredness. This is in line with other studies, which demonstrated that drug users struggle with internalizing and externalizing problems including diﬃculty in sleep maintenance, sleep eﬃciency, and sleep duration (46, 68). Family social support buﬀers the eﬀects of internalizing problems observed in substance users. Therefore, family involvement may moderate the risk factors associated with substance use and related emotional and behavioral challenges (69). However, if family members of substance users have chronic conditions, then they are less likely to buﬀer the eﬀects of internalizing problems. This possibly explains the relationship of chronic conditions in family members with various sleep complaints and overall poor sleep because family members with chronic conditions may be less likely to constructively contribute to moderate the eﬀects of internalizing problems (69). As most study participants were khat users, it is possible to speculate that repeated use of khat, alcohol, and tobacco may have adverse consequences on attention and psychomotor function. Chronic amphetamine use promoted deﬁcits in psychomotor functioning, attention, and sleep disruption (52, 53). Moreover, the concurrent use of nicotine and amphetamine in female rats demonstrated potentiation of behavioral response with an increase in the dopamine concentrations in the striatal slices rich in dopaminergic receptors (44). Frontiers in Psychiatry \| www.frontiersin.org REFERENCES 9. Barikani A, Javadi M, Raﬁei S. Sleep quality and blood lipid composition among patients with diabetes. Int J Endocrinol Metab. (2019) 17:e81062. doi: 10.5812/ijem.81062 1. Liu Y, Williamson V, Setlow B, Cottler LB, Knackstedt LA. The importance of considering polysubstance use: lessons from cocaine research. Drug Alcohol Depend. (2018) 192:16–28. doi: 10.1016/j.drugalcdep.2018. 07.025 1. Liu Y, Williamson V, Setlow B, Cottler LB, Knackstedt LA. The importance of considering polysubstance use: lessons from cocaine research. Drug Alcohol Depend. (2018) 192:16–28. doi: 10.1016/j.drugalcdep.2018. 07.025 10. Khandelwal D, Dutta D, Chittawar S, Kalra S. Sleep disorders in type 2 diabetes. Indian J Endocrinol Metab. (2017) 21:758– 61. doi: 10.4103/ijem.IJEM_156_17 2. Walsh JK, Coulouvrat C, Hajak G, Lakoma MD, Petukhova M, Roth T, et al. Nighttime insomnia symptoms and perceived health in the America Insomnia Survey (AIS). Sleep. (2011) 34:997–1011. doi: 10.5665/SLEEP. 1150 11. Ancoli-Israel S. The impact and prevalence of chronic insomnia and other sleep disturbances associated with chronic illness. 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LIMITATIONS OF THE STUDY The current study’s results must be viewed in light of the study’s cross-sectional nature. Also, the present study only focused on poor sleep and associated sleep disturbances and metacognition. Other factors that may impact sleep were not considered. The collection of data was done by using a validated questionnaire, which lacked objective sleep metrics. Also, illicit drug users were questioned about their drug use (duration in months) with a recall period of 1 month which may be associated with recall bias. Future studies should explore longitudinal study design in a cohort group of individuals who may use CNS stimulants and depressants together and compare to single-drug users or normal subjects. Nevertheless, our study is the ﬁrst to report a high prevalence of sleep complaints and their association Alcohol, on the other hand, is a cytochrome p450 inhibitor; chronic alcohol use may downregulate GABAergic receptors and stimulate the glutamatergic receptors, thereby leading to hyperarousal impulsive behavior state and further increase vulnerability to aﬀective and substance use disorders (54, 55). Sleep disorders may confer vulnerability to future neuropsychiatric complications and cognitive dysfunction (56). Concurrent use of alcohol and methamphetamine (METH) showed impairment of learning and memory compared to METH alone in rats (57). Additional evidence in rats demonstrated that the use of alcohol and METH produced synergy to impair hippocampal-mediated spatial memory compared to using METH alone (58). Alcohol administration May 2022 \| Volume 13 \| Article 792460 Frontiers in Psychiatry \| www.frontiersin.org 6 Sleep Correlates in Polysubstance Users Manzar et al. The patients/participants provided their written informed consent to participate in this study. with dependence, metacognitive deﬁcit, and socio-demographic factors in polysubstance users. From the data presented in this study, it is not possible to discern diﬀerences in associated factors of poor sleep/poor sleep-related symptoms in discrete groups of polysubstance users. Future studies with case–control designs involving distinct groups of polysubstance users with longitudinal data collection may help in identifying relationships between sub-groups of polysubstance users and sleep. ETHICS STATEMENT The authors are grateful to the Researchers Supporting Project number (RSP-2021/382), King Saud University, Riyadh, Saudi Arabia for funding this research. The authors are grateful to the Deanship of Scientiﬁc Research, Majmaah University, for funding the research (R-2022-113). The studies involving human participants were reviewed and approved by Human Institutional Ethics Committee, College of Health Sciences, Mizan-Tepi University, Mizan-Aman, Ethiopia. DATA AVAILABILITY STATEMENT The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. AUTHOR CONTRIBUTIONS MM, MS, HH, AMA, DN, ET, SS, SRP, and AB conceptualized the study and its methodology and were involved in data collection and curation also. MM and MK did the data analysis and wrote and edited the manuscript. AHA, MS, HH, AMA, and AB were involved in supervision. All authors reviewed and approved the manuscript. CONCLUSION This study demonstrates that poor sleep, khat dependence, and metacognitive deﬁcits are highly prevalent in community polysubstance users. 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Hoﬀman R, Al’absi, M. Khat use and neurobehavioral functions: suggestions for future studies. J Ethnopharmacol. (2010) 132:554– 63. doi: 10.1016/j.jep.2010.05.033 43. Nakajima M, Dokam A, Kasim AN, AlsooﬁM, Khalil NS, Al’absi M. REFERENCES The eﬀect of escalating dose, multiple binge methamphetamine regimen and alcohol combination on spatial memory and oxidative stress markers in rat brain. J Alcohol Drug Depend. (2014) 2:159. doi: 10.4172/2329-6488.1000159 37. Ellinwood EHJr, Eibergen RD, Kilbey MM. Stimulants: interaction with clinically relevant drugs. Ann N Y Acad Sci. (1976) 281:393–408. doi: 10.1111/j.1749-6632.1976.tb27948.x 38. Timko C, Han X, Woodhead E, Shelley A, Cucciare MA. Polysubstance use by stimulant users: health outcomes over three years. J Stud Alcohol Drugs. (2018) 79:799–807. doi: 10.15288/jsad.2018.79.799 59. Herbeck DM, Brecht M-L. Substance use and mental health characteristics associated with cognitive functioning among adults who use methamphetamine. 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W4255753930.txt	https://www.ubiquitypress.com/site/chapters/10.5334/bam.e/download/342/	null		MOOCs	Ubiquity Press eBooks	2,014	cc-by	7,026	CH A PT ER 5 MOOCs Beware of the man who works hard to learn something, learns it, and finds himself no wiser than before. He is full of murderous resentment of people who are ignorant without having come by their ignorance the hard way. —Kurt Vonnegut Introduction Having looked at a long established practice of open access publishing in Chapter 3 and a relatively stable approach of OERs in Chapter 4, this chapter will consider the rapid and rather volatile world of MOOCs. No subject in educational technology in recent years has generated as much excitement amongst educational entrepreneurs and angst amongst established academics as MOOCs. If open access represents the clearest case for the argument that openness has been successful, then MOOCs are probably the best example of the second strand of this – t hat the battle for the future direction is now occurring. It was MOOCs after all, and not OERs, open access or open scholarship, that caused veteran elearning expert Tony Bates (2014) to despair, ‘I can’t express adequately just how pissed off I am about M OOCs – not the concept, but all the hubris and 90 The Battle for Open nonsense that’s been talked and written about them. At a personal level, it was as if 45 years of work was for nothing.’ Why should this be so? What is it about MOOCs that causes despair and excitement in equal measure? This will be the subject of the next two chapters, concentrating first on MOOCs themselves, and then on the media interest around them in the Chapter 6. MOOCs can stand as a microcosm of the issues in open education, because it is with open courses that they are brought into sharpest relief. This rapid growth of MOOCs can be demonstrated by comparing their internet interest with that of OERs. A simple use of Google Trends reveals how interest in MOOCs has grown, comparative to OERs (see Figure 4). While OERs have had steady growth since 2009, indicating an increased awareness, MOOCs arrive seemingly from nowhere in late 2012 and rapidly overtake OERs. This plot emphasises the point made at the end of the previous chapter regarding the sudden media interest in MOOCs. However, to put it in perspective, we can also plot MOOCs against a subject that has wider public awareness. Zuckerman (2012) jokingly suggests using the US celebrity Kim Kardashian to act as an indicative measure of internet attention. Figure 5 shows this comparison, and because Google Trends normalises the Y-scale so that it is showing relative interest rather than absolute number of searches, the rather sobering evidence is that in this plot, MOOCs don’t even register. There are two interesting aspects of MOOCs from the perspective of the battle for open. The first is what they are, the opportunities and threats they pose and the type of openness they afford. The second is the media interest in them and why they find resonance with a certain type of narrative. This chapter will deal with the first of these, looking at the history, benefits, Source: Google Trends http://www.google.com/trends/explore#q=moocs%2C%20OERs&cmpt=q. (7th September 2014) Figure 4: Google Trends plot of relative interest in MOOCs and OERs. OERs MOOCs MOOCs 91 Source: Google Trends http://www.google.com/trends/explore#q=moocs%2C%20Kardashian&cmpt=q. (7th September 2014) Figure 5: Google Trends plot of relative interest in MOOCs vs. Kim Kardashian MOOCs Kim Kardashian 92 The Battle for Open MOOCs 93 commercialisation and pedagogy of MOOCs. The next chapter will examine the second issue, that of narrative, in detail. MOOC Background MOOCs are a topic where a number of people can lay claim to being the instigator. What counts as a MOOC is open to interpretation. People had released content previously, either as part of the OER movement or independently, and this could be in the form of a whole course. However, there was a coalescence of interest around running open courses from a number of people associated with the open education movement. David Wiley ran a campus based course in 2007 and made it open to anyone online to participate, as did Alec Couros, operating an ‘open boundary’ course. However, the title of founder is often given to Connectivism and Connective Knowledge (CCK08), run by George Siemens and Stephen Downes, in 2008. It was commentary on this course that gave rise to the term MOOC, jointly attributed to Dave Cormier and Bryan Alexander. There are familiar names in this list of early MOOC providers because MOOCs can be seen as a logical extension of the open education movement. What characterised these early MOOCs was an interest in the possibilities that being open and networked offered. The subject matter of these early courses was related to the mode of presentation, so courses were in topics such as open education, digital identity or networked pedagogy. As with early elearning courses, which would often be about the subject of elearning itself, these early stages of experimentation focused on subjects where the medium was the message. But as with elearning, this soon broadened out to encompass all topics. 94 The Battle for Open Another characteristic of these early MOOCs was that they were associated with individuals, not institutions. They were seen as George and Stephen’s course, rather than a Stanford or MIT course. This meant that they were experimental in terms of technology, both by necessity and design. These MOOCs used a combination of open technologies, such as WordPress and Twitter, some institutional hosting through tools such as Moodle, and even some self-created tools such as Stephen Downes’s gRSShopper. Learning to use these tools and to make connections across the open internet was seen as a key aim for these early MOOCs. Then in 2011, MOOCs took a very different turn when Sebastian Thrun launched the Stanford Artificial Intelligence course, with over 120,000 enrolled learners. This attracted much attention from the media and venture capitalists. With the cost of formal education soaring, the idea that you could take courses from the top universities for free seemed irresistible. Harvard and MIT created EdX, Coursera was launched by Daphne Koller and Andrew Ng with venture capital funding and Thrun founded Udacity. The year 2012 was deemed ‘Year of the MOOC’ by the New York Times (Pappano 2012) as most major US universities signed up to one or other of the main providers, or launched their own courses. MOOC mania was not restricted to North America: in the UK the OU launched FutureLearn in 2013; in Germany it was iVersity; and in Australia, Open2Study. Coursera is the most prominent of the MOOC providers, and it has over 500 courses from 107 universities and over 5 million learners enrolled (Protalinski 2013). The pace of uptake, hype and development seemed breathless in comparison with most educational projects. These new MOOCs were very different from the early ones pioneered by the open education movement. They tended to be institutional, based on a proprietary platform and driven by a MOOCs 95 strongly instructivist pedagogy. Whereas the initial MOOCs had emphasised the importance of networking, many new MOOCs were focused on video instruction and automatic assessment. The distinction was made between cMOOCs for the early, connectivist type MOOCs and xMOOCs for the new, didactic models (Siemens 2012). Before we examine the impact of this commercial aspect on the nature of openness in MOOCs, it is worth considering some of the positive aspects of the rapid increase in profile for open education and elearning in general. For many educational technologists who had strived for years to get fellow academics or senior managers interested in different aspects of open education, MOOCs provided a means of getting attention and funding. As Siemens (2014) puts it, ‘if education was grunge, MOOCs were its Nirvana,’ the breakthrough act that gained attention. It might be incorrect to cast the global education movement as a fringe movement such as grunge rock, but MOOCs certainly accelerated the attention and interest in open education. Such increased profile can be both a blessing and a curse, particularly when it follows on the back of hype about revolution in higher education. But even setting aside the possibly d ubious benefits of suddenly becoming the popular child in class, MOOCs are important because they raise a number of issues for educators, and – c rucial to the theme of this b ook – t hese issues arise directly as a result of the open nature of MOOCs. In the following section, three of these are addressed. These are not the only issues raised by MOOCs, nor is this an exhaustive coverage of them – course design and pedagogy could form a book in itself. The intention here is to illustrate how the open nature of MOOCs causes fundamental questions to be asked about accepted education practice. 96 The Battle for Open MOOCs and Quality The first such issue is that of quality and how it is measured. Formal higher education has developed a set of quality measures based on a specific relationship between the education provider and the student. That relationship is fundamentally altered in a MOOC, and so these existing measures are not applicable. Let us consider why we measure quality. Largely it is to verify that aims and intentions have been met. The aims of the institution may be to have a sufficient number of students, for them to stay with and pass the course, and for the institution’s reputation to be upheld. The educator in charge of the course may have similar aims, along with those of a professional interest in exploring the possibilities afforded by MOOCs. The student will have the aims of learning what they set out to, passing the course, enjoying the experience and gaining useful skills. We therefore develop quality measures and procedures that monitor these intentions. These could be student completion rates, student satisfaction scores, external assessment of course c ontent, checks against external benchmarks, etc. In a MOOC many of these intentions are altered, either radically or subtly. At the moment it’s not entirely clear what the intentions of institutions are in relation to MOOCs – is it to attract more formal students, to provide a public good, to make money? In this early stage it might be a confused mixture of all of these, combined with a need to appear to be doing something. For educators, the intention might be experimentation with curriculum or pedagogy, improvement of their personal reputation or personal development. A more interesting difference arises if the intentions of the learner are considered. While some of the original aims may remain, for instance, it may help in career development, others are MOOCs 97 exaggerated or absent. The need to pass the course, for instance, is drastically reduced, because progress on to subsequent courses is not dependent on it, and most importantly, because there is no financial commitment and the personal interest in learning is heightened. In conventional courses there will be a wide range of different types of learner, but in MOOCs, the presence of what are termed ‘leisure learners’ is much higher than normal. They’re nearly all leisure learners – they don’t have to do this after all, it’s something that is competing with leisure pursuits. A whole new class of learners exist in MOOCs that you rarely see in formal education. These are what we might term drive-by learners (after Groom’s 2011 ‘drive-by assignments’). These are learners who are signing up because they can. It costs nothing to sign up; they can take a look, see if they like anything and move on. They may dip in and out over the course, taking bits they find engaging, or they may not even turn up at all. In formal education the financial and emotional commitment is much higher, making drive-by learners very rare. Kizilcec, Piech and Schneider (2013) used analytics to differentiate four types of MOOC learners: completing, auditing, disengaging and sampling. Although a comparison of these four types with formal learners has not been completed, one could assume that the commitments required to continue in formal education reduces the likelihood of sampling and auditing students, with the emphasis on completing. If we consider these new types of learners and their intentions, then the existing quality measures don’t map across satisfactorily. For instance, very few of these learners have course completion as a major goal. And progression on to other courses is not yet a metric in a pick-and-choose world, although we will undoubtedly see increasing pressures to make MOOC learners persist with a particular brand of MOOC provider, just as we see this with 98 The Battle for Open computer or phone providers. With such a broad range of learners, MOOCs find themselves up against a tough comparison with formal education. To use Weinberger’s (2007) phrase, higher education ‘filters on the way in’, whereas MOOCs ‘filter on the way out’. The quality measures are therefore very different. Student satisfaction rates for a system that has completely open enrolment and filters on the way out are unlikely to compare favourably with a very different system where there has been a filtering already. Filtering on the way out and operating in the open does, however, allow for new types of quality measures. These could be altmetrics-type measures (what kind of ‘buzz’ does it create, what is the public reaction of participants) or analytics (how many people come back, what is the dwell time, bounce rate, etc.). But the comparisons should be with other MOOCs, not with formal education. Quality, and what is measured, is therefore just one example of established practices that the attention on MOOCs should make us reconsider. MOOCs and Cost A second issue that MOOCs raise for formal education is that they force an examination of the costs associated with teaching. Estimates of how much it takes to produce a MOOC vary, with Udacity budgeting US$200,000, EdX US$250,000 (DeJong 2013) and University of North Carolina estimating US$150,000 for their Coursera MOOC (Goldstein 2013). Once created, the idea is that they can be run at next to no cost, although this will depend on how closely involved the lead academic is in each presentation. Clearly if you are not charging fees for people to study on a course, then its presentation costs need to be low if it is to be a sustainable model. As we saw with OERs, there are different models of MOOCs 99 sustainability, and seed funding is often required, but eventually such approaches need to stand on their own. The costs of elearning in general (not MOOCs) has been analysed by a number of researchers (e.g. Bates 1995, Weller 2004). Costs can be divided into production, i.e., those costs associated with creating the course material and any associated resources, rights, etc., and presentation costs, those associated with the delivery of the course. Generally the production costs are fixed, particularly in elearning, so they don’t vary with the number of students, while presentation costs are variable, so they increase with the number of students. The key difference for MOOCs is that in order to achieve the scale they desire, while remaining free to study, this model is not viable. Presentation costs for MOOCs need to be close to zero. The basic model of MOOCs is that of unsupported learning; in cMOOCs this support is replaced by a peer network, in xMOOCs, by automatic feedback. At the Open University, ratios for course production and presentation costs over five presentations, averaged across disciplines, are estimated to be about 1:3. That is, the presentation costs are the most expensive element, once the initial production costs have been invested. This is largely made up of salaries paid to part-time tutors to support students, but also other generic and specific student support services, e.g. support for students with disabilities, pastoral support, helpdesk costs, running regional centres, etc. This illustrates that by far the biggest cost is that of tuition. Paying people to support learners is the costly part of education. In order for MOOCs to be viable they need to remove much of these presentation costs. The question that MOOCs make higher education ask of itself is, what value is this set of costs to learners? Many of the services it represents are the key to l ong-term success 100 The Battle for Open for learners. The need for these may not be evenly distributed, though. Some learners hardly ever avail themselves of these, don’t require tuition and do very well studying on their own. Other learners require a lot of support for various reasons and probably have more than their ‘fair’ share of these services (i.e., more than they’ve actually paid for). And most are in the middle; they make use of them sometimes, depending on circumstances. For distance education in particular, this first group, the confident, independent learners will probably cope well with MOOCs. They probably represent the 10% or so who complete MOOCs. Then there are some for whom no amount of support can help them through, either study isn’t for them or this is the wrong time. But sitting in the middle is a substantial group who need varying levels of support to ‘survive’ a protracted course of study. But that doesn’t necessarily mean that universities shouldn’t look at ways of reducing the cost of presentation. This highlights the dilemma for u niversities – m any students may not think they need these services, but they are essential for long-term success. It’s akin to a universal credit, such as a state pension. Some need it more than others, but if you remove the principle of all paying into it, then it becomes prohibitively expensive for those who do need it. So the question that MOOCs make both universities and students address is – h ow much do we value support? It’s a profound question for the future direction of education. MOOCs and Course Design The third and final issue we will consider relates to course design. As mentioned in Chapter 1, being open creates a number of different opportunities for pedagogy. There are many different possibilities and motivations for being open, and as mentioned MOOCs 101 earlier, open pedagogy would make a good book subject on its own. This section will focus on just one aspect, to again illustrate how the open nature of MOOCs raises different issues which then have a consequent impact on standard educational practice. One of the oft-cited problems with MOOCs is their low completion rate. Some argue that to talk of completion rates in MOOCs is to miss their point. Downes (2014) has commented, ‘Nobody ever complained that newspapers have low completion rates.’ Learners take what they want from a MOOC in the same way that readers take what they want from a newspaper. Others state that MOOCs can’t really back up their revolutionary claims when only about 10% of learners complete a MOOC (Lewin 2013). Jordan and Weller (2013a) have done some work plotting completion rates taking the various sources of publicly available data. The average completion rate (and there are different ways of defining completion) was 12.6%. A study by the University of Pennsylvania found lower completion rates of around 4% (Perna et al. 2013). Figure 6 plots the attrition rates of active users, i.e., those that come into the course and do something such as watching a video, across disciplines: The pattern in Figure 6 is very consistent across all disciplines. Given this fairly robust pattern of behaviour, there are two course design responses. Design for Retention The first response is to say that completion is a desired metric. There may be courses where it is desirable that as many people as possible complete. For example, a remedial maths course will require learners to complete a majority of the topics. The Bridge2Success project used a MOOC- like approach to aid 0 1 2 3 4 6 7 8 Weeks since start of course 5 9 Source: Jordan and Weller 2013a. Published under a CC-BY license. Figure 6: Attrition rates of active participants in MOOCs across disciplines. 0 20 40 60 80 100 Percentage of enrolled students active 10 11 12 13 102 The Battle for Open MOOCs 103 learners with maths so they could gain a place on an employment program, so completion was very important (Pitt et al. 2013). In this case the course design needs to address the ‘problem’ of drop-out rates. There might be a number of ways of attempting this: by adding in more feedback, using badges to motivate people, creating support structures, supplementing with face-to-face study groups, breaking longer courses into shorter ones, etc. Design for Selection The second design approach is to decide that completion isn’t an important metric. The course designer accepts the MOOC attrition rates in Figure 7 and designs the experience with that in mind. In this design approach the designer might break away from the linear course model, to allow people to engage in the ‘newspaper’ type selection that Downes refers to. A course might be structured around themes, for instance, and each one around largely independent activities. In this case course completion really doesn’t matter, since learners take what they want. As a slight aside, it is likely that MOOC completion rates are being defined in such a way that gives them a low output compared with formal education, largely because the manner in which enrolment is defined is so broad. In formal education there are different ways of defining who has enrolled on a course, but it usually allows a c ooling-off period. Students are not counted as being enrolled if they drop out in the first two weeks or fail to turn up at all. So, taking MOOC enrolment figures to be the number who signed up for a MOOC even if they never come into it is always going to give harsh figures. A better figure might be the number of students active after 1 week. This is the baseline figure as those are the students who have actually started the course. 0 20 40 60 80 0 1 2 3 5 6 7 8 9 Weeks since start of course 4 Source: Jordan and Weller 2013b. Published under a CC-BY license. Figure 7: Average number of active learners over time. Percentage of enrolled students active 100 10 11 12 y = 90.087x-0.836 R² = 0.95102 13 104 The Battle for Open MOOCs 105 Another graph Jordan and Weller (2013b) plotted showed the average number of students active across weeks (Figure 7), starting with the initial enrolment figures. At the end of week 1, there are about 55% of students still active from the initial registration point. Many of those who registered will not even have come into the course once, so it is misleading to say they have dropped out. If this 55% figure is taken as the actual enrolment statistic as our starting figure, then the average completion rate rises to around 23%. With open entry learners on an unsupported course, this figure might not be as catastrophic as the numbers often quoted. There is a flip side to redefining completion rates in this way, in that it drastically reduces the impressive enrolment figures used to justify MOOC investments. What this example and the preceding two demonstrate is that there are beneficial, or at least significant, issues raised for formal education by MOOCs. This is one of the strengths of openness – it causes us to examine assumptions in standard practice, which can be improved or altered. How educators design, cost and assess the quality of all courses, not just open ones, becomes altered by digital, networked applications, but it is the addition of the catalyst of openness that really accelerates the changes and possibilities. It is this positive impact of MOOCs that I want to focus on before examining their possible downsides. The next section will examine how MOOCs could relate to higher education and perform a complementary function. MOOCs as Complement to Formal Education Much of the hype around MOOCs has positioned them as being in competition to formal education. While this adversarial framing may make good sense in terms of a media narrative, as we 106 The Battle for Open will see in the next chapter, it underplays both the actual impact of MOOCs and the adaptability of education. An alternative perspective is to view MOOCs as being similar to OERs, and complementary to formal education. Five such possible functions for MOOCs are set out below. Open up a portion of c ourses – A n online (or blended) course could be structured so that a portion of it functions as a s tand- alone MOOC. This allows students to see if it’s the type of course they want to study, to make connections and experience studying. This type of trialling has been found to be quite significant with OERs (e.g. Perryman, Law and Law 2013). It has several benefits for the institution and the learner. Firstly, it acts as a shop window, so it can increase student recruitment. Secondly, it can increase student retention, since those learners who will struggle can find this out for free and either take a different subject, study at a different level or take preparatory material. Thirdly it can widen participation, reaching audiences that the institution may have struggled to reach before. However, it should also be said that without support, the experience may be negative for some students and put them off from studying further. Open boundary c ourses – A s we have already encountered, some courses that have a campus based cohort can be made open to all. The digital storytelling course DS106 and the photography course Phonar are good examples of such courses. As well as the advantages set out above, this has particular benefits in certain subject areas. Photography is one such area where exposure to a wider audience, including professionals and experienced hobbyists, is beneficial. But for all students there is a benefit in developing a network of peers beyond their immediate cohort. MOOC collaboration – I nstitutions could collaborate on MOOCs which are useful for a range of their students. The same logic that MOOCs 107 underpinned learning objects comes into play here: Why teach the same subject at several places, when one high-quality MOOC can be created for all students to take that is recognised by all participating institutions? MOOC recognition – B y formally recognising certain MOOCs, it is possible that some institutions could shorten some of the courses they offer. For example, a learner could demonstrate that they have successfully completed a determined number of MOOCs, then they could enter an undergraduate degree in the second year and complete in two years. For the students it means fees are reduced by at least a third, which might make degree study more attractive. For campus universities they are selling the ‘campus experience’ more, without it being as prohibitively expensive. There would be reservations about developing some higher level, graduate skills with this approach, but it is feasible that a few institutions might adopt it to differentiate themselves. Curriculum experimentation and expansion – F ormal online courses are an increasingly large investment, which means course approval becomes more rigorous. The demands placed on a formal course are lessened for a MOOC (although they do not disappear), which allows for experimentation. And because MOOCs appeal to a global audience, what may not be a viable course for a campus, fee paying constituency may well be viable to a global community of informal learners. The result is that curriculum experimentation becomes less risky. It also means institutions can offer a broader curriculum, because they can offer their own curriculum but also recognise MOOCs from others. For example, ‘Hydro-engineering and Russian’ may be offered by a university that covers the engineering element, while Russian language is delivered via t hird-party MOOCs which are accredited and supported by the host university. 108 The Battle for Open These possible scenarios illustrate how MOOCs could benefit formal education and operate alongside it in a sustainable model. However, much of the recent coverage of MOOCs has not focused on these possibilities, and instead has stressed the concept of MOOC as a replacement for university. This is partly a function of the commercial nature of many MOOC entrants, and it is this aspect that will be explored next. The Commercialisation of MOOCs Soon after Sebastian Thrun’s MOOC caught the attention of the media, a number of commercial MOOC providers were established with venture capital funding. The most significant of these were Thrun’s own Udacity and another Stanford based start-up, Coursera, led by Daphne Koller and Andrew Ng. After an initial investment of $22 million, Coursera gained a further $43 million in 2013 (Kolowich 2013a). The business model of MOOC providers is not always clear. Coursera have stated that they have earned US$1 million in revenue through selling certificates of completion, which cost between US$30 and US$100 (Heussner 2013). They also announced an employee matching service, Careers Service, whereby employers could pay a fee to be matched with the best performing MOOC students (Young 2012). These elements of headhunting and certification were combined by creating a paid- for ‘Signature Track’ model, whereby students pay a fee to have verifiable identity, records and certification (Coursera 2013a). In May 2013 Coursera also announced that they were partnering with 10 campus universities to offer campus based MOOCs (Coursera 2013b), where students on campus could take a MOOC with local support. This positioned them as an elearning MOOCs 109 courseware provider, which Mike Caulfield (2013) had suggested was the intention all along. In the meantime Sebastian Thrun announced that Udacity were close to finding the ‘magic formula’ for education (Carr 2013). Then in an interview in November 2013, driven by the completion rates outlined above, he announced that Udacity had a ‘lousy product’ and they were repositioning themselves to provide corporate training (Chafkin 2013). Such a pivot drew a considerable degree of comment and derision given the bold claims Thrun had made previously, with Siemens (2013) perhaps summing it up most succinctly: ‘Make no mistake – this is a failure of Udacity and Sebastian Thrun. This is not a failure of open education, learning at scale, online learning, or MOOCs. Thrun tied his fate too early to VC funding. As a result, Udacity is now driven by revenue pursuits, not innovation.’ It is Siemens’s last point that is worth pursuing in the context of MOOCs – t he influence of venture capital funding. We should not be surprised that Coursera have attempted a range of business models, such an approach is not unusual with internet start-ups. It does suggest, however, that they are not entirely sure what the role of MOOCs is. Koller (2012) has promoted the democratisation of learning that MOOCs and Coursera offer as a social good, and their figures are impressive, with over 17 million enrolments by September 2013 (Coursera 2013c) – although this number should be treated with caution regarding what constitutes an enrolment, as mentioned previously. For comparison, there are only 2,300,000 students in higher education in the whole of the UK (HESA 2013). It might seem churlish therefore to criticise Coursera and other MOOC providers for providing access to free education. This section will not address issues such as pedagogy, which some have levelled as a criticism against MOOCs. While some of these 110 The Battle for Open accusations may be valid, they often betray either a snobbishness regarding all online learning or an over-estimation of the variety and face to face contact that many students experience. Instead the focus here will be on the open aspect of MOOCs. Although early findings (Kolowich 2013b) suggest that successful learners tend to be experienced learners with existing degrees, it may well be that given time and increased familiarity with MOOCs, Koller may be justified in her vision about the democratisation of learning. However, it is unlikely that such an altruistic goal is the intention of the venture capitalists who have invested $85 million in Coursera. As MOOC companies have shifted their models to try and recoup these costs, they have moved further away from an open model: their contents are not openly licensed, so they cannot be reused by others; enrolment is often restricted to limited periods, so content cannot be accessed without enrolling; and many MOOC providers are limiting the universities they partner with to elite institutions. The Signature Track model of Coursera may be cheap compared with formal education, but it is not an open model, nor is the blended learning, campus based delivery. Udacity’s transformation to a corporate elearning company demonstrates how quickly this shift from global provider of open education can occur if it is not founded in principles of openness. There has been a precedent for the Udacity move in FlatWorld Knowledge. FlatWorld was set up as an open access textbook publisher that allowed educators to modify the free online version and sold the physical product for a set price. In 2012 they announced that they were dropping free access to textbooks (Howard 2012), although they would remain an ‘affordable solution’. The reason behind this was that their open business model simply wasn’t generating sufficient revenue. Affordable textbooks are to be welcomed, but that is a very different entity MOOCs 111 from an open textbook. As Siemens suggests, close alliance to revenue funding will come to dominate the concerns of start- ups, and openness is usually the first casualty when this happens. Given the costs of creating a MOOC, and the return that universities will start requiring for the investment of their staff, it is debatable whether MOOCs can be sustainable as a stand-alone business. As with OERs, they may be sustainable as an adjunct to existing university practice or for national agencies, charities and professional bodies who have an interest in engaging learners. Unless they are rooted in openness, however, it is unlikely that this will remain a central tenet of their identity. It may well be that MOOC providers transform themselves into l ow-cost education alternatives by offering a combination of quite sophisticated unsupported courses and automatic assessment. This would have a profound impact on access to education and higher education itself, but it would be a different proposition to their original ‘open as in free’ model, and it would have more in common with the open entry model of distance education personified by open universities. Whether elite universities would continue to subsidise a low-cost provider through provision of courses then becomes questionable, once the open aspect has been removed. Conclusions MOOCs didn’t appear overnight from nowhere, although one might be forgiven for thinking so from the coverage they received. Figure 8 from Yuan and Powell (2013) provides a clear indication of the contributing influences for MOOCs. While some MOOC providers, such as the Harvard and MIT founded EdX, can be seen as part of a continuum with OERs, others have developed along commercial lines. To learners on Source: Yuan & Powell 2013. Published under a CC-BY license. Figure 8: MOOCs and Open Education Timeline. 112 The Battle for Open MOOCs 113 MOOCs, these ideological differences may not have much of an impact – a Coursera MOOC does not feel radically distinct from an EdX one. As we have seen, though, they may have longer-term implications on the directions that MOOCs take. The initial MOOCs were largely experimental, explicitly designed to take advantage of the possibilities that being open and networked offered. Openness was thus a key component in their design. As MOOCs became associated more with institutions, they acquired what we might term a ‘brand burden’. If MOOCs are to be seen as a global shop window, then their identity becomes closer to that of broadcast rather than network, with high-value production quality. Any failure of a MOOC can lead to considerable negative publicity for the institution, as the example of the Georgia Tech Coursera offering on Fundamentals of Online Education demonstrated (Kolowich 2013c). This course had problems with students using Google Docs to register and had to be suspended, mainly as a result of the scale of users. This shift from acceptable experimentation to part of the institution’s communications policy may have benefits in terms of sustainability, as MOOCs can be costed relative to the marketing benefit they gain, which is a model understood by universities. It may however have some negatives such as: • MOOCs become prohibitively expensive – A good MOOC requires such high-end production that it is not economically viable given the low return. • Only elite institutions offer MOOCs – G iven the expense, only those institutions who have the money or the skills to produce b roadcast-quality content will provide them. • MOOCs become pedagogically conservative – Part of the problem with the Georgia Tech course was that 114 The Battle for Open it was experimenting with a new approach, and if the cost of failure is too high then it becomes better not to attempt anything risky or innovative. • Fear of MOOC failure becomes a barrier to adoption – Public failure can lead to damage for an individual’s and an institution’s reputation, so many will consider the risk too great. In a relatively short space of time, MOOCs would have moved from being a means that allow educators to experiment with technology and pedagogy to another form of broadcast controlled by a few. This loss of experimentation may also arise as a result of there being a few dominant MOOC providers. Instead of discovering new models of open education, running a MOOC on the Coursera (or EdX or FutureLearn) platform becomes seen as the way to run a MOOC. Diversity in the market is undesirable for commercial providers; they want to become the Microsoft or Google of MOOCs, since that leads to the best revenue. Indeed, becoming the dominant provider may be the only route to high revenue returns in the MOOC field. In the opening chapter I argued that the tensions in open education could be deemed a battle, because there was real value associated with being a victor. A loss of experimentation and market dominance for open courses would be an example of one such outcome. This perceived loss of control over the platform for open courses has led to a ‘Reclaim Open’ initiative from MIT and UC Irvine. The Reclaim Open (2013) site bemoans that ‘recent high- profile forays into online learning for higher education seem to replicate a traditional lecture-based, course-based model of campus instruction, instead of embracing the peer-to-peer connected nature of the web.’ The site promises that ‘Reclaim Open MOOCs 115 Learning intervenes in this debate by supporting and showcasing innovation that brings together the best of truly open, online and networked learning in the wilds of the Internet.’ This can be viewed as a c ounter-movement to the growing dominance of certain models of MOOCs, which their technology platforms come to embody. The Reclaim Open initiative views engagement with various forms of technology as a route through which educators can take ownership of what it means to be open. Whether one supports Reclaim Open or not, their existence is an indication of the stage we are in for the battle for open, and suggests that ownership of the term is slipping, or has slipped, away. One does not see a ‘reclaim exams’ or ‘reclaim libraries’ movement. If the analysis performed at the end of the last chapter for OERs against the open principles from Chapter 2 is repeated for MOOCs, this reveals some of the reasons for this underlying disquiet about MOOCs: • Freedom to reuse – MOOC contents are not usually openly licensed, so they cannot be reused in different contexts (some providers have started to use CC licences now) • Open access – MOOCs are open to all to sign up • Free cost – t his has been the main focus of MOOCs • Easy u se – the MOOC platforms have developed e asy- to-use interfaces, although as noted above, the completion rates for this type of learning are low • Digital, networked content – although MOOCs are obviously online and digital, they are often not fully networked, in that they can exist within a closed platform • Social, community based approaches – some MOOCs are based around a very c ommunity-driven approach, 116 The Battle for Open whereas others are more instructivist and individual paced • Ethical arguments for openness – the democratisation of learning has been made as an ethical argument for MOOCs, but less on openness itself • Openness as efficient m odel – a part from some cMOOCs, MOOCs are not usually developed in the open; instead they tend to be developed as proprietary products from within universities This is not to discount the impact that companies such as Udacity and Coursera have had. They have raised the profile of elearning and open education considerably and innovated on technological fronts at a much more rapid pace than universities manage. The presence of commercial interests in the field can create a healthy mix of competition, innovation and different perspectives. For learners who are studying free courses the reservations universities and academics have regarding MOOCs may seem like an inevitable case of turkeys not voting for Christmas. However, it would be to the detriment of learners in the long term if one MOOC platform came to dominate or if, having undermined many higher education establishments, MOOCs then began to charge for courses. Part of the reluctance (or resentment even) regarding MOOCs has been less focused on the actual concept or the providers, but rather as a reaction to the hyperbole and media flurry that has accompanied them. It is important to separate these two aspects out as the inevitable backlash sets in. This is in response to the exaggerated promise made for MOOCs rather than the more nuanced reality they may offer. Examining the nature of this narrative will reveal much regarding the battle for open, and this is the subject of the next chapter.
https://openalex.org/W4391960724	https://www.e-revistes.uji.es/index.php/recerca/article/download/7207/8049	es		The idea of the infinite: a defunding of the total and a foundation of the ethical	Recerca	2,024	cc-by	11,224	RECERCA, REVISTA DE PENSAMENT I ANÀLISI, NÚM. 29(1). 2024. ISSN electrónico: 2254-4135 – pp. 1-24 DOI: http://dx.doi.org/10.6035/recerca.7207 La idea de infinito: un desfundar lo total y fundar lo ético The idea of the infinite: a defunding of the total and a foundation of the ethical GABRIEL LEIVA RUBIO (Universitat de Barcelona)1 Artículo recibido: 27 de febrero de 2023 Solicitud de revisión: 7 de julio de 2023 Artículo aceptado: 8 de febrero de 2024 Leiva Rubio, Gabriel (2023). La idea de infinito: un desfundar lo total y fundar lo ético. Recerca. Revista de Pensament i Anàlisi, 29(1), pp. 1-24. doi: http://dx.doi.org/10.6035/recerca.7207 Resumen Este ensayo practica una hermenéutica a Totalidad e infinito a partir de cinco epígrafes, abocados todos a explorar los múltiples sentidos de la propuesta levinasiana en torno al fundamento trascendental de lo ético. El primer apartado busca analizar la relación entre lo que Lévinas designa como la faz del ser y el concepto de totalidad; en el epígrafe siguiente se explicita la diferencia existente, en el interior de la comprensión temporal de lo total, entre lo histórico y lo escatológico; en el tercer epígrafe se analizan los móviles que llevan a Lévinas a sustituir lo metafísico por lo ético, revelando a este segundo saber como filosofía primera; y, finalmente, el cuarto y el quinto epígrafe quedan dedicados a entender dos conceptos que, entrelazados, condensan la matriz conceptual desde donde Lévinas acentúa la originalidad de su propuesta: el rostro del Otro y la idea de infinito. Palabras clave: Lévinas, Totalidad e infinito, ética, el rostro del Otro, la idea de infinito. Abstract This essay practices a hermeneutics analysis of Totality and Infinity from five epigraphs, all dedicated to exploring the multiple meanings of the Levinasian proposal about the transcendental foundation of the ethical. The first section seeks to analyze the relationship between what Lévinas designates as the visage of being and the concept of totality; in the following epigraph, the difference existing, within the temporal understanding of the total, between 1 leivargab@gmail.com 2 RECERCA · DOI: http://dx.doi.org/10.6035/recerca.7207 · ISSN electrónico: 2254-4135 - pp. 1-24 the historical and the eschatological is made explicit; the third section analyzes the motives that lead Lévinas to substitute the metaphysical for the ethical, revealing this second knowledge as the first philosophy; and finally, the fourth and fifth sections are dedicated to understanding two concepts that, intertwined, condense the conceptual matrix from which Lévinas emphasizes the originality of his proposal: the face of the other and the idea of infinity. Key Words: Levinas, Totality and Infinity, ethics, face of the other, the idea of infinity. INTRODUCCIÓN «Si Dios no existe, todo está permitido.»2 «Si Dios existe, todo está permitido.»3 Según la intuición, parece que al menos uno de los dos exergos que encabezan este ensayo está errado. O bien Dostoievski, o bien Žižek. Aunque también existe la posibilidad de que ambos estén equivocados y, si se pasara por encima de la esa lógica que traen consigo todas las intuiciones, podría darse el caso de que ambos tengan razón. Aún y cuando no es el objetivo determinar aquí la validez o no de tales sentencias, sí resulta interesante, para los objetivos de la presente investigación, llamar la atención sobre el substancial vínculo que existe en esas afirmaciones, entre lo divino y lo que se ha dado en llamar lo permitido. Dostoievski, en boca de Iván Karamazov, reconoce en su famosa frase que al ser sustraída la vida humana de ese metarelato (Dios) que funda, justifica y explica cada forma de obrar (lo permitido), entonces toda acción y proceder sería lícito y, por ende, el humano quedaría enteramente liberado de las responsabilidades y consecuencias que conllevan cada uno de sus actos. Žižek, por su parte, plantea el mismo movimiento del novelista ruso, pero en sentido inverso: la existencia de Dios como la condición de posibilidad explicativa de que cualquier responsabilidad y consecuencia derivadas de la acción humana recaerían sobre Dios, lo cual posibilitaría al hombre la liberación de toda responsabilidad y consecuencia asociadas a su accionar. Aunque ambos autores parten de posiciones diametralmente opuestas, los dos coinciden en un mismo cauce de reflexión: las consecuencias que traería la desaparición del fundamento de la praxis ética. A la pregunta ¿cómo debo ac2 3 Dostoievski, 2000: 941-942. Žižek, 2020: 206. GABRIEL LEIVA RUBIO. La idea de infinito: un desfundar lo total y fundar lo ético. tuar?, y en esto sí que convergen ambas sentencias, la respuesta sería la misma: ¡como sea! ¡Todo está permitido! Desde luego que es posible considerar más variantes al respecto. Por ejemplo, desde la óptica sartreana, la existencia o no de Dios importa poco al basamento y justificación de cualquier acto y consecuencia que se derive de la praxis ética humana. Esto es así porque en Sartre la responsabilidad recae por entero sobre los hombros del hombre, sobre su acción y su decisión.4 Entonces, donde Dostoievski suspende las consecuencias de la praxis ética basándose en la posible inexistencia de Dios como fundamento, y Žižek afirma la misma conclusión precisamente a partir de la existencia de Dios, Sartre desaparece de la fórmula a lo divino y deposita sobre el humano la entera responsabilidad de sus decisiones. De cualquier manera, la jugarreta metafísica de un fundamento que constituya o destituya la praxis ética es un motivo recurrente en la historia de la filosofía. Mas este fundamento de lo ético puede ser entendido a partir de dos formas particulares: o como un fundamento objetivo, o como un fundamento trascendental. El primero se refiere, sobre todo, a esa fundación de lo ético a partir de un espíritu objetivado en leyes, en una comunidad o un estado. Y dicho fundamento objetivo tiene muchísimo que ver con lo que Hegel llama eticidad (Sittlichkeit), ese desarrollo histórico de la razón que se ha externalizado y configurado bajo un grupo de normas y costumbres que han apuntalado una suerte de saber ético mediado por el derecho y las instituciones políticas. El fundamento de lo ético sería, bajo esta forma, un cúmulo de sustancias objetivas que externalizan y median necesariamente toda praxis humana. En cuanto a la segunda alternativa, el fundamento trascendental de lo ético, habría que decir que esta variante no puede ser entendida como la mediación resultante del propio desarrollo y externalización de la razón humana, sino como algo ajeno a la razón y sus creaciones. Un algo que explica y fundamenta, además de lo objetivo, también la propia naturaleza humana y toda su potencialidad. Este fundamento trascendental sería entonces la posibilidad de explicar lo objetivo prescindiendo de cualquier mediación intelectual; por lo tanto, habría que identificarlo con lo inmediato y necesario, pues de ser me4 «Yo emerjo solo y en la angustia frente al proyecto único y primero que constituye mi ser. Todas las barreras, todos los pretiles se desploman por la conciencia de mi libertad: no tengo recurso, no puedo apelar a ningún valor contra el hecho de ser yo quien sostiene en el ser los valores. Nada puede asegurarme contra mi acto, cortado como estoy del mundo y de mi esencia por esa nada que soy; yo tengo que realizar el sentido del mundo y de mi esencia: yo decido sobre ellos, solo, injustificable y sin excusa» (Sartre, 1937: 77). 3 4 RECERCA · DOI: http://dx.doi.org/10.6035/recerca.7207 · ISSN electrónico: 2254-4135 - pp. 1-24 diato, no podría resultar el fundamento último de lo ético, sino un momento más del desarrollo de la misma razón. Es a esta segunda variante de lo ético (la que se funda en un elemento trascendental) la que este artículo toma como objeto de investigación. Para su ejemplificación y comprensión, resulta decisiva la paradigmática obra Totalidad e infinito de Emmanuel Lévinas, donde aparecen, con una potencia tremenda, ideas tales como el rostro, la idea de infinito o lo absolutamente Otro, que enmarcan una propuesta definida por su originalidad a partir de la experiencia fundamentalmente trascendental que se da en la absoluta relación de lo Uno y el Otro; experiencia que, necesariamente, prescinde de esa otrora forma de filosofar donde lo ético se circunscribe a los límites que fija el pensamiento, engendrando así un saber totalizador que hace del Otro un objeto más de la razón. Para dar cumplimiento a su objetivo principal, este ensayo practica una hermenéutica a Totalidad e infinito a partir de cinco epígrafes, abocados todos a explorar los múltiples sentidos de la propuesta levinasiana y, con ello, comprender la necesidad de un fundamento trascendental para lo ético. El presente artículo queda estructurado por: un primer apartado que busca analizar la relación entre lo que Lévinas designa como la faz del ser y el concepto de totalidad; en el epígrafe siguiente se explicita la diferencia existente, en el interior de la comprensión temporal de lo total, entre lo histórico y lo escatológico; en el tercer epígrafe se analizan los móviles que llevan a Lévinas a sustituir lo metafísico por lo ético, revelando a este segundo saber como filosofía primera; y, finalmente, el cuarto y el quinto epígrafe quedan dedicados a entender dos conceptos que, entrelazados, condensan la matriz conceptual desde donde Lévinas acentúa la originalidad de su propuesta: el rostro del Otro y la idea de infinito. 1. LA TOTALIDAD COMO FAZ DEL SER En una primera aproximación, puede decirse que Totalidad e infinito5 se propone dos objetivos: realizar una crítica al concepto de totalidad y demostrar la necesaria emergencia del concepto de infinito. Siendo así, se hace necesario, desde un punto de vista metodológico, analizar ambos conceptos como preámbulo a cualquier otra consideración, para así entender no solo determi5 De ahora en adelante, TI . GABRIEL LEIVA RUBIO. La idea de infinito: un desfundar lo total y fundar lo ético. naciones esenciales de uno y otro concepto, sino la forma en que estos se relacionan entre sí. Para cualquier disquisición sobre el concepto de totalidad es fundamental la primera mención que Lévinas hace del término: La faz del ser que aparece en la guerra se decanta en el concepto de totalidad que domina la filosofía occidental. En ella (la faz del ser) los individuos son meros portadores de fuerzas que los dirigen a sus espaldas. Toman prestado un sentido a esta totalidad (sentido invisible fuera de ella). La unicidad de cada presente es sacrificada incesantemente a un porvenir convocado a despejar su sentido objetivo. Porque solo el último sentido cuenta, solo el último acto transforma los seres en sí mismos (Lévinas, 2002: 48). Aun y cuando en esta valoración inicial Lévinas no explicita ni define el concepto de totalidad de forma positiva,6 es posible percibir cómo comienza a abordarlo a partir de ciertas nociones que necesariamente conviene aclarar: faz del ser, fuerzas que dirigen a los individuos a sus espaldas, porvenir convocado y el último sentido. La primera de estas nociones (faz del ser) resulta particularmente importante, pues justamente a partir de ella es que Lévinas justifica buena parte de las nociones que quedan aún por aclarar. Pero ¿qué es la faz del ser? Con el uso del término faz, Lévinas parece referirse a un cierto aspecto unilateral del ser, una determinación ontológica que no colma su sentido y fundamentación. La expresión que usa el propio Lévinas, la face de l’être,7 deja la potencia de lo ontológico reducida a una condición relativa que aniquila, de antemano, el sentido y la significación que tan importante concepto tendrá en consideraciones ulteriores. La expresión o el concepto que acompaña a la faz del ser, en esta ocasión, haciéndolo un concepto relativo, es la guerra. Hecha la precisión debe volverse al interrogante: ¿a qué se refiere Lévinas cuando indica que «La faz del ser que aparece en la guerra se decanta en el concepto de totalidad que domina la filosofía occidental» (Lévinas, 2002: 48)? Unos párrafos antes del fragmento que se analiza, Lévinas apunta algo fundamental: «el ser se revela como guerra al pensamiento filosófico; la guerra no solo lo afecta como el hecho más patente, sino como la patencia misma —o la verdad— de lo real» (Lévinas, 2002: 47). De tal manera, el ser es presentado «Lévinas introduce bruscamente términos que adquieren su significado más por evocación y oposición que por definición» (Hand, 2009: 37). 7 El fragmento original en francés dicta: «La face de l'être qui se montre dans la guerre, se fixe dans le concept de totalité qui domine la philosophie occidentale» (Lévinas, 1987: 6). Por otra parte, la edición inglesa dispone: «The visage of being that show itself in war is fixed in the concept of totality, which dominates Western philosophy» (Lévinas, 1979: 21). 6 5 6 RECERCA · DOI: http://dx.doi.org/10.6035/recerca.7207 · ISSN electrónico: 2254-4135 - pp. 1-24 aquí por Lévinas de forma heracliteana, cual danza bélica donde el logos se hace presente como el medio que persiste entre términos opuestos que se enfrentan. Siendo así, derivar al ser de la guerra es hacer exterior su condición determinante, su propia dinámica. La guerra se presenta, de esta forma, como la experiencia por antonomasia del ser puro. Pero a esta conclusión debería agregarse que dicha forma de explicar al ser —como guerra— tiene que ser totalizante, universal, extensiva a todos los aspectos de lo real; de lo contrario, no estaría dando parte del ser, que no es sino el concepto omniabarcante que refrenda y resume todo lo que es, sino de un fragmento del todo. Y es sobre este último aspecto —tomar una parte por el todo—, que Lévinas quiere llamar la atención. El concepto de totalidad que, según Lévinas, «domina la filosofía occidental» sería entonces el medio ideal a partir del cual se pretende explicar todo lo real desde una fórmula ontológica que tuviese la potencia para agotarlo y explicarlo todo. La totalidad, entonces, no tendría márgenes, límites, un algo fuera de sí porque, de ser así, no sería totalizadora, universal. De ahí que no pueda haber sentido al margen de dicha totalidad. Precisada tal cuestión puede entonces entenderse con más claridad eso que Lévinas señalaba sobre los individuos como «meros portadores de fuerzas que los dirigen a sus espaldas» (Lévinas, 2002: 48). Y es que este tipo de filosofar totalizante, esa faz del ser que en la guerra se decanta por la totalidad tiene, necesariamente, que concederle un lugar a las partes, que, fijas o movientes, termine por integrarse al dinamismo de su realidad. Lo que Lévinas designa alegóricamente como «fuerzas que los dirigen a sus espaldas» no remite en lo absoluto a la voluntad de un Schopenhauer ni al inconsciente freudiano, sino a ese lugar predeterminado o anticipado que asigna la totalidad a las partes; lugar donde los individuos, los entes y las cosas juegan un rol apriorístico que, esencialmente, facilite la organicidad y el funcionamiento de lo total. De ahí que el propio Lévinas asegure que «el acontecimiento ontológico que se perfila en esta negra claridad es la movilización de los seres […], llevada a cabo por un orden objetivo al que no se pueden sustraer» (Lévinas, 2002: 47). Otorgarle ese sentido a los individuos sería entonces una pirueta formal imposible de realizar dentro de los límites de la totalidad, puesto que el sentido correspondería a la totalidad y no a los individuos. Estos últimos tendrían sentido, solamente, si esa totalidad y su orden se lo permitieran. Pero, a su vez, el sentido de los individuos (de lo humano), quedaría como un algo relativo al sentido último de lo total, de ahí que al final del fragmento Lévinas apunte que «solo el último acto transforma los seres en sí mismos». GABRIEL LEIVA RUBIO. La idea de infinito: un desfundar lo total y fundar lo ético. 2. LO HISTÓRICO Y LO ESCATOLÓGICO Una vez explicitada la relación de identidad percibida por Lévinas entre la ontología dominante de occidente y el concepto de totalidad, se hace pertinente desentrañar una determinación fundamental de este concepto: la temporalidad. En el análisis previo, intencionalmente se obvió la implicación fundamental de esta visión totalizadora que, según Lévinas, ha realizado la ontología dominante de la filosofía occidental. Dicha implicación es la que manifiesta Lévinas cuando indica: «la unicidad de cada presente es sacrificada incesantemente a un porvenir convocado a despejar su sentido objetivo». Es decir, lo presente, el ahora, queda desechado como parte de una paradoja que convoca al individuo a una suerte de tiempo atemporal, donde el futuro, entendido como superación del presente, despeja el sentido de cualquier tiempo otro. Pero tal futuro no es tampoco tiempo, sino la idea de un telos que pareciera adherirse con fuerza al nervio de lo total. El esquema pasado, presente y futuro queda devorado así por un esquema mayor, que no solo contiene y explica el esquema anterior, sino que le otorga sentido y fundamento.8 Si bien la discusión más pormenorizada de Lévinas sobre lo temporal no tiene lugar en TI, sino en cuatro conferencias pronunciadas durante el curso 1946-1947 que luego fueron recogidas bajo el titulo El tiempo y el Otro,9 en el prefacio de TI hay una relación conceptual dicotómica directamente relacionada con el problema del tiempo: la historia y la escatología. La escatología, en tanto que el más allá de la historia arranca los seres a la jurisdicción de la historia y del porvenir, los interpela en su plena responsabilidad y a ella los convoca. Sometiendo a juicio la historia en su conjunto, exterior a las mismas guerras que marcan su fin, restituye a cada instante su plena significación en ese mismo instante: todas las causas están maduras para ser entendidas. No es el juicio final el que importa, sino el juicio de todos los momentos en el tiempo en que se juzga a los vivos. La idea escatológica de juicio implica que los seres tienen una identidad «antes» de la eternidad, antes de la consumación de la historia, antes de que los tiempos sean cumplidos, mientras que aún hay tiempo, implica que los seres existen en relación, pero a partir de sí y no a partir de la toA este respecto, y en relación con la comprensión contemporánea de la temporalidad, señala Sebastián L. Vidal algo interesante: «El tiempo se ha convertido en objetivo de la crítica social y en su interior se ven ahora algunas de las claves de las relaciones sociales de nuestra época, dejando de ser condición ontológica de posibilidad para ser síntoma y blanco de la analítica social» (Vidal, 2023: 2). 9 El tiempo y el Otro fue el nombre que le dio Lévinas a cuatro conferencias que impartió en el primer año de funcionamiento del College Philosophique (fundado por Jean Wahl), las cuales fueron editadas como ensayos y aparecieron en 1948 en la obra colectiva titulada Las Opciones, el Mundo, la Existencia. Para consultar este texto, véase Lévinas, 1993. 8 7 8 RECERCA · DOI: http://dx.doi.org/10.6035/recerca.7207 · ISSN electrónico: 2254-4135 - pp. 1-24 talidad. La idea del ser que desborda la historia hace posible entes a la vez comprometidos en el ser y personales, convocados a responder por su proceso y, en consecuencia, ya adultos, pero, por esto mismo, entes que pueden hablar, en lugar de prestar sus labios a la palabra anónima de la historia (Lévinas, 2002: 49). Lévinas señala aquí una doble separación de lo temporal a partir de su comprensión. Por una parte, lo histórico y, por otra, lo escatológico como lo meta-histórico. El primer aspecto de la temporalidad, ese que Lévinas designa como juicio histórico, es aquel que pretende historiar al ser y determinar, a partir de su génesis y construcción, su sentido definitivo. Esta visión, tendenciosa y teleológica, es inmediatamente desechada por el autor, puesto que dicho enfoque analiza la temporalidad como totalidad, haciéndola un objeto más de investigación, y excluyendo así la subjetividad en el gesto aprisionador de un objetivismo esquemático incapaz de captar el impulso vital del ser y su relación con lo demás. Siendo así, el tiempo histórico (juicio histórico) reduciría y relativizaría la subjetividad en un esquema que la hace objetiva, y que, a la postre, termina por totalizarla. Eso que Lévinas designa como juicio escatológico, a partir del cual los seres, los individuos y la historia quedan supeditados a la completa responsabilidad del Yo frente al Otro, restituye la imprescindible importancia que tiene el instante, lo mismo como medida irreductible a cualquier objetivación que como elemento vinculante entre los individuos. Lo escatológico, el cara a cara con lo trascendental, hace que los individuos desborden la línea temporal sobre la que la historia posa su sombra, dejando la entera responsabilidad de sus existencias a un proceso que empieza y termina en su interioridad. Es por ello que Lévinas define la interioridad como «la posibilidad misma de un nacimiento y de una muerte que no extraen de ningún modo su significación de la historia» (Lévinas, 2002: 79).10 Y es que la escatología no solo prescinde de ese sistema teleológico del que se vale la totalización histórica para afirmar su presencia de forma absoluta, sino es capaz de situar al ser más allá de los con10 «[…] para que la totalización de la historia no sea el último designio del ser, es necesario que la muerte que, para los sobrevivientes es fin, no sea solamente este fin; es necesario que haya en el morir otra dirección que la que lleva al fin como a un punto de impacto en la duración de los sobrevivientes. La separación indica la posibilidad para un ente de instalarse y tener su propio destino, es decir, de nacer y morir sin que la ubicación de este nacimiento y de esta muerte en el tiempo de la historia universal contabilice su realidad» (Lévinas, 2002: 79); «El Juicio viril de la historia, […] es cruel. Las normas universales de ese juicio hacen callar la unicidad en la que se sostiene la apología y de donde saca sus argumentos. Lo invisible, al ordenarse en totalidad, agravia la subjetividad, porque, por esencia, el juicio de la historia consiste en traducir toda apología en argumentos visibles y en silenciar la fuente inagotable de la singularidad de donde surgen y en la cual ningún argumento podría dar razón» (Lévinas, 2002: 257). GABRIEL LEIVA RUBIO. La idea de infinito: un desfundar lo total y fundar lo ético. tornos invisibles de esa misma totalidad, fuera del tiempo historiográfico que esta instaura. En consecuencia, la trascendencia que reviste la escatología devuelve la imagen de una excedencia que la totalidad no logra apresar, «como si la totalidad objetiva no completara la verdadera medida del ser, como si otro concepto debiera expresar esta trascendencia con relación a la totalidad, noenglobable en una totalidad y tan original como ella» (Lévinas, 2002: 79). Resulta esclarecedor percatarse de que Lévinas insiste en criticar el manejo que hace la filosofía occidental del saber metafísico. A este respecto, Merold Wesphal señala una doble intención de la crítica levinasiana: Después de Heidegger, el nombre de todo lo malo de la tradición filosófica occidental ha sido generalmente lo metafísico (en su modo onto-teológico) o la metafísica de la presencia. Lévinas hace de la ontología su chivo expiatorio y reserva el nombre de metafísica para la postura que él afirma. La razón de esta inversión terminológica es doble. En primer lugar, quiere preservar la conexión entre la metafísica y la trascendencia, y cree que lo que solemos llamar metafísica occidental ha sido, en gran medida, el intento de eliminar o, al menos, domesticar la trascendencia. En segundo lugar, cree que la primacía de la categoría de ser ha desempeñado un papel importante en este proyecto que ha convertido a la «razón» en aliada de la guerra. El ser es epistemológicamente problemático en la medida en que el ser se entiende como aquello que se entrega al entendimiento humano para ser pensado, haciendo de los horizontes humanos la medida del significado y la verdad (Wesphal, 2008: 13) Es precisamente en esta dimensión exterior a la historia, en ese no-lugar que recoge y explica aquella faz del ser —en márgenes que la totalidad reduce a su dinámica y que no puede contener—, donde se concentra la llamada de atención levinasiana. La crítica del lituano-francés a la metafísica dominante en occidente se traduce en diatriba a esa pretensión ontológica que intenta explicar todo lo que es mediante el ejercicio esquemático del logos. Pues, para Lévinas, hay algo que opera fuera de los límites de lo epistémico y que, aun siendo metafísico, no practica la totalización del ser. Este tipo de experiencia no queda circunscrita a los explícitos márgenes de la totalidad, sino a una suerte de «significación sin contexto» (Lévinas, 2002: 50), a una experiencia irreductible a dicho saber totalizador. Y, para concretar tal planteo, apunta Lévinas: «es necesaria una relación original y originaria con el ser» (Lévinas, 2002: 48).11 Es decir, tiene que haber una filosofía primera que explique lo me11 Es en este sentido y «de acuerdo con sus colegas negativistas franceses y con el trasfondo de Nietzsche y Heidegger, donde Lévinas desarrolla su propia narrativa crítica de la filosofía occidental como voluntad de poder disfrazada de Logos. Sus intentos de totalizar el mundo y nuestra experiencia de él, para hacer que todo encaje en sus esquemas conceptuales, son vistos como una serie de intentos para hacer que el mundo 9 10 RECERCA · DOI: http://dx.doi.org/10.6035/recerca.7207 · ISSN electrónico: 2254-4135 - pp. 1-24 tafísico totalizante, una metafísica que, más que una visión objetivante, sea una óptica, que más que entender viva la irreductible experiencia del logos. Es en este sentido que Lévinas concibe lo ético. 3. LA ÉTICA, FILOSOFÍA PRIMERA Previo a profundizar cómo y por qué Lévinas entiende que esta ética suya supera la visión de esa metafísica tradicional de occidente, resulta conveniente detenerse en un importante llamado de atención levinasiano: Contener más de lo que se es capaz no significa abarcar o englobar con el pensamiento la totalidad del ser o, al menos poder, con posterioridad, dar cuenta de ello por la función interior del pensamiento constituyente. Contener más de lo que se es capaz es, en todo momento, hacer estallar los cuadros de un contenido pensado, superar las barreras de la inmanencia, pero sin que esta caída en el ser se reduzca de nuevo a un concepto de caída (Lévinas, 2002: 53). Es decir, la ética desborda el efecto totalizador del pensamiento, no por el mero hecho de contenerlo, sino precisamente porque lo ético tiene la potencia de hacer salir al pensamiento de sí mismo, de romper su hermética inmanencia. De ahí que lo ético no pueda ser asimilado o contenido, puesto que: no se limita a preparar el ejercicio teórico del pensamiento que monopolizaría la trascendencia. La oposición tradicional entre teoría y práctica desaparecerá a partir de la trascendencia metafísica en la que se establece una relación con lo absolutamente otro o la verdad, cuya vía real es la ética. Hasta ahora, la relación entre teoría y práctica no se concebía de otro modo que como una solidaridad o una jerarquía: la actividad se funda en conocimientos que la iluminan; el conocimiento exige a los actos el dominio de la materia, de las almas y de las sociedades —una técnica, una moral,12 una política— que procuran la paz necesaria para su ejercicio puro. Nosotros vamos más lejos y, a riesgo de que parezca que confundimos teoría y práctica, tratamos una y otra como modos de la trascendencia metafísica. La confusión aparente es intencional y constituye una de las tesis de este libro (Lévinas, 2002: 55). sea seguro para un Yo sin obstáculos por cualquier Otro que no sea su propio otro, es decir, la condición necesaria para su propia posibilidad, algo para ser usado, poseído, disfrutado. Mientras que la metafísica es el deseo que apunta hacia algo totalmente distinto, hacia lo Absolutamente Otro. La ontología occidental ha reducido sistemáticamente el Otro a lo Mismo» (Wesphal, 2008: 13). 12 «Lévinas usa las expresiones moralidad y ética intercambiablemente, no obstante, prefiere el uso de ética que se deriva del griego ‘ethos’, aunque algunas veces no distingue ética de moralidad, sobre todo cuando quiere diferenciar ética como “filosofía primera” de las reglas específicas de la moralidad» (Bernstein, 2004: 253). GABRIEL LEIVA RUBIO. La idea de infinito: un desfundar lo total y fundar lo ético. Lo ético no es entonces un logos superior, un esquema otro donde la totalidad encuentra su adecuación y sistematización. Lo ético aparece en Lévinas, sobre todo, como una suspensión o ruptura de esa dualidad esquemática de teoría y praxis, desborde de lo esquemático en una trascendencia que es la raigambre más fundante de la relación entre lo Uno y lo absolutamente Otro. Es con este gesto como Lévinas impugna esa diferencia radical en la generalidad del concepto que supedita y subsume al Otro en los dominios irrestrictos de la identidad del logos. Lévinas se percata de que la alteridad se resiste al esquema del pensamiento, precisamente por estar constituida ella de un enigma indescifrable. Es por ello que Lévinas protesta contra la injusticia de un pensamiento que, incapaz de experimentar pacíficamente la alteridad del Otro, ha ejercido su poder hasta anularla. Es desde este punto de vista que la violencia teórica parece haber justificado la violencia ética. La tarea pendiente de la filosofía sería entonces salvaguardar lo humano de aquella faz del ser que termina siempre decantándose por la guerra. ¿Cómo? Proponiendo la experiencia del Otro y como alteridad absoluta. En resumen, donde la totalidad ontologizante dibujaba una identidad irrestricta y esquemática, Lévinas propone una filosofía de la alteridad, pero no de una que pueda relativizarse o reducirse a una identidad dialécticamente mediada, sino una alteridad trascendente (metafísica) que no permita negociación alguna con el entendimiento y sus representaciones. La alteridad humana no se piensa a partir de la alteridad puramente formal y lógica por la que se distinguen unos de otros los términos de toda multiplicidad [...] La noción de alteridad trascendente se nos aparece como una diferencia que contrasta con todas las demás diferencias, no solamente como una cualidad diferente de todas las demás, sino como la cualidad misma de la diferencia (Lévinas, 2002: 74). El Yo, el Otro y la relación que los une, no deben caer bajo el encanto representacional del pensamiento. Ni el Yo puede disolverse en el Otro, ni el Otro en el Yo, así como tampoco la relación que los liga (Nosotros) puede tornarse un esquema que trascienda explicativamente su relación, pues, de ser así, llegaría la totalidad, es decir, la guerra.13 La alteridad levinasiana es trascendente, sí, pero entendiendo esta trascendencia no como un predicado formal, sino como un presupuesto metafísi13 «Si la Alteridad acaba desapareciendo y solamente queda lo mismo, irrumpe la Barbarie, la negación de la Diferencia, de la Exterioridad, de la Trascendencia» (Mélich, 1998: 14). 11 12 RECERCA · DOI: http://dx.doi.org/10.6035/recerca.7207 · ISSN electrónico: 2254-4135 - pp. 1-24 co que antecede y funda cualquier gesto o pretensión representacional del entendimiento.14 De forma análoga a la alteridad, el Yo no puede quedar sujeto ni a un esquema ideal que satisfaga condiciones a priori, ni a relativismos de ningún tipo. Por el contrario, el Yo, el Otro y la relación que los junta no puede ser un constructo ideal (o material) que permanece siempre idéntico a sí mismo, sino un proceso de recuperación que anude definitivamente lo Uno a lo Otro de forma tal que resista a la pretensión esquemática del logos. Ser yo es, fuera de toda individuación a partir de un sistema de referencias, tener la identidad como contenido. El yo no es un ser que permanece siempre el mismo, sino el ser cuya existencia consiste en Identificarse, en recobrar su identidad a través de todo lo que le acontece. Es la identidad por excelencia, la obra original de la identificación (Lévinas, 2002: 60). Si bien Lévinas no explica a qué se refiere cuando dice «tener la identidad como contenido», es evidente que la identidad que refiere, la del Yo (ipseidad), es un movimiento. Pero no un movimiento que se da por la oposición de contrarios (danza bélica inagotable), puesto que esto supondría, nuevamente, una configuración ideal que, o bien es el causante y el centro de una totalidad, o bien es una parte de aquella.15 Ambas lógicas, según lo hasta aquí analizado, no hacen sino entregar al pensamiento la llave de la verdad absoluta, haciendo derivar la verdad de la misma instancia que la genera. Paradoja absurda y reiterada. De esta instancia del análisis se derivan dos interrogantes que parece imposible ignorar: si el pensamiento no basta para abarcar al Yo, al Otro y a la «La relación metafísica no podría ser, propiamente hablando, una representación, porque lo Otro se disolvería allí en el Mismo: toda representación se deja interpretar esencialmente como constitución trascendental. El Otro con el que el metafísico está en relación y que reconoce como otro no está simplemente en otro sitio. Le ocurre como con las ideas de Platón, que, según la fórmula de Aristóteles, no está en ningún lugar. El poder del Yo no franqueará la distancia que indica la alteridad de lo Otro. Ciertamente mi intimidad más íntima me aparece extraña u hostil; los objetos usuales, los alimentos, el mismo mundo que habitamos, son otros con relación a nosotros. Pero la alteridad del yo y del mundo habitado es solo formal. Cae bajo mis poderes en un mundo en el que habito […]. Lo Otro metafísico es otro como una alteridad que no es formal, con una alteridad que no es un simple revés de la identidad, ni de una alteridad hecha de resistencia al Mismo, sino con una alteridad anterior a toda iniciativa, a todo imperialismo del Mismo» (Lévinas, 2002: 62). 15 «Si el Mismo se identificase por simple oposición a lo Otro, formaría ya parte de una totalidad que englobaría al Mismo y lo Otro» (Lévinas, 2002: 62). «En el “conócete a ti mismo” no se logra su última etapa. No porque el “conócete a ti mismo” sea limitado o de mala fe, sino porque, por sí mismo, solo es libertad, es decir, arbitrario e injustificado y, en este sentido odioso; es yo, egoísmo. El ateísmo del yo señala la ruptura de la participación y, por lo tanto, la posibilidad de buscarse una justificación, es decir, una dependencia frente a una exterioridad sin que esta dependencia absorba el ser dependiente, sostenido por hilos invisibles» (Lévinas, 2002: 111). 14 GABRIEL LEIVA RUBIO. La idea de infinito: un desfundar lo total y fundar lo ético. relación que los liga, ¿cómo es posible entonces articular algún tipo de saber edificante, operante, práctico? ¿Puede articularse alguna idea que anude a los hombres entre sí de manera efectiva sin que esto suponga la armazón de una nueva totalidad que, a la postre, termine por derivar en la guerra? O, en otros términos, ¿puede hablarse de algo una vez ha quedado suspendido el pensamiento para articular una relación entre el Yo y el Otro? 4. EL ROSTRO DEL OTRO O EL QUIEBRE DE LO TOTAL Para intentar responder a las interrogantes que cierran el epígrafe anterior es fundamental partir de un presupuesto: una posible suspensión del elemento intelectivo como fundamento necesario para desarticular la posible emergencia de la totalidad. Es decir, donde «…el pensamiento consiste en hablar» (Lévinas, 2002: 64), en discursar o estructurar, ha de haber algún medio, no totalizador o lógico, que ligue lo Uno (el Mismo) y lo Otro en una relación no estructurada o esquemática. Y esta relación, que suspendiendo al pensamiento es capaz de ligar lo Uno y el Otro, Lévinas la nombra religión.16 Del origen etimológico del término religión (re-ligāre, ‘volver a ligar’) y del uso que le asigna Lévinas en su discurso, se derivaría que la relación con el Otro, al margen del pensamiento, está sucediendo, o debe suceder, por segunda vez. Es decir, ese volver a ligar de lo religioso presupondría un extravío y la necesaria recuperación de eso que yace perdido. Desde este sentido, teológicamente contornado, Lévinas articula su noción. Es decir, el Yo y el Otro deben religarse prescindiendo de todo aquello que el Yo ha hecho con su racionalidad para hacerse del Otro. Y esta religión del Otro es la tarea de todo el proyecto ético levinasiano. Entender en el Otro la fundamentación del Yo entraña una potencia enorme, pero volver al Otro, a partir de la mera presentación del Otro, es algo inconmensurable. Aun habiéndose aclarado esto, quedan irresueltas las preguntas ya planteadas. ¿Cómo tiene lugar esta religión, este volver a ligar el Yo con el Otro sin que irrumpa el pensamiento y su ejercicio totalizador? Lévinas responde: El modo por el cual se presenta el Otro, que supera la idea de lo Otro en mí, lo llamamos, en efecto, rostro. Este modo no consiste en figurar como tema ante mi mirada, en expo16 «Reservamos a la relación entre el ser mundano y el ser trascendente que no lleva a ninguna comunidad del concepto ni a ninguna totalidad —relación sin relación— el término de religión» (Lévinas, 2002: 103). 13 14 RECERCA · DOI: http://dx.doi.org/10.6035/recerca.7207 · ISSN electrónico: 2254-4135 - pp. 1-24 nerse como un conjunto de cualidades formando una imagen. El rostro del Otro destruye en todo momento y desborda la imagen plástica que él me deja, la idea a mi medida y a la medida de su ideatum: la idea adecuada. […] El rostro, contra la ontología contemporánea, aporta una noción de verdad que no es el develamiento de un Neutro impersonal, sino una expresión (Lévinas, 2002: 74-75). El rostro aparece en el vocabulario de Lévinas como la exterioridad de la presencia del Otro (de ahí que el subtítulo de TI sea Un ensayo sobre la exterioridad), la irrupción de ese enigma inconmensurable a partir del cual el Yo y lo Otro se relacionan y reconocen. El rostro parece ser la evidencia absoluta de que la verdad no proviene del ser, esa neutralidad impersonal del saber ontológico, sino de una presencia, que desnuda y desborda en su acto de aparición cualquier posible representación del Otro por parte del Yo. El rostro se aparece entonces como el medio de expresión de la alteridad y de su valor inconmensurable, centro metafísico que derriba las pretensiones de la ontología: la externalidad del Otro en su forma relacional. El rostro levinasiano no forma parte de una teoría o esquema. Tampoco es un tema ante mi mirada, ni un conjunto de cualidades que forman una imagen. El rostro no puede representarse, sino presentarse y ejercer sobre el Yo la potencia incontenible de la verdad del Otro. La noción de rostro como verdad del Otro hace de la verdad un movimiento relacional imposible de refrendar en términos gnoseológicos, ontológicos o estéticos. El rostro es metafísico, y los es precisamente en la medida en que es ético. Es, en definitiva, la presentación de lo trascendente sin la mediación del pensamiento, trayendo consigo la posibilidad de la experiencia que asegura la verdad de lo humano contra la totalización. Pero, además, el rostro también desborda la historia, precisamente a partir de esa relación enigmática que instaura entre el Yo y el Otro y que el juicio histórico de la totalidad no puede contener ni explicar.17 El rostro contiene algo que, paradójicamente, no puede ser contenido, una característica que lo constituye como el núcleo de la propuesta de Lévinas y que lo determina esencialmente. Constituye la presentación de una idea que contiene, como determinación esencial, desbordarse 17 «A diferencia de una filosofía de la trascendencia que sitúa en otra parte la verdadera vida a la cual accedería el hombre al escaparse de aquí, en los instantes privilegiados de la elevación litúrgica, mística o al morir —y a diferencia de una filosofía de la inmanencia en la que se aprehendería verdaderamente el ser cuando todo “Otro” (causa de guerra), englobado por el Yo, se desvaneciera al término de la historia—, nos proponemos, en cambio, describir en el desarrollo de la existencia terrestre, de la existencia económica como la llamamos, una relación con el Otro que no acaba en una totalidad divina o humana, una relación que no es una totalización de la historia, sino la idea de lo infinito. Tal relación es la metafísica misma» (Lévinas, 2002: 76). GABRIEL LEIVA RUBIO. La idea de infinito: un desfundar lo total y fundar lo ético. a sí misma y que, una vez definida, deja necesariamente de ser lo que es.18 A resultas, aparece la idea de lo infinito: Nuestros análisis están dirigidos por una estructura formal: la idea de lo Infinito en nosotros. Para tener la idea de lo Infinito, es necesario existir separado. Esta separación no puede producirse solo como un eco de la trascendencia de lo Infinito. Si no, la separación se sostendría en una correlación que restauraría la totalidad y haría ilusoria la trascendencia. Ahora bien, la idea de lo Infinito es la trascendencia misma, el desbordamiento de una idea adecuada. Si la totalidad no puede constituirse, es porque lo Infinito no se deja integrar. No es la insuficiencia del Yo la que impide la totalidad, sino lo Infinito del Otro (Lévinas, 2002: 103). En este trascendental fragmento, Lévinas asienta dos de sus tesis principales: la idea de lo infinito (la trascendencia misma), que impide la constitución de la totalidad porque se resiste a su integración; y el viraje de lo metafísico, que pondera la verdad en lo infinito del Otro y no en la potencia del Yo. Partiendo de las consideraciones anteriores hay dos ideas importantes que destacar sobre la idea de infinito: a) no se deja integrar a la totalidad por su propia determinación esencial; b) no queda contenida en el Yo, lo que hace imposible que la ipseidad se haga cargo del sentido de lo total (subjetivismo). Gracias a este movimiento, Lévinas invalida cualquier relativización del Otro, lo mismo por la totalidad (sistema, esquema), que por la potencia imaginativa o intelectual del Yo. El Otro, gracias a la idea del infinito, queda a salvo de esquemas, evitando así que pueda ser usado, intelectualizado, reducido.19 Salvaguardar al Otro es posible en el proyecto levinasiano, precisamente, gracias a la irreductibilidad enigmática de su rostro y a la potencia infinita de su latencia. De ahí que Lévinas concluya: «la idea de totalidad y la idea de lo Infinito difieren precisamente en esto: la primera es puramente teorética, la otra es moral» (Lévinas, 2002: 106). Nuevamente, la ética es la verdadera metafísica. «[…] si experiencia significa precisamente relación con lo absolutamente otro —es decir, con lo que siempre desborda al pensamiento— la relación con lo infinito lleva a cabo la experiencia por excelencia» (Lévinas, 2002: 51). 19 «La idea de infinito que el rostro encierra es para Lévinas la clave por la que el pensamiento se pone en relación con lo que va más allá de su capacidad» (Hand, 2009: 42). 18 15 16 RECERCA · DOI: http://dx.doi.org/10.6035/recerca.7207 · ISSN electrónico: 2254-4135 - pp. 1-24 5. LA IDEA DE LO INFINITO La idea de lo infinito es, probablemente, la más definitoria del corpus lévinasiano. Pues, donde resultan imprescindibles las nociones de rostro, Otro, ética o trascendencia, el concepto de infinito implica la condición de posibilidad, no solo del quiebre de la totalidad, sino de cada una de las nociones anteriores. Se hace entonces imprescindible intentar una aproximación hermenéutica a este concepto a fin de desentrañar sus determinaciones y su alcance en la obra objeto del presente análisis. La primera mención al concepto de infinito propone: ¿Y la filosofía misma no se define, a fin de cuentas, como una tentativa de vivir en la evidencia, al oponerse a la opinión del prójimo, a las ilusiones y a la fantasía de su propia subjetividad? A menos que la evidencia filosófica remita por sí misma a una situación que no pueda ya expresarse en las categorías de la «totalidad». A menos que el no-saber en el que comienza el saber filosófico, no coincida con la nada a secas, sino solo con la nada de los objetos. Sin sustituir la filosofía por la escatología, sin «demostrar» filosóficamente las «verdades» escatológicas: se puede ascender a partir de la experiencia de la totalidad a una situación en la que la totalidad se quiebra, cuando esta situación condiciona la totalidad misma. Tal situación es el resplandor de la exterioridad o de la trascendencia en el rostro del otro. El concepto de esta trascendencia, rigurosamente desarrollado, se expresa con el término infinito (Lévinas, 2002: 50-51). Con ello, Lévinas evoca la clásica doxa y la pretensión de la filosofía de vivir por encima de esta. Sin embargo, al desechar la doxa, lo mismo por ser un conocimiento cambiante, aparente, o por no constituir, en sí mismo, un movimiento que llegue a la verdad a partir de la propia inmanencia del pensamiento y la evidencia que este requiere para fundar su logos, puede incurrirse en la formulación de una totalidad. La filosofía, cree Lévinas, no solo ha buscado la verdad en la inmanencia del pensamiento, sino que ha necesitado expresarla en marcos de referencia que la atrapen, lo cual ha conducido a que la verdad pareciese tener que adecuarse a la filosofía y no viceversa. De ahí la totalización, el esquema, la paralización y la indeterminación del ser, el dualismo insostenible entre teoría y praxis. Es por ello que Lévinas no se decanta por eliminar la evidencia de la que históricamente se ha nutrido la filosofía, sino, por el contrario, le exige a la filosofía y a su intrínseco movimiento de saber que se ajuste, irrestrictamente, a dicho movimiento. Es decir, le exige consecuencia al movimiento del saber filosófico, una consecuencia que co- GABRIEL LEIVA RUBIO. La idea de infinito: un desfundar lo total y fundar lo ético. mienza y termina necesariamente en ese reencuentro definitivo con el original y auténtico sentido del ser. Y obsérvese que el aludido reencuentro, por su propia naturaleza, exige una relación con el ser que no quede circunscrita a los límites fijados por la totalidad; que no tome a la Nada absoluta como el origen impoluto desde donde el filosofar ha de iniciar su marcha. Lévinas, protestando contra esa pretensión que cargan siempre las demostraciones filosóficas (fundadas en la evidencia), le demanda a la filosofía la honestidad y la modestia de la no demostración. Le exige que constate, a partir de la misma totalidad instaurada por su movimiento, la existencia de una situación que la antecede, metafísicamente hablando. Una situación que la quiebra y la hace saltar en pedazos, y que, traducida al ser como experiencia, es lo que Lévinas llama «el resplandor de la exterioridad o de la trascendencia en el rostro del otro» (Lévinas, 2002: 51). Y, dicho resplandor, necesariamente, ciega la mirada, anula la visión. Se trata, entonces, de un resplandor que no puede traducirse en palabras ni ser expresado en juicios de razón. Así como el prisionero de la famosa alegoría platónica queda cegado por la luz del sol una vez ha salido de las sombras, el filósofo ha de temblar de vergüenza20 ante la imposibilidad de explicar la potencia del rostro del Otro. Y esta experiencia inexplicable que desborda y termina por quebrar la armazón de lo total en el resplandor del rostro del Otro es lo infinito. Por esta razón, el rostro del Otro no se deja abarcar, ni por la doxa, ni por ese pensamiento que se funda en las evidencias. Muy por el contrario, la idea de lo infinito que resplandece en el rostro del Otro, esa que no se deja englobar, porque de hacerlo no sería infinita,21 ha de ser la condición de posibilidad de toda opinión y de todo pensamiento.22 Se añade a los atributos de lo infinito el también ser la condición de posibilidad de la verdad del Otro, del Yo y del Nosotros, expresada como relación esencial que liga lo Uno a lo Otro. Dicho condicionante queda explicitado «La libertad que puede tener vergüenza de sí misma funda la verdad» (Lévinas, 2002: 106); «a causa de la presencia ante el rostro del Otro, el hombre no se deja engañar por su glorioso triunfo de viviente y, distinto del animal, puede conocer la diferencia entre el ser y el fenómeno, reconocer su fenomenalidad, el defecto de su plenitud, defecto inconvertible en necesidades y que, más allá de la plenitud y del vacío, no podrá llenar» (Lévinas 2002: 197). 21 «La limitación solo se produce en una totalidad, mientras que la relación con el Otro hace estallar el tope de la totalidad» (Lévinas, 2002: 189). 22 «[…] se desvanece como el viento cuando el pensamiento la toca, o se revela como ya interior a este pensamiento. En la idea de lo infinito se piensa lo que permanece siempre exterior al pensamiento. Condición de toda opinión, es también condición de toda verdad objetiva» (Lévinas, 2002: 51). 20 17 18 RECERCA · DOI: http://dx.doi.org/10.6035/recerca.7207 · ISSN electrónico: 2254-4135 - pp. 1-24 cuando Lévinas señala que «la idea de lo infinito es el espíritu antes de presentarse a la distinción de lo que descubre por sí mismo y de lo que recibe de la opinión» (Lévinas, 2002: 51). Siendo así, la opinión y el pensamiento, como momentos de reconocimiento, quedan amparados bajo una idea que les aporta basamento y sentido. El Otro, el Yo y el Nosotros son entonces un misterio que se resiste a ser revelado por el pensamiento, un enigma anclado, únicamente, a la irrestricta inmensidad del infinito. Provoca, cuando menos, estupor desvelar que en el origen y la esencia del espíritu —esa condición de posibilidad de lo pensado, potencia excelsa de conceptos, especulaciones y entramados de ideas— subyace un algo que trasciende cualquier entendimiento suyo: La relación con lo infinito no puede, ciertamente, expresarse en términos de experiencia, porque lo infinito desborda el pensamiento que lo piensa. En este desbordamiento se produce precisamente su infinición misma, de tal suerte que será necesario aludir a la relación con lo infinito de otro modo que en términos de experiencia objetiva. Pero si experiencia significa precisamente relación con lo absolutamente otro —es decir, con lo que siempre desborda al pensamiento— la relación con lo infinito lleva a cabo la experiencia por excelencia (Lévinas, 2002: 51). Pero ¿cómo explicar la relación de lo Uno y lo Otro si ambos, como condición fundacional, son irreductibles al pensamiento? En otras palabras, ¿cómo puede relacionarse una ipseidad, cuya naturaleza interior ha sido fundada en una idea que se resiste a ser definida, con otra ipseidad de igual condición? Si el Yo permanece como un enigma tanto a la opinión como al pensamiento, ¿cómo pueden relacionarse el Uno y el Otro? Sobre este decisivo dilema Lévinas se pronuncia, según es usual en él, de manera indirecta: Este libro presentará la subjetividad, recibiendo al Otro, como hospitalidad. En ella se lleva a cabo la idea de lo infinito. La intencionalidad, en la que el pensamiento sigue siendo adecuación al objeto, no define la conciencia en su nivel fundamental. Todo saber en tanto que intencionalidad supone ya la idea de lo infinito, la inadecuación por excelencia (Lévinas, 2002: 53). Siguiendo la peculiar lógica discursiva de Lévinas, no ha de esperarse una definición positiva que explique de modo claro y directo el acto que liga o relaciona lo Uno con lo Otro. En lugar de tales precisiones, apela a lo que no puede ser dicho de esta relación. Si bien alude a esta relación bajo el concepto de hospitalidad, este término, lejos de que explicar la relación, lo que hace es GABRIEL LEIVA RUBIO. La idea de infinito: un desfundar lo total y fundar lo ético. nombrarla.23 No quiere decir esto que Lévinas se declare incapaz de explicar esta relación en términos conceptuales, sino que este recibimiento del Otro por parte del Yo no debe ser explicado, deducido o racionalizado, puesto que esto supondría retornar, nuevamente, a esa totalización que ejerce el pensamiento cuando toma al Otro como objeto de su impronta. Y es que, donde el pensamiento impugna el saber, Lévinas sostiene el deber. Lo metafísico es ético y no ontológico, porque el Otro desborda cualquier idea del Yo, y lo ético es metafísico, porque el deber es para con una idea más allá de lo óntico, una idea que se resiste a ser englobada por otra. Además, el modo en que se relacionan lo Uno y lo Otro no debe ser reducido a una relación entre términos cualesquiera. No se trata de explicar una fórmula o un principio, o de pensar que el Uno se encuentre en un sitio determinado con respecto al Otro.24 La diferencia entre el Yo y el Otro es tan vacía como la diferencia entre el Yo y el mundo en la medida que es formal y, por tanto, ausente de contenido: lo Otro metafísico es otro como una alteridad que no es formal, con una alteridad que no es un simple revés de la identidad, ni de una alteridad hecha de resistencia al Mismo, sino con una alteridad anterior a toda iniciativa, a todo imperialismo del Mismo. Otro con una alteridad que constituye el contenido mismo de lo Otro. Otro de una alteridad que no limita al Mismo, porque al limitar al Mismo, lo Otro no sería rigurosamente Otro (Lévinas, 2002: 62). Lo Otro se mantiene idéntico a sí mismo. No entra en una relación formal con el Yo, con el Mismo. No hay mediación lógica entre el Yo y lo Otro, pues, de lo contrario, lo Otro no sería absolutamente Otro, sino un algo relativo. A este respecto Bernhard Waldenfels señala acertadamente: «el Otro está inmediatamente presente y auto-presente. La alteridad mantiene el carácter de un fenómeno, o, más exactamente, el otro, al estar separado de la totalidad de los El término hospitalidad, con muy pocas menciones a lo largo de TI (apenas ocho menciones), bien puede equipararse al de reconocimiento o recogimiento del Otro por el Yo. El concepto de hospitalidad aparece, precisamente, cuando esa separación entre lo Uno y lo Otro que marca la interioridad reconoce que el Yo es también el Otro, desbordando así la inmanencia del pensamiento y devolviendo una trascendencia ético-metafísica que solo puede ser reconocida a través de la expresión del rostro del Otro. «El acontecimiento metafísico de la trascendencia, el recibimiento del Otro, la hospitalidad» (Lévinas, 2002: 265); «Al pensamiento metafísico en el que alguien finito tiene la idea de lo infinito —en el que se produce la separación radical y, simultáneamente, la relación con el otro— le hemos reservado el término de intencionalidad, de conciencia […], recibimiento del rostro, hospitalidad y no tematización» (Lévinas, 2002: 303). 24 «El Otro con el que el metafísico está en relación y que reconoce como otro no está simplemente en otro sitio» (Lévinas, 2002: 62). 23 19 20 RECERCA · DOI: http://dx.doi.org/10.6035/recerca.7207 · ISSN electrónico: 2254-4135 - pp. 1-24 seres, es el fenómeno por excelencia cuya epifanía incluye la ausencia» (Waldenfels, 2004: 73-74). El Otro está entonces absolutamente separado25 en una suerte de presencia ausente que la totalidad no puede percibir. Pero, con estas precisiones, lejos de aclarar las preguntas anteriores, pareciese que se complejiza el asunto ya que, donde la ipseidad fungía como un misterio indescifrable para sí misma y para el entendimiento, ahora el Otro se aparece como absolutamente separado y como ausencia inmediatamente presente. Pareciera que la relación entre el Yo, lo Uno o el Mismo y la alteridad de absolutamente Otro no puede ser resuelta. Pero Lévinas dice: Somos el Mismo y el Otro. La conjunción no indica aquí ni adición ni poder de un término sobre otro. […] la relación del Mismo y del Otro —a la cual pareciéramos imponer condiciones tan extraordinarias— es el lenguaje. El lenguaje lleva a cabo, en efecto, una relación de tal suerte que los términos no son limítrofes en esta relación, que el Otro, a pesar de la relación con el Mismo, sigue siendo trascendente al Mismo. La relación del Mismo y del Otro —o metafísica— funciona originalmente como discurso, en el que el Mismo, resumido en su ipseidad de «yo» —de ente particular único y autóctono— sale de sí (Lévinas, 2002: 63). El lenguaje es la propuesta levinasiana de espacio relacional donde se da la identidad de lo Uno y lo Otro. Pero esta identidad del somos que explica Lévinas mediante el lenguaje no es una identidad formal donde lo Uno y lo Otro son términos idénticos o contiguos, sino que el lenguaje, mediante el gesto del decir, los logra reunir en una identidad que no solo mantiene la trascendencia y autonomía del Otro, sino que logra que el Yo salga de sí.26 Es esta la definitiva esencia del lenguaje: la relación con el Otro27 sin que el Otro pierda lo que es de suyo: El Otro no se me opone como otra libertad, es decir, parecida a la mía y, por lo tanto, hostil a la mía. Otro no es otra libertad tan arbitraria como la mía, ya que en ese caso franquearía inmediatamente lo infinito que me separa de ella para entrar bajo el mismo concepto. Su alteridad se manifiesta en un señorío que no conquista, sino que enseña. La enseñanza no es una especie de género llamado dominación, una hegemonía que funciona en seno de una totalidad, sino la presencia de lo Infinito que hace saltar el círculo cerrado de la totalidad (Lévinas, 2002: 189). «El metafísico está absolutamente separado» (Lévinas, 2002: 59). «La originalidad de la relación, a nuestro parecer, consiste en la autonomía del ser separado» (Lévinas, 2002: 87). 27 «[…] la esencia del lenguaje es la relación con el Otro» (Lévinas, 2002: 220). 25 26 GABRIEL LEIVA RUBIO. La idea de infinito: un desfundar lo total y fundar lo ético. La alteridad del Otro enseña, precisamente, porque no quiere conquistar, puesto que la mera presencia de su ausencia desborda esa potencia ontológica que hasta el momento la ipseidad creía ilimitada. El lenguaje, presente en esa relación, no es entonces herramienta o medio para adecuar el mundo a la realidad de los pensamientos, o un aparato de descripción hecho de proposiciones lógicas que determinan la validez o no de una argumentación cualquiera, sino la expresión viva de lo Otro como infinitamente Otro en su proceso de reconocimiento. De ahí que Josep Maria Esquirol indique que: El Decir es la expresión de la anarquía, de una situación donde la asimetría nada tiene que ver con el dominio, sino con la acogida y el recibimiento. Asimetría de la responsabilidad, y no asimetría del dominio. Mientras la retórica de la persuasión está al servicio del poder, el decir anárquico no se preocupa por asegurar los hitos discursivos: no insiste en el: «es que yo he dicho que...»; lo Dicho queda desplazado hacia un segundo plano (Esquirol, 2012: 115). De tal forma, la relación entre el Yo y el Otro en el lenguaje no traduce una experiencia de totalidad. El Otro permanece impasible ante la potencia de la inteligencia del Yo porque su condición de absoluto no desaparece en dicho encuentro.28 El cara a cara no supone entonces un desafío de conquista, ni una lucha a muerte por el reconocimiento —como pensaba Hegel—, sino una proximidad hospitalaria que se resiste a cualquier operatoria lógica.29 Puede inferirse entonces que el lenguaje que relaciona lo Uno y lo Otro no consiste en hablar, tarea que le es propia al pensamiento, sino en restablecer esa otrora potencia metafísica de lo ético a partir de la presentación expresiva del rostro del Otro. El infinito que se presenta en el rostro del Otro como exterioridad es expresado desde ese decir que enseña y ampara. Es importante notar que la relación que instaura el lenguaje acontece preservando los términos no como opuestos, sino como idénticos a sí mismos. El Otro se relaciona con el Yo de manera metafísica, no porque el Otro sea una idea que colma y explica lo total, sino, precisamente, porque este Otro es ético, «Lo Otro metafísicamente deseado no es “otro” como el pan que como, o como el país en que habito, como el paisaje que contemplo, como a veces, yo mismo a mí mismo, este yo, este otro. De estas realidades, puedo nutrirme y, en gran medida, satisfacerme, como si me hubiesen simplemente faltado. Por ello mismo, su alteridad se reabsorbe en mi identidad de pensante o de poseedor. El deseo metafísico (sin embargo) tiende hacia lo totalmente otro, hacia lo absolutamente otro» (Lévinas, 2002: 57). 29 «Una relación cuyos términos no forman una totalidad solo puede producirse, pues, en la economía general del ser como tránsito del Yo al Otro, como cara a cara, como perfilando una distancia en profundidad —la del discurso de la bondad, del Deseo— irreductible a aquello que la actividad sintética del entendimiento establece entre los términos diversos —mutuamente— que se ofrecen a su operación sinóptica» (Lévinas, 2002: 63). 28 21 22 RECERCA · DOI: http://dx.doi.org/10.6035/recerca.7207 · ISSN electrónico: 2254-4135 - pp. 1-24 es decir, irreductible a un sistema que condicione o explique su esencia.30 El infinito que resplandece en el rostro del Otro sitúa al hombre ante la rotunda constatación de que su saber, su razón y su pensamiento se encuentran necesariamente precedidos por una situación que les resulta inexplicable, donde los límites que contemplaba hasta entonces su irrestricta libertad han sido borrados por la hospitalidad y el recogimiento de que es capaz la potencia de la alteridad en su inmediata presentación. CONCLUSIÓN Habiéndose realizado el análisis hermenéutico de los principales conceptos que estructuran y definen la propuesta levinasiana en TI, puede concluirse que lo ético es un acto de protesta contra el concepto de totalidad, determinado este, esencialmente, por su inmanencia. Lo ético aparece aquí como un algo exterior que se resiste a ser categorizado, definido, reducido a un límite; constituyendo como un concepto cuya cualidad esencial es, paradójicamente, contener más de lo que es capaz. Y es esta la función que Lévinas le confiere al rostro del Otro: ser evidencia de que el infinito inconmensurable que lo dota de sentido es, a la vez, la determinación esencial e inabarcable de su más íntima naturaleza. Por eso, el Otro no es un otro cualquiera, sino un absolutamente Otro, un enigma con el que nada puede hacer el pensamiento. Habría que coincidir en que Lévinas no concibe lo ético entonces como un sistema de ideas que regulan la conducta y la responsabilidad del hombre. No toma entonces la pura subjetividad como fundamento de lo ético (Autonomie en Kant),31 ni como espíritu finito objetivado en instituciones y leyes políticas (Sittlickheit en Hegel).32 Lo ético no se regula, ni desde adentro, ni desde «el establecimiento de este primado de lo ético, es decir, de la relación de hombre a hombre […], primado de una estructura irreductible en la cual se apoyan todas las demás (y en particular todas las que, de una manera original, parecen ponernos en contacto con lo sublime impersonal, estético u ontológico)...» (Lévinas, 2002: 102). 31 «[…] la autonomía de la voluntad es el único principio de todas las leyes morales y de los deberes conforme a ellas; toda heteronomía del albedrío, en cambio, no solo no funda obligación alguna, sino que más bien es contraria al principio de la misma y de la moralidad de la voluntad» (Kant, 1994: 52, § 8). Esta noción de autonomía debe ser vinculada con otro supuesto: la ley fundamental de la razón práctica, donde Kant explica su famoso imperativo categórico: «Obra de tal modo que la máxima de tu voluntad pueda valer siempre, al mismo tiempo, como principio de una legislación universal» (Kant, 1994: 47, § 7). 32 La expresión alemana Sittlichkeit no tiene una traducción directa al castellano, pero en el ámbito académico se creó el término eticidad. En ocasiones, el término se ha traducido al francés y al inglés como ‘vida ética’. De Zan pro30 puso traducir eticidad como ‘mundo ético’. J. M. Ripalda planteó la traducción ‘ética comunitaria’. Las dificul- GABRIEL LEIVA RUBIO. La idea de infinito: un desfundar lo total y fundar lo ético. afuera, porque, sencillamente, no es regulable; ni se circunscribe más que a la expresión rotunda e irreductible del infinito manifestándose en el rostro del Otro. De ahí que lo ético, más que un punto de vista, resulte una óptica o una visión sin imagen. Lo ético rompe entonces con lo dual, suspendiendo tanto lo teórico como lo práctico en una trascendencia metafísica que acontece a partir de la inconmensurable presencia del Otro. Donde lo ético —en los términos de la filosofía tradicional de occidente— reducía o relativizaba la alteridad a un sistema que ponderaba al ser como la quintaesencia del todo, Lévinas imprime un giro que hace de lo ético lo preponderante, lo esencial y primigenio. La filosofía primera como ética y la ética como filosofía primera. Pero tal giro no supone un nuevo sistema de ideas que abarque y explique el anterior. No se trata de un logos más omniabarcante que su antecesor, sino de un enigma inextricable del cual la razón no puede dar parte. En definitiva, Lévinas no quiere explicar al Otro sino salvarlo del esquema totalizador del pensamiento, ese que termina siempre decantándose por la guerra. Él se propone salvar al Otro de la guerra. De ahí que la relación ética entre el Yo y lo Otro no sea una relación conceptual o intelectual entre mismidades y sí una relación entre alteridades absolutamente infinitas e irreductibles a cualquier esquema de pensamiento. Es por esta razón que Lévinas llega a la idea de infinito, idea que, una vez pensada, el propio pensamiento tiene que reconocer su inoperancia, puesto que la idea de infinito desborda cualquier definición que sobre ella se haga.33 BIBLIOGRAFÍA Bernstein, Richard J. (2004) Evil and the temptation of theodicy. En Critchley, Simon y Bernasconi, Robert (ed.). The Cambridge Companion to Lévinas. Cambridge: Cambridge University Press. De Zan, Julio (2009). La filosofía social y política de Hegel. Trabajo y propiedad en la filosofía práctica. Buenos Aires: Ediciones del Signo. tades de traducción han obligado a mantener esta nueva palabra y conferirle significado, en referencia a la vida ética o mundo ético (véase De Zan, 2009: 171). 33 «[…] la idea de lo infinito tiene esto de excepcional: que su ideatum deja atrás su idea, ya que, para las cosas, la coincidencia total de sus realidades, objetivas y formales, no está excluida» (Lévinas, 2002: 72). 23 24 RECERCA · DOI: http://dx.doi.org/10.6035/recerca.7207 · ISSN electrónico: 2254-4135 - pp. 1-24 Dostoievski, Fiodor (2000). Los hermanos Karamazov. Barcelona: Cátedra. Esquirol, Josep M. (2012). La primera palabra, o la esencia del lenguaje como amparo. Ágora, 31(1), 103-120. doi: https://doi.org/10.15304/ag .31.1.227 Hand, Seán (2009). Emmanuel Lévinas. London: Routledge, Taylor & Francis Group. Kant, Immanuel (1994). Crítica de la razón práctica. Salamanca: Sígueme. Lévinas, Emmanuel (1979). Totality and Infinity, An essay of exteriority. Boston: Martins Nijhoff Publishers. Lévinas, Emmanuel (1987). Totalité et Infini. Essai sur l'extériorité. Kluwer Academie. Lévinas, Emmanuel (1993). El Tiempo y el Otro. Introducción de Félix Duque. Barcelona: Paidós, UAB. Lévinas, Emmanuel (2002). Totalidad e infinito. Salamanca: Sígueme. Mélich, Joan-Carles (1998). Totalitarismo y fecundidad. La filosofía frente a Auschwitz. Barcelona: Anthropos. Sartre, Jean Paul (1937). L’Etre et le Neant. Paris: Gallimard. Vidal Labajos, Sebastian (2023). Tiempo, subjetividad y dominación social en las sociedades contemporáneas: de la dominación abstracta a la ética neoliberal del tiempo. Recerca. Revista de Pensament i Anàlisi, 28(2), 1-25. doi: http://dx.doi.org/10.6035/recerc a.6749 Waldenfels, Bernhard (2004). Lévinas and the face of the other. En Critchley, Simon y Bernasconi, Robert (ed.). Cambridge Companion to Lévinas (63-82). Cambridge: Cambridge University Press. Wesphal, Merold (2008). Lévinas and Kierkegaard in dialogue. Bloomington: Indiana University Press. Žižek, Slavoj (2020). La realidad de lo virtual (The Reality of the Virtual). Documental dirigido por Ben Wright (2007). Transcripción y traducción de la intervención de Žižek: Sergio Aguilar Alcalá. Revista Iberoamericana de Comunicación, 38, 182-208.
https://openalex.org/W4280521191	https://zenodo.org/records/6548815/files/Shavkatjonova%20Muhlisa%20Asqarali%20qizi.pdf	Quechua	null	OʻZBEK TILINING SOHALARDA QOʻLLANISHI VA UNING JAMIYAT HAYOTIDA TUTGAN OʻRNI.	Zenodo (CERN European Organization for Nuclear Research)	2,022	cc-by	638	OʻZBEK TILINING SOHALARDA QOʻLLANISHI VA UNING JAMIYAT HAYOTIDA TUTGAN OʻRNI. https://doi.org/10.5281/zenodo.6548815 Shavkatjonova Muhlisa Asqarali qizi Andijon davlat universiteti Boshlangʻich taʼlim yoʻnalishi 202- guruh Annotatsiya. Insonning eng muhim xususiyatlaridan biri sizlash qobiliyatidir. Ravon nutq orqali ifodalangan fikr tushunarli va yoqimlidir. Nutq harakatlari murakkab aʼzolar tizimi orqali amalga oshiriladiki , bunda bosh miya faoliyati asosiy rol oʻynaydi , nutq faqat inson uchun xos boʻlgan alohida va yuqori darajadagi aloqa shaklidir. Nutqiy aloqa jarayonida kishilar fikr almashadilar va bir - birlariga ta'sir etadilar.Nutqiy aloqa til orqali amalga oshiriladi. Nutq talaffuzi mexanizmi harakatini tushunish uchun nutq apparatining tuzilishini yaxshi bilishi zarur. Nutq buzilishi uzoq yillar davomida oʻrganib kelayotgan sohadir. Nutqning normal faoliyati holatiga uning psixofiziologik mexanizmlarning zaiflashuviga bogʻliq holda til normalaridan chetlashuvi bilan belgilanadi. Kirish soʻzlar: soʻzlash qobiliyati , ravon nutq , til , nutq. Kirish soʻzlar: soʻzlash qobiliyati , ravon nutq , til , nutq. Nutq tilni ishga solish jarayonidir. Subyektning nutqiy faoliyati ichki vositasi va uni amalga oshirish usullari sifatida til va nutqni oʻz ichiga oladi. Nutq bu psixik xodisa , kishilarning shaxslararo oʻzaro muayyan hamkorligi sharoitlarda rivojlanadigan , individual xususiyatlarga ega boʻlgan shaxs mulkidir. U tildan farqli ravishda ijtimoiy - tarixiy sharoitlarda rivojlanadigan xalq ijtimoiy mulki hodisasidir. Ravon nutq kishilarning muloqotini va oʻzaro bir - birlarini tushunishlarini taʼminlovchi mazmunan keng yoyilgan fikrdir. Ravon nutqni fikrlar dunyosidan ajratib boʻlmaydi : ravon nutq - bu fikrlar ravonligidir , unda bolaning mantiqiy fikrlash , oʻzi qabul qilayotganlarini mulohaza qilish va ularni toʻgʻri ifodalash qobiliyati aks etadi. Ravon nutqni shakllantirish , uning vazifasini oʻzgartirish murakkablashib borayotgan bola faoliyatioqibati boʻlib , u bolaning atrofdagilar bilan muloqotga kirishish sharoiti , muloqot shakliga bogʻliq boʻladi. Ravon nutqni shakllanishi ilk yoshdan boshlab asta - sekin roʻy beradi. Bolalar hayotining dastlabki yetti yilida atrofdagi kishilar bilan muloqot vositasi sifatida nutqning paydo boʻlishi va uni rivojlantirish jarayoniga alohida eʼtibor qaratish zarur. Har bir kishi hayotida ravon nutq muhim ahamiyatga ega boʻlib , u uchta asosiy vazifani bajaradi : individuallararo , ichki individual va umuminsoniy. Til millatning noyob xazinasi boʻlib , doim ogʻzaki va yozma holda namoyon boʻlgan. Boy , yorqin , maroqli nutq soʻzlayotgan kim boʻlishidan qatʼiy nazar , nuri hisoblanadi. Tilning oliyjanob imkoniyatlar nutq orqali nutq jarayonida ochiladi. Nutq boʻlmas ekan tilning cheksiz imkoniyatlari yuzaga chiqmay qolaveradi. Soʻz va gʻazal sultoni A.Navoiy til va nutq munosabatlarini shunday izohlaydi. " Til shuncha sharafi bilan nutqning qurolidir. Agar nutq nomaqbul boʻlib chiqsa tilning manfaatidir " . Demak til qancha zoʻr boʻlmasin , u nutq uchun qurol sifatida xizmat qilar ekan. FOYDALANILGAN ADABIYOTLAR: 1. X.Muhiddinova , Z. Salisheva , X. Poʻlatova. Oʻzbek tili. -- T.2012 2. M.X.Ahmedova , K.A.Gayubova. Oʻzbek tili. -- T.2019 3. hattp://kutubxona.ziyonet.uz OʻZBEK TILINING SOHALARDA QOʻLLANISHI VA UNING JAMIYAT HAYOTIDA TUTGAN OʻRNI. Agar til oʻq boʻlsa , nutq kamondir. Oʻqning qudrati , kamonning qudratiga bogʻliq. Bugungi kunda Respublikamizda taʼlim tizimini chuqur isloh qilish masalalari jamiyatimiz rivojlanishining asosiy qismida aylandi. Bu islohotlarni amalga oshirishda Oʻzbekiston Respublikasini yanada rivojlantirish boʻyicha " Harakatlar strategiyasi " dasturiy amaldir. Darhaqiqat , ona tilini oʻqitishdan asosiy maqsad - oʻquvchilarda oʻz ona tilida fikrni toʻgʻri , aniq , ravshan va goʻzal ifodalay olish koʻnikmalarini rivojlantirishimiz lozim. Oʻquvchilarga til hodisalari alohida hodisalar sifatida emas , balki nutq imkoniyatlarini kengaytiruvchi vositalar sifatida oʻrganish maqsadga muvofiq. Masalan , oʻquvchilar fonetikadan " Nutq tovushlari " mavzusini nutq madaniyati va oʻzbek tilshunosligining dolzarb muammolarini oʻrganar ekanlar , asosiy diqqat eʼtibor tovushlarning trio talaffuzi va imlosini oʻzlashtirish bilan birga , soʻz boyligini oshirishga qaratilishi lozim. Xulosa. Oʻquvchilarda adabiy til va sheva elementlarini yaxshi farqlash koʻnikmalarini shakllantirishga erishish adabiy til meʼyorlarini oʻrgatishga keng yoʻl ochadi. Oʻquvchi nutqini rivojlantirish murakkab jarayon. Unga monelik qilayotgan omillar faqat taʼlim mazmuni yoki oʻqituvchigagina bogʻliq emas. Unga monelik qilayotgan omillar faqat taʼlim mazmuni yoki oʻqituvchigagina bogʻliq emas. Bu jamiyatga ham bogʻliq masala. Barcha imkoniyatlar toʻlaligini ishga solinsa fikrni ogʻzaki va yozma ravishda toʻgʻri , ravon ifodalashga oʻrgatish jarayoni oson kechadi. Oʻquvchi nutqini rivojlantirish masalalari oʻz yechimini topadi.
https://openalex.org/W4390110556	https://ojs.academicon.pl/np/article/download/7231/8311	Polish	null	Rec.: Marian Plezia, Dokoła reformy szkolnej St. Konarskiego. Studia klasyczne pijarów polskich, Lublin 1953	Nasza Przeszłość	1,966	cc-by-sa	32,496	R E C E N ZJE i S P R A W O Z D A N IA R E C E N ZJE i S P R A W O Z D A N IA 28» R E C E N Z J E I S P R A W O Z D A N I A t. zw. krakowskim okresie, prawdziwy rozkwit badań archiwalnych pod protektoratem Polskiej Akademii Umiejętności, która w r. 1886 zorga nizowała naukową Ekspedycję Rzymską. Autor wylicza 27 uczonych polskich, uczestników Ekspedycji, którzy gromadzili olbrzymi materiał w „Tekach Watykańskich". y Wreszcie w V okresie-(1914— 1939) badania w 'Archiwum Watykań skim prowadzi szereg uczonych polskich. W „Repertorium" Autor zestawił w 135 numerach prace uczonych polskich, korzystających z materiałów rzymskich, przy tym uwzględnił prace autorów innych narodowości, którzy wprost lub ubocznie trak towali sprawy polskie (jak np. Theiner, Pierling, Boudou, Rouet de Joumel, Smurło i in.). W porządku alfabetycznym nazwisk podano ty tuły dzieł, zaznaczając przy każdym z nich działy Archiwum Watykań skiego, z których czerpano głównie materiał, nadto — dla ułatwienia — sygnatury książek- w Bibliotece Watykańskiej. T R Z Y TO M Y „STU D IÓ W TEO LO G ICZNYCH ". „Studia Teologiczne" wydawane w W ilnie od r. 1932 przez grono Profesorów Wydziału Teologicznego Uniwersytetu Stefana Batorego pod naczelną redakcją niżej podpisanego, zostały wznowione w czasie wojny w Rzymie przez Ks. Prałata Waleriana Meysztowicza, b. profesora wileńskiego Wydziału Teologicznego. Z jego inicjatywy i jego staraniem ukazały się trzy nowe tomy „Studiów Teologicznych" w kolejnej nume racji jako tomy X I, X II i X III, zawierające w dawnym formacie i daw nej szacie zewnętrznej następujące prace: Zaznaczyć trzeba, że „Repertorium " nie jest kompletne. Autor był zmuszony warunkami pracy podczas wojny światowej ograniczyć się do korzystania z Biblioteki Watykańskiej, która aczkolwiek bogata nie posiada wszystkich prac, opartych na źródłach z Archiwum W aty kańskiego, stąd w „Repertorium" powstały luki. Autor, zaznaczając, iż zamierza dać „opusculum potius utile, quam doctum“ (str. 9), miał rację. Jest to raczej repertorium dzieł, dotyczących spraw polskich, znajdujących się głównie w Bibliotece Watykańskiej. Tak np. z licz nych prac ks. Fijałka opartych na materiałach archiwalnych rzymskich, ..Repertorium1' wymienia tylko wydany razem z Prof. W. Semkowiczem ..Kodeks dyplomatyczny Katedry i kościołów diecezji wileńskiej. X I. Meysztowicz Valerianus, Repertorium bibliographicum pro rebus polani cis Archirń Secreti Vaticani, Vaticani 1943. Str. 20+Tabula serie- rum Archivi Secreti Vaticani siglarumąue ąuibus in „Repertorio" sig- nantur. X II. Meysztowicz Valerianus, De Archivo Nuntiaturae Varsaviensis quod nunc in Archivo Secreto Vaticano servatur, Vaticani 1944. Str. 99. X III. Savio Petrus, De actis Nuntiaturae Poloniae quae partem Archivi Secretariatus Status constituunt, Vaticani 1947. Str. 156. W szeregu prac Wł. Abrahama pominięta została choćby praca ..Polska a chrzest Litw y" („Polska i Litw a w dziejowym stosunku"). Nie można było pominąć „Polskiego Słownika Biograficznego1, który wszak w wielu życiorysach opiera się lub przynajmniej rejestruje ma teriały Archiwum Watykańskiego. X III. Savio Petrus, De actis Nuntiaturae Poloniae quae partem Archivi Secretariatus Status constituunt, Vaticani 1947. Str. 156. 1. We wstępie do „Repertorium bibliographicum" dał ks. Meyszt wicz w zarysie historię studiów uczonych polskich w Archiwum W a tykańskim, rozróżniając pięć okresów. I okres rozpoczął się w r. 1577, gdy Sirletus, prefekt Biblioteki i Archiwum Watykańskiego z po lecenia Stefana Batorego zarządził sporządzenie odpisów dokumen tów dotyczących Polski. Poszukiwania archiwalne, prowadzone następ nie dla Zygmunta III, zostały na długi czas przerwane z powodu wojen i w okresie saskim. Dopiero w r. Nasza Przeszłość T R Z Y TO M Y „STU D IÓ W TEO LO G ICZNYCH ". 1754— 1796 w Warszawie nuncjuszów, podając najważniejszą bibliografię i dając charakterystykę ich działalności w Polsce. (N aj mniej znaną dla braku akt jest nuncjatura Serry). Wszyscy ci nuncju sze po powrocie do Rzymu zostali odznaczeni purpurą kardynalską (A r chetti, przedstawiony do kapelusza kardynalskiego przez Katarzynę, otrzym ał biret czerwony w Grodnie z rąk Stanisława Augusta). Wszyscy ci dyplomaci watykańscy świetnie władali łaciną i francuszczyzną, języ kiem polskim, niestety, żaden. cjatur odnoszą się poszczególne teki archiwalne. Tablice sporządzone są dokładnie i będą służyć wielką pomocą dla badaczy naukowych w ich poszukiwaniach archiwalnych. Jak się dowiadujemy z opisu Autora, cenne to archiwum po ostatnim rozbiorze Polski nuncjusz Litta odesłał z W arszawy do Wiednia, skąd dopiero w r. 1859 przewieziono je do R zy mu. Najwięcej zasług w uporządkowaniu archiwum położył Garampi, najwybitniejszy i najuczeńszy z pośród nuncjuszów tego okresu, któ rego działalność w Polsce zasługuje na specjalną monografię. Na uwagę historyków zasługuje audytor Caleppi, późniejszy kardynał, oraz Ghi- giotti, który za czasów nuncjatury Garampiego piastował urząd sekre tarza królewskiego do spraw włoskich. Dla lepszej orientacji przy ko rzystaniu z akt archiwum Autor skreślił krótkie życiorysy 7 rezydują cych od r. 1754— 1796 w Warszawie nuncjuszów, podając najważniejszą bibliografię i dając charakterystykę ich działalności w Polsce. (N aj mniej znaną dla braku akt jest nuncjatura Serry). Wszyscy ci nuncju sze po powrocie do Rzymu zostali odznaczeni purpurą kardynalską (A r chetti, przedstawiony do kapelusza kardynalskiego przez Katarzynę, otrzym ał biret czerwony w Grodnie z rąk Stanisława Augusta). Wszyscy ci dyplomaci watykańscy świetnie władali łaciną i francuszczyzną, języ kiem polskim, niestety, żaden. prawie każde zawiera materiały do spraw polskich. Dwa działy są spe cjalnie poświęcone Polsce: archiwum Nuncjatury Warszawskiej — Nun- ziatura di Varsavia, mało przez historyków wykorzystane i, i archiwum Nuncjatury Polski — Nunziatura di Polonia. Tych dwóch różnych dzia łów nie należy mieszać. Zbiorom archiwalnym Nuncjatury Warszawskiej jest poświęcony tom X II „Studiów". Zawiera on wstęp, ogólny opis archiwum Nuncja tury Warszawskiej, krótkie życiorysy siedmiu rezydujących od 1754— — 1796 w Polsce nuncjuszów (Serra, Visconti, Durini, Garampi, Archetti, Saluzzo, Litta) (str. 9— 43), w 20 tablicach wykaz woluminów i tek z dokumentami (str. 45— 89). Archiwum Nuncjatury Warszawskiej skła da się ze 195 woluminów i tek, mieszczących kopie ekspediowanych w latach 1757— 1794 z Polski do Sekretariatu Stanu dokumentów i ory ginały nadsyłanych listów z Rzymu czy też od różnych osobistości do Nuncjatury. 1 Materiały z X V I cz. tego archiwum „Ordini Regolari", jakie ze brałem podczas pobytu w Rzymie, wraz z cennym zbiorem fotografii dokumentów do historii klasztorów w Polsce i W. Ks. Lit. w okresie rozbiorów Polski, zostały przez Gestapo zniszczone po uwięzieniu moim ■oraz Kolegów z Wydziału Teologicznego. T R Z Y TO M Y „STU D IÓ W TEO LO G ICZNYCH ". Znajdują się również akta lub ich odpisy z lat wcześniej szych. Istnieje kilka katalogów tego archiwum, ostatni z nich sporządził Perali w r. 1930. Rozszerzeniem Peraliego są tablice ks. Meysztowicza z dokładnym i przejrzystym wykazem w 9 rubrykach poszczególnych tek, liczby kart, tytułów, lat, w których powstały odnośne akta, przez kogo były pisane i zbierane, wreszcie treść każdej z tek. Rezydencją nuncjatury był dom przy klasztorze Teatynów przy ul. Długiej, pod koniec nuncjatury Garampiego wynajęto na mieszkanie nuncjusza pałac hr. Dembowskiego przy ul. Senatorskiej. Tom kończy się dokładnym wykazem imion i nazwisk, co jest rzeczą konieczną w tego rodzaju publikacjach. Całość tego archiwum rozpada się na 20 działów, każdemu z nich poświęcona jest tablica, poprzedzona krytycznymi uwagami. A więc: 1) preliminaria dla informacji nuncjuszów o Kościele Polskim, sporzą dzone przez Garampiego; 2) Kolegia papieskie w Polsce i W. Ks. Lit., bezpośrednio zależne od Stolicy Apostolskiej, jak Alumnat Papieski w Wilnie, kolegia w Krasławiu, Swierzniu, w e Lw ow ie i Brunsberdze; 3) sejmy; 4) dysydenci i schizmatycy; 5) ziemie oderwane od Polski pod czas I rozbioru; 6) listy Sekretariatu Stanu do nuncjuszów; 7) regesta listów nuncjatury do Sekretariatu Stanu (wyjaśniające stosunki między Stolicą Apostolską i Polską); 8) korespondencje Kongregacji Propagandy W iary z nuncjuszami; 9) listy Kurii rzymskiej do nuncjuszów; 10) rege sta listów nuncjuszów do Kurii rzymskiej; 11) regesta listów z nuncja tury do różnych osobistości; 12) listy różnych osób do nuncjuszów; 13) akta odnoszące się do metropolity Felicjana Wołodkowicza; 14) „M i- scellanea“ ; 15) misje; 16) zakony; 17) biskupstwa i diecezje; 18) „affari sospesi"; 19) specjalny dział dotyczący Teatynów (warszawskich); 20) regesta nuncjuszów od r. 1587 do 1713. Ozdobę książki stanowi 15 doskonale odbitych na kredowym pa pierze reprodukcji portretów 7 nuncjuszów, Stanisława Augusta, 4 pa pieży, pomników Garampiego, Saluzza i Litty, wreszcie widok ul. Dłu giej w Warszawie z rezydencją nuncjatury, — szkoda, że w wykazie ilustracji nie podano bliższych objaśnień. Tom X II „Studiów Teologicznych" poświęcony jest ostatniemu w Polsce Nuncjuszowi Arcybiskupowi Cortesiemu. ~ 3. Archiwum Nuncjatury Polski — Nunziatura di Polonia składa się z 396 + 24 Additamenta woluminów akt, zbieranych przez Sekretariat Stanu od r. 1567— 1806. Znajdują się tam oryginały listów i dokumentów Wysyłanych przez nuncjuszów rezydujących w Polsce i przez inne osoby do Rzymu oraz regesta i kopie ekspediowanych listów przez Sekretariat do Polski. Obszernym katalogiem tego archiwum w opracowaniu Mons. T R Z Y TO M Y „STU D IÓ W TEO LO G ICZNYCH ". 1782 rozpoczął się II okres, gdy Albertrandy, wysłany przez Stanisława Augusta, w ciągu trzyletniego swego pobytu w Rzym ie zgromadził w 110 tekach olbrzymi zbiór doku mentów, zebranych w archiwach rzymskich. Zbiór ten przewieziony z Warszawy do Krzemieńca, następnie do Paryża, znalazł się wreszcie w Bibliotece X X . Czartoryskich w Krakowie. W III okresie (1842— 1881) główne zasługi w poszukiwaniach archi walnych położyli Aleksander hr. Przeździecki i Theiner. Po otwarciu tajnego archiwum Watykańskiego dla uczonych w r. 1881 nastąpił w IV, Bardzo pożytecznym dodatkiem przy końcu „Repertorium1* jest dokładna tablica orientacyjna wszystkich działów czyli poszczególnych części („series“ — ..fondi1') Archiwum Watykańskiego, z których każda ma swą nazwę i numerację akt (archiwum tajne, Camera Apostolica, Istrumenta Miscellanea ed affini, Registra Avenionensia, Dataria Apo stolica, olbrzymi dział Sekretariatu Stanu, Segreteria dei Brevi, Acces- sioni vecchie — recenti — recentissime, Archivio S. Angelo). Tom ten „Studiów" poświęcił Autor śp. Ks. Profesorowi Leonowi Puciacie, Przyjacielowi i Mistrzowi, który zmarł 12. V II. 1943 r-, wkrót ce po wypuszczeniu z obozu w Szałtupiu na Litwie, gdzie byli więzieni przez Gestapo profesorowie Wydziału Teologicznego Uniwersytetu Ste fana Batorego i Seminarium Duchownego w Wilnie. 2. Archiwum Watykańskie składa się z przeszło 250 oddzielny działów, stanowiących jakby odrębne archiwa — „fondi", z których 2. Archiwum Watykańskie składa się z przeszło 250 oddzielny działów, stanowiących jakby odrębne archiwa — „fondi", z których Nasza Przeszłość 19 Nasza Przeszłość 19 R E C EN ZJE i S P R A W O Z D A N IA R E C EN ZJE i S P R A W O Z D A N IA 290 291 cjatur odnoszą się poszczególne teki archiwalne. Tablice sporządzone są dokładnie i będą służyć wielką pomocą dla badaczy naukowych w ich poszukiwaniach archiwalnych. Jak się dowiadujemy z opisu Autora, cenne to archiwum po ostatnim rozbiorze Polski nuncjusz Litta odesłał z W arszawy do Wiednia, skąd dopiero w r. 1859 przewieziono je do R zy mu. Najwięcej zasług w uporządkowaniu archiwum położył Garampi, najwybitniejszy i najuczeńszy z pośród nuncjuszów tego okresu, któ rego działalność w Polsce zasługuje na specjalną monografię. Na uwagę historyków zasługuje audytor Caleppi, późniejszy kardynał, oraz Ghi- giotti, który za czasów nuncjatury Garampiego piastował urząd sekre tarza królewskiego do spraw włoskich. Dla lepszej orientacji przy ko rzystaniu z akt archiwum Autor skreślił krótkie życiorysy 7 rezydują cych od r. T R Z Y TO M Y „STU D IÓ W TEO LO G ICZNYCH ". Piotra Savio, archiwisty Archiwum Watykańskiego, jest ostatni z w y mienionych w yżej tomów „Studiów Teologicznych" p. t. „De actis Nun tiaturae Poloniae quae partem archivi Secretariatus Status constituunt“. Tom ten, poprzedzony łacińskim wstępem ks. Meysztowicza, wydany jest w języku włoskim. Dodane są na końcu dwie tablice z wykazami, do jakich lat i nun Katalog składa się z dwóch zasadniczych części: z inwentarza archi wum Nuncjatury Polski (str. 7— 78) i ze „schedario cronologico“ — w y RE C EN ZJE i S P R A W O Z D A N IA 292 R E C E N ZJE i S P R A W O Z D A N IA 293 kazu poszczególnych akt, ułożonego w porządku chronologicznym (str. 79— 156). Inwentarz archiwum — jak objaśnia Mons. Savio — uwzględ nia przy poszczególnych tomach zasadniczo trzy krótkie notatki: histo ryczną — a więc tytuł, data, sygnatura, nomenklatura, nazwiska nun cjuszów i ministrów Sekretariatu Stanu, znajdują się tam wzmianki 0 pochodzeniu danej teki z aktami oraz o indeksie, o ile się znajduje. Druga notatka dotyczy rodzaju i dat korespondencji. Trzecia wreszcie podaje wym iary poszczególnych tek archiwalnych, liczbę kart i pism. Przed każdym tomem podana została sygnatura nowa jak również dawna. przy poszukiwaniach źródłowych w Archiwum Watykańskim, to też ukazanie się tych tomów należy powitać ze szczerym uznaniem ) Ks. Czesław Falkowski Ks. J ó z e f U m i ń s k i , Powstanie biskupstwa poznańskiego i za leżnego odeń archidiakonatu czerskiego. Wiadomości Kościelne (Organ Archidiecezji Wrocławskiej), Wrocław 1952, N r 1— 2, s. 35— 64. Ks. J ó z e f U m i ń s k i , Powstanie biskupstwa poznańskiego i za leżnego odeń archidiakonatu czerskiego. Wiadomości Kościelne (Organ Archidiecezji Wrocławskiej), Wrocław 1952, N r 1— 2, s. 35— 64. Zagadnienie powstania biskupstwa poznańskiego, łączące się ściśle z kwestią początków chrześcijaństwa w Polsce, należy niewątpliwie do najbardziej spornych zagadnień w literaturze historycznej. Szczupłość źródeł nie pozwalająca na definitywne i całkiem pewne rozwiązanie tej kwestii pociągała za sobą (i pociąga nadal) wysuwanie licznych, niekiedy różniących się krańcowo między sobą, hipotez. O trudnościach związa nych z próbami wyjaśnienia początków biskupstwa poznańskiego świad czy najlepiej fakt, że różnice poglądów dotyczą tym razem prac szeregu najwybitniejszych historyków . Wykaz chronologiczny akt został ułożony w 9 grupach: 1) listy i de pesze szyfrowane nuncjuszów do Sekretariatu Stanu (od r. ) Przyp. Redakcji. Recenzja ks. prof. Falkowskiego, obecnie Ordy nariusza Łomżyńskiego, napisana jeszcze w r. 1948, z przyczyn od nas niezależnych nie mogła ukazać się wcześniej. Zamieszczamy ją w niniej szym tomie, tym bardziej, że w czasopismach polskich nie ukazała się recenzja o „Studiach Teologicznych1. 1 Najważniejszą literaturę przedmiotu z wymienieniem kwestii spo nych zestawia M. Z. J e d l i c k i , Kronika Thietmara, Poznań 1953, s. 74 przypis 111 (koniec przypisu na s. 79). Do najważniejszych kwestii spor nych zaliczyłbym: 1) stanowisko prawne pierwszego biskupstwa pol skiego (misyjne czy diecezjalne), 2) gdzie była jego siedziba (Poznań czy Gniezno) oraz 3) z czyjej inicjatywy ono powstało i jaki był stosu nek cesarstwa do nowoutworzonego biskupstwa. Natomiast nie można za liczyć do kwestii spornych zagadnienia stosunku pierwszej organizacji kościelnej w Polsce do arcybiskupstwa magdeburskiego, gdyż zostało ono wyjaśnione w sposób nie podlegający dyskusji przez P. K e h r a , Das Erzblstum Magdeburg und die erste Organisation der christlichen, Kirche in Polen, Abhandlungen der Preussischen Akademie der Wissen- .schaften, Jahrg. 1920. Phil.-Hist. Klasse Nr 1, Berlin 1920. Tylko w czę ści słuszne jest twierdzenie M. Z. J e d l i c k i e g o , op. cit., s. 76 (w przy pisie), że uczeni, którzy przyjm owali diecezjalny charakter pierwszego biskupstwa polskiego, opowiadali się za jego przynależnością do arcybiskupstwa magdeburskiego. Ostatnio (jeszcze przed ukazaniem .się pracy J e d l i c k i e g o ) ks. J. N o w a c k i , Groby królewskie w ka tedrze poznańskiej, Miesięcznik Kościelny Archidiecezji Poznańskiej, R. V II N r 7/8, 1-952, s. 170 nn. silnie akcentuje diecezjalny charakter pierwszego biskupstwa w Polsce, bynajmniej nie przyjmując jego za leżności od Magdeburga. T R Z Y TO M Y „STU D IÓ W TEO LO G ICZNYCH ". 1561); 2) listy 1 depesze szyfrowane Sekretariatu Stanu do nuncjuszów (od r. 1560); 3) korespondencje ministrów (od r. 1564); 4) korespondencje biskupów (od r. 1563); 5) listy do nuncjuszów (od r. 1561); 6) listy panujących, do stojników i osób prywatnych do Papieża i do Sekretariatu Stanu (od r. 1541); 7) pisma informacyjne; 8) sprawy; 9) papiery i druki różne. N ie trzeba dowodzić, iż wykaz chronologiczny jest ogromnym ułatwieniem w poszukiwaniu akt rozrzuconych w różnych tekach, zwłaszcza wobec braku przy końcu dzieła wykazu nazwisk i rzeczy. Studium ks. Umińskiego stanowi nową próbę rozwiązania tego tak bardzo spornego zagadnienia. Autor postawił sobie za cel udowodnie nie, że nie tylko biskup Jordan, ale także biskup Unger nie byli bisku pami diecezjalnymi (poznańskimi), lecz tylko dworskimi. Według autora termin „biskupi dworscy11 oznacza tych biskupów, „którzy przebywali na Brakiem natomiast pracy, zwłaszcza w drugiej jej części, jest nieu stalona pisownia nazwisk polskich oraz częste ich zniekształcanie. Dla przykładu wystarczy przytoczyć kilka błędów: „Volschi’* (str. 104) za miast Wolski, „Miskowski" lub „Myskowski“ zamiast Myszkowski, na str. 107 poprawnie „Korsak** i „Corsach1, „Dzialiński" zamiast Działyń- ski, na str. 115 „Hulsen" i „Hilzen, „eonte Girolamo Rozdrazew" (str. 118) i „eonte Cristoforo Rozdrazoff11 (str. 119) i „mons. Girolamo Rozra- zewski" (str. 120), „Crassinski1 i „Coshca‘‘, „Brezniczki“ (str. 121) zamiast Brzeżnicki; biskup wił. Jerzy Tyszkiewicz figuruje jako „Tyszkiowic (str. 122), metropolita Leon Załęski — jako „Leone Szlubic Zalecki (Za- beschi) (str. 126), Szaniawski — jako „Szaniowski** (str. 127) itp. ) Przyp. Redakcji. Recenzja ks. prof. Falkowskiego, obecnie Ordy nariusza Łomżyńskiego, napisana jeszcze w r. 1948, z przyczyn od nas niezależnych nie mogła ukazać się wcześniej. Zamieszczamy ją w niniej szym tomie, tym bardziej, że w czasopismach polskich nie ukazała się recenzja o „Studiach Teologicznych1. j „ g y 1 Najważniejszą literaturę przedmiotu z wymienieniem kwestii spo nych zestawia M. Z. J e d l i c k i , Kronika Thietmara, Poznań 1953, s. 74 przypis 111 (koniec przypisu na s. 79). Do najważniejszych kwestii spor nych zaliczyłbym: 1) stanowisko prawne pierwszego biskupstwa pol skiego (misyjne czy diecezjalne), 2) gdzie była jego siedziba (Poznań czy Gniezno) oraz 3) z czyjej inicjatywy ono powstało i jaki był stosu nek cesarstwa do nowoutworzonego biskupstwa. Natomiast nie można za liczyć do kwestii spornych zagadnienia stosunku pierwszej organizacji kościelnej w Polsce do arcybiskupstwa magdeburskiego, gdyż zostało ono wyjaśnione w sposób nie podlegający dyskusji przez P. T R Z Y TO M Y „STU D IÓ W TEO LO G ICZNYCH ". W ielu z nich pozostało już na zawsze na Mazowszu tworząc tam „jak .gdyby kolonię Poznania" (s. 63). W międzyczasie powstało biskupstwo poznańskie, które swą pieczą objęło także i ziemię czerską. W chwili założenia biskupstwa mazowieckiego czyli płockiego związek tej części Mazowsza z diecezją poznańską był już na tyle utwierdzony, że pozo stawiono ją przy biskupstwie poznańskim 5. wstaniem tzw. archidiakonatu czerskiego. W brew poglądom W. Abra hama i M. Gębarowicza, którzy opowiadali się za powstaniem archidia konatu czerskiego w 1124 r., autor stara się dowieść, że związanie Ma zowsza czerskiego z diecezją poznańską nastąpiło przed r. 1076, tj. przed utworzeniem biskupstwa płockiego, w przeciwnym bowiem razie zosta łoby ono włączone do przeznaczonego głównie dla Mazowsza, biskup stwa płockiego. To powiązanie ziemi czerskiej z diecezją poznańską było zdaniem ks. Umińskiego następstwem schronienia się Wielkopolan {w tym i pewnej liczby duchownych poznańskich) w tej części Mazowsza podczas reakcji pogańskiej w trzydziestych latach X I w. Można się tego domyślać na podstawie przekazu Galla, nadmieniającego o uciekinie rach z Wielkopolski, że ultra flum en Wysla in Masouiam fugiebant. W ielu z nich pozostało już na zawsze na Mazowszu tworząc tam „jak .gdyby kolonię Poznania" (s. 63). W międzyczasie powstało biskupstwo poznańskie, które swą pieczą objęło także i ziemię czerską. W chwili założenia biskupstwa mazowieckiego czyli płockiego związek tej części Mazowsza z diecezją poznańską był już na tyle utwierdzony, że pozo stawiono ją przy biskupstwie poznańskim 5. j p y p p Tak w grubym skrócie przedstawiają się początki biskupstwa po znańskiego i archidiakonatu czerskiego w świetle w yw odów autora. Trudno przesądzać, czy wyniki studium ks. Umińskigeo zostaną przy jęte przez naukę historyczną. Jeszcze w tym samym roku (1952) co roz prawa autora ukazała się praca ks. J. N ow ackiegoe, omawiająca te same zagadnienia (w oparciu o ten sam materiał źródłowy), a docho dząca do krańcowo odmiennych wyników. Jest to chyba najlepszym do wodem, jak skomplikowana i trudna do rozwiązania jest sprawa począt ków biskupstwa poznańskiego. Właśnie dlatego moje zastrzeżenia w od niesieniu do rezultatów pracy ks. Umińskiego traktuję w formie jak naj bardziej dyskusyjnej. W ydaje się, że autor poszedł zbyt daleko, kwestio nując istnienie biskupstwa poznańskiego nie tylko przed r. 1000, ale także i po założeniu arcybiskupstwa gnieźnieńskiego. 2 Za biskupów dworskich uważa ks. U m i ń s k i m. in. tych, któ rych współcześni nazywali biskupami polskimi — episcopi polonienses. Zagadnienie biskupów dworskich poruszył po raz pierwszy ks. D a - v i d, zob. U m i ń s k i , op. cit., s. 39/40 (w przypisie). Ten ostatni jednak wysunął tezę o biskupach dworskich niezależnie od ks. D a v i d a. różniąc się poza tym od niego w pewnych szczegółach. Sprostować na leży przyjęty przez ks. U m i ń s k i e g o (s. 39) pogląd D a v i d a ja koby Franko ..eplscopus bellagradensis“ był polskim biskupem dwor skim, działającym na Pomorzu, por. krytykę tego poglądu przez F. P o h o r e c k i e g o , Roczniki Historyczne, t. VI, 1930, s. 111— 113. 3 Zgodnie z przeważającym dotychczas poglądem autor odrzuca (s. 43— 44) tezę o istnieniu biskupstwa poznańskiego przed r. 1000, głów nie w oparciu o stwierdzenie przez Thietmara legalności założenia arcy biskupstwa gnieźnieńskiego. 4 Międzyrzecz leży na terenie discezji poznańskiej. 5 Autor słusznie stwierdza (s. 64), że utworzenie odrębnego, for malnego archidiakonatu (czerskiego) nastąpiło dopiero później. 6 Zob. końcową część przypisu nr 1. 7, Thietmar, jak to wykazała literatura, był niewątpliwie stronni czy w przedstawieniu początków organizacji kościelnej w Polsce. N ie uzasadnia to jednak odrzucenia wiadomości Thietmara, że Unger był biskupem poznańskim. T R Z Y TO M Y „STU D IÓ W TEO LO G ICZNYCH ". dla Jordana i Brunona) właściwsza od dotych czas używanego określenia „biskupi misyjni", ponieważ „nie wyklucza wcale misyjnego charakteru ich działalności, a zato obrazuje trafniej ich pozycję w stosunku do zwierzchności państwowej" (s. 39)2. Zasadni cza część pracy (s. 44— 54) dotyczy krytyki dotychczasowego poglądu, że biskup Unger był od chwili powstania metropolii gnieźnieńskiej poznań skim biskupem diecezjalnym 3. Dowodu na to, że Unger nie był bisku pem poznańskim jeszcze w kilka lat po zjeździe gnieźnieńskim, dopa truje się autor w przeniesieniu zwłok Pięciu Męczenników z M iędzy rzecza 4 do Gniezna. Zdaniem ks. Umińskiego, gdyby istniało w tym czasie biskupstwo poznańskie, ter nie zezwoliłoby ono na przeniesienie relikw ii Pięciu Męczenników do Gniezna, ponieważ stanowiły one dla biskupstwa rzecz zbyt cenną, aby można się było jej tak łatwo pozbyć. Nazwanie Ungera w Vita Quinque Fratrum „episcopus terrae" uznaje autor za równoznaczne z „episcopus poloniensis", co oznacza jego zda niem biskupa dworskiego. Jeżeli zaś biskupstwo poznańskie nie po wstało podczas zjazdu gnieźnieńskiego, to nie mogło ono powstać ani za panowania Bolesława Chrobrego ani za rządów Mieszka II, ponieważ nie było jeszcze wtedy potrzeby założenia nowej diecezji „tuż pod bo kiem Gniezna11 (s. 55). Okoliczności sprzyjające wyodrębnieniu z arcy biskupstwa gnieźnieńskiego biskupstwa poznańskiego mogły zaistnieć dopiero w latach 1038— 1076, to jest w okresie między powrotem z w y gnania Kazim ierza Odnowiciela a wznowieniem metropolii gnieźnień skiej. Był to okres kiedy arcybiskupstwo gnieźnieńskie faktycznie nie istniało, „nie było więc nikogo, ktoby mógł się sprzeciwiać wyodręb nieniu z Gniezna nowego biskupstwa poznańskiego" (s. 56). Za najpraw dopodobniejsze uważa autor założenie biskupstwa poznańskiego około r. 1050. Początki nowozałożonego biskupstwa wiąże ks. Umiński z po- wstaniem tzw. archidiakonatu czerskiego. W brew poglądom W. Abra hama i M. Gębarowicza, którzy opowiadali się za powstaniem archidia konatu czerskiego w 1124 r., autor stara się dowieść, że związanie Ma zowsza czerskiego z diecezją poznańską nastąpiło przed r. 1076, tj. przed utworzeniem biskupstwa płockiego, w przeciwnym bowiem razie zosta łoby ono włączone do przeznaczonego głównie dla Mazowsza, biskup stwa płockiego. To powiązanie ziemi czerskiej z diecezją poznańską było zdaniem ks. Umińskiego następstwem schronienia się Wielkopolan {w tym i pewnej liczby duchownych poznańskich) w tej części Mazowsza podczas reakcji pogańskiej w trzydziestych latach X I w. Można się tego domyślać na podstawie przekazu Galla, nadmieniającego o uciekinie rach z Wielkopolski, że ultra flum en Wysla in Masouiam fugiebant. T R Z Y TO M Y „STU D IÓ W TEO LO G ICZNYCH ". K e h r a , Das Erzblstum Magdeburg und die erste Organisation der christlichen, Kirche in Polen, Abhandlungen der Preussischen Akademie der Wissen- .schaften, Jahrg. 1920. Phil.-Hist. Klasse Nr 1, Berlin 1920. Tylko w czę ści słuszne jest twierdzenie M. Z. J e d l i c k i e g o , op. cit., s. 76 (w przy pisie), że uczeni, którzy przyjm owali diecezjalny charakter pierwszego biskupstwa polskiego, opowiadali się za jego przynależnością do arcybiskupstwa magdeburskiego. Ostatnio (jeszcze przed ukazaniem .się pracy J e d l i c k i e g o ) ks. J. N o w a c k i , Groby królewskie w ka tedrze poznańskiej, Miesięcznik Kościelny Archidiecezji Poznańskiej, R. V II N r 7/8, 1-952, s. 170 nn. silnie akcentuje diecezjalny charakter pierwszego biskupstwa w Polsce, bynajmniej nie przyjmując jego za leżności od Magdeburga. Autor przepisywał nazwiska ze starych akt i zachował ówczesną pi sownię, dodając czasem przy nazwisku polskim łacińskie brzmienie, np. „Lorenzo Gosliczky (Goslicius)1, „Miskowski (Mischovius, Miscovius)“ , należało jednak ustalić przyjętą pisownię znanych nazwisk historycznych. Tom X III „Studiów, jak zaznaczono w e wstępie, wydano z fundu szu Polskiego Instytutu Historycznego w Rzymie. Katalog ten stanowi pierwszą część pracy Mons. Piotra Savio, część druga, znajdująca się w druku, zawiera ułożone w analogicznym porządku inwentarze akt Ambasady Petersburskiej, Nuncjusza i Ministra w Wiedniu oraz Rezy denta Pruskiego. g T)okumenty te również przedstawiają cenny i ciekawy materiał dla historyków polskich. g T)okumenty te również przedstawiają cenny i ciekawy materiał dla historyków polskich. y p Zarówno katalog Nuncjatury Warszawskiej jak i katalog Nuncja tury Polski oddadzą nauce historycznej duże usługi, będąc ułatwieniem y p Zarówno katalog Nuncjatury Warszawskiej jak i katalog Nuncja tury Polski oddadzą nauce historycznej duże usługi, będąc ułatwieniem R E C E N ZJE i S P R A W O Z D A N IA R E C E N Z JE i S P R A W O Z D A N IA 294 295 polskim dworze, zostali przez dwór zaproszeni lub przyjęci, dostawali od dworu utrzymanie i zaopatrzenie i służyli potrzebom religijnym czy państwowym kraju według interesów i wskazań dworu' (s. 37). Zda niem ks. Umińskiego nazwa „dworscy" jest w odniesieniu do szeregu biskupów polskich (n. p. 5 Autor słusznie stwierdza (s. 64), że utworzenie odrębnego, for malnego archidiakonatu (czerskiego) nastąpiło dopiero później. Ma to wynikać z tego, że Gall pisał pierwszą księgę na terenie Mazowsza Płockiego. Dla Galla, przebywającego po wschodniej stronie Wisły, ultra flumen Wysla ma oznaczać Mazowsze po zachodniej stronie Wisły. W brew autorowi trzeba twierdzić, że nie ma najmniejszych do wodów na to, aby Gall pisał jakąkolwiek część swej kroniki na terenie Mazowsza Płockiego oraz, że tłumaczenie autora pozostaje sztucznym, nawet w wypadku przyjęcia jego tezy o miejscu pobytu Galla. Mylny jest również pogląd ks. U m i ń s k i e g o o wczesnym przybyciu G alla do Polski (na przełomie X I i X II w.), zob. w tej sprawie T. G r u d z i ń s k i , Ze studiów nad kroniką Galla, cz. I, Zapiski TN T, t. X V II, 1952, s. 73— 74 oraz cz. II, Zapiski TN T, t. X X , 1954, s. 51— 52. l* Pogląd, że mogło tu chodzić również o Mazowsze lewobrzeżne reprezentuje T. G r u d z i ń s k i , op. cit., cz. II, s. 65— 66. 14 W sprawie przynależności kościelnej ziemi czerskiej do utw rzonego w r. 1000 biskupstwa poznańskiego por. J. N o w a c k i , op. cit., s. 185— 186. T R Z Y TO M Y „STU D IÓ W TEO LO G ICZNYCH ". Za autorem należy przyjąć, że związek Mazowsza czer skiego z diecezją poznańską datuje się od chwili jej utworzenia, które jednak wbrew autorowi kładę na r. 1000,4. 8 Autor słusznie zwrócił uwagę na pewną trudność wytłumaczenia równoczesnego powstania dwóch diecezji na terenie Wielkopolski i to w bliskiej od siebie odległości. Jest rzeczą ciekawą, że przeciwstawne sobie pod względem założeń prace ks. ks.: U m i ń s k i e g o i N o w a c k i e g o zgodnie przyjmują nierównoczesne powstanie arcybiskupstwa gnieźnieńskiego i biskupstwa poznańskiego. Przyznając słuszność oby dwom autorom w uznaniu za niezrozumiałe równoczesne utworzenie obydwóch diecezji wielkopolskich, trzeba jednak zaznaczyć, że nie może to stanowić argumentu wykluczajcego ich równoczesnego założenia w r. 1000. Jeżeli by przyjęło się powstanie obydwóch diecezji w różnym czasie, to już więcej prawdopodobieństwa ma za sobą pogląd ks. N o- w a c k i e g o o utworzeniu biskupstwa poznańskiego w 968 r. Autor ten udowodnił w sposób dość przekonywujący, że Poznań był siedzibą pierwszego biskupstwa w Polsce, usuwając tym samym jedną ze spor nych kwestii związanych z początkami organizacji kościelnej w Polsce. Pozostaje jednak nadal kwestią otwartą, czy ustalenie się siedziby bi skupiej w Poznaniu dokonało się drogą prawną (kanoniczną), jak przyj muje ks. N o w a c k i , ery też via -facti. W tym ostatnim wypadku bi skupstwo w Poznaniu pozostawała nadal biskupstwem misyjnym. Na wet w wypadku przyjęcia misyjnego charakteru pierwszego biskup stwa polskiego, związanego jednak via facti z Poznaniem, znajdujemy w pewnym stopniu wytłumaczenie dość niezrozumiałego równoczesnego utworzenia obydwóch diecezji wielkopolskich. Zakładając arcybiskup- stwo w Gnieźnie nie było można łatwo przejść nad faktem istnienia w polskim Poznaniu długoletniej siedziby biskupstwa misyjnego. Nie zgadzając się z podstawową tezą autora o powstaniu biskupstwa poznańskiego dopiero około r. 1050, podkreślam dyskusyjność moich za strzeżeń, zdając sobie doskonale sprawę ze złożoności całego zagadnie nia jak i z trudności jego ostatecznego rozwiązania. Autor dal nam nowe i zupełnie oryginalne ujęcie problematyki początków biskupstwa poznańskiego, które powinno stać się zachętą do dalszych w tym zakre sie studiów. Nie bez znaczenia jest również poruszone przez ks. Umiń skiego zagadnienie biskupów dworskich, wymagające osobnego omó wienia. Kazimierz Jasiński T a d e u s z S i l n i c k i , Biskup Nanker, Warszawa 1953, wyd. Pax, s. 134. T R Z Y TO M Y „STU D IÓ W TEO LO G ICZNYCH ". Odrzuciwszy w ca łości przekaz Thietmara o wyłączeniu Ungera, biskupa poznańskiego — Vungero P o s n a n i e n s i excepto 7 — spod jurysdykcji arcybiskupstwa gnieźnieńskiego autor pozbawił się wszelkiej podstawy źródłowej, tak niezbędnej przy każdym badaniu historycznym. Utworzenie biskupstwa poznańskiego około r. 1050, nie ma, co należy ze szczególnym naciskiem podkreślić, żadnego poparcia źródłowego. Jest ono wynikiem domysłów R E C EN ZJE i S P R A W O Z D A N IA RE C EN ZJE i S P R A W O Z D A N IA 296 297 autora, wprawdzie logicznie skonstruowanych8, jednakże nie kontrolo wanych w najmniejszym stopniu materiałem źródłowym. Jedyne źródło, które ks. Umiński zużytkował na poparcie swej tezy — Żyw ot Pięciu Braci 9 — nie dostarcza, moim zdaniem dowodu na nieistnienie w tym czasie biskupstwa poznańskiego, trudno bowiem z niego wyciągna.ć ja kieś pewniejsze wnioski zarówno za jak i przeciw istnieniu biskupstwa poznańskiego l0. Stąd też pozostać należy przy tezie, że biskupstwo po znańskie powstało w r. 1000 Również geneza archidiakonatu czer skiego w przedstawieniu autora budzi szereg wątpliwości. Błędną jest przesłanka na której opiera ks. Umiński twierdzenie, że zwrot Galla autora, wprawdzie logicznie skonstruowanych8, jednakże nie kontrolo wanych w najmniejszym stopniu materiałem źródłowym. Jedyne źródło, które ks. Umiński zużytkował na poparcie swej tezy — Żyw ot Pięciu Braci 9 — nie dostarcza, moim zdaniem dowodu na nieistnienie w tym czasie biskupstwa poznańskiego, trudno bowiem z niego wyciągna.ć ja kieś pewniejsze wnioski zarówno za jak i przeciw istnieniu biskupstwa poznańskiego l0. Stąd też pozostać należy przy tezie, że biskupstwo po znańskie powstało w r. 1000 Również geneza archidiakonatu czer skiego w przedstawieniu autora budzi szereg wątpliwości. Błędną jest przesłanka na której opiera ks. Umiński twierdzenie, że zwrot Galla ultra flum en Wysla in Masouiam oznacza Mazowsze położone na lewym brzegu Wisły (s. 62),s. Gdyby nawet tłumaczenie to okazało się słu szne 13, to i tak pozostają jeszcze wątpliwości co do tego, czy ludność wielkopolska uciekała na Mazowsze czerskie; raczej przyjąć należy, że kierowała się ona na bliższe Mazowsze płockie. Zresztą nawet przyjęcie osiedlenia się na Mazowszu czerskim licznej kolonii wielkopolskiej nie może w sposób pewny dowodzić genezy archidiakonatu czerskiego. Słu sznie natomiast sprzeciwia się autor powstaniu archidiakonatu czer skiego w 1124 r. , , p , , , l* Pogląd, że mogło tu chodzić również o Mazowsze lewobrzeżne reprezentuje T. G r u d z i ń s k i , op. cit., cz. II, s. 65— 66. p j , p , , 14 W sprawie przynależności kościelnej ziemi czerskiej do utw rzonego w r. 1000 biskupstwa poznańskiego por. J. N o w a c k i , op. cit., s. 185— 186. Ma to wynikać z tego, że Gall pisał pierwszą księgę na terenie Mazowsza Płockiego. Dla Galla, przebywającego po wschodniej stronie Wisły, ultra flumen Wysla ma oznaczać Mazowsze po zachodniej stronie Wisły. W brew autorowi trzeba twierdzić, że nie ma najmniejszych do wodów na to, aby Gall pisał jakąkolwiek część swej kroniki na terenie Mazowsza Płockiego oraz, że tłumaczenie autora pozostaje sztucznym, nawet w wypadku przyjęcia jego tezy o miejscu pobytu Galla. Mylny jest również pogląd ks. U m i ń s k i e g o o wczesnym przybyciu G alla do Polski (na przełomie X I i X II w.), zob. w tej sprawie T. G r u d z i ń s k i , Ze studiów nad kroniką Galla, cz. I, Zapiski TN T, t. X V II, 1952, s. 73— 74 oraz cz. II, Zapiski TN T, t. X X , 1954, s. 51— 52. T R Z Y TO M Y „STU D IÓ W TEO LO G ICZNYCH ". Silnickiego jest zbyt opisowa, zbyt mało ana lityczna. Punktem wyjścia jest dla Autora z reguły jakiś ustalony już fakt, wydarzenie. Fakt tsn obszernie wyjaśnia snując w związku z nim rozważania częstokroć zbyt oderwane od źródeł i literatury. Dla przykładu przyjrzyjm y się rozdziałowi X I, w którym omówiono sprawę przeniesienia Nankera z Krakowa do Wrocławia. Obok wielu cieka wych uwag wypowiedzianych przy tej okazji, uderzyć nas musi brak rozważenia treści podstawowego w tym wypadku źródła, mianowicie bulli, a właściwie szeregu buli papieskich wydanych przy okazji zmiany na stanowisku biskupa krakowskiego, a przecież przede wszystkim na tej drodze musimy szukać wyjaśnienia samego faktu. Potrzebne byłoby dalej zestawienie i zanalizowanie wszystkich odnoszących się do tego wydarzenia relacji źródłowych. Uznanie policzka, jakim według Dłu gosza znieważył Łokietek Nankera za legendę tylko dla racji prawno- kanonicznych bez rozważenia między innymi całej relacji Długoszowej o Nankerze i próby dojścia do jej źródeł, nie może nas zadowolić. W tymże ustępie uwagę naszą zwróci także pominięcie przez Autora rozważań na temat roli Jana X X II i to mimo tego, że Ptaśnik (Denar ■św. Piotra, s. 184) temu właśnie papieżowi przypisywał decydującą rolę przy przenosinach. Co zrobiła przed 1945 r. nauka historyczna dla zbadania postaci Nankera? Z najważniejszych prac można tu wspomnieć studia C. Griin- hagena (1864), T. Wojciechowskiego (1900), Ptaśnika (1907—8), Fijałka (1916), T. Silnickiego (1939). Dotknięto w nich różnych okresów życia i działalności biskupia ustalając w iele faktów o podstawowym znacze niu, ale zarysowały się również poważne rozbieżności w rozumieniu i ocenie zwłaszcza ostatnich kilkunastu lat życia biskupa, począwszy od zagadkowego przeniesienia go w 1326 r. ze stolicy krakowskiej na wrocławską. Trzeba przyznać, że działalność Nankera jako biskupa miała miejsce w bardzo skomplikowanych warunkach społeczno-poli- tycznych, a że jednocześnie względnie skąpe źródła nie oświetlają jej wystarczająco jasno i jednoznacznie, nic dziwnego, że wywołała kon trowersje. Postulaty badawcze musiały tu iść przede wszystkim w kie runku zestawienia i poddania gruntownej analizie m ożliwie pełnej podstawy źródłowej. Jednocześnie, skoro aktualna się stała szczegól nie w ostatnim dwunastoleciu sprawa kultu Nankera, zadaniem stało się poddanie badaniu wszelkich dotychczasowych przejawów tego kultu. W tym zakresie współczesna historiografia może się poszczycić bardzo poważnym dorobkiem, który doskonale można było zastosować na gruncie polskim m. in. odnośnie do biskupa Nankera. Niestety, ani w jednym ani w drugim kierunku nie za wiele zdołaliśmy przez kil kanaście lat zrobić. Nie spełniły bowiem w pełni, w sensie ściśle nau kowym, zadania dwie poważniejsze prace prof. T R Z Y TO M Y „STU D IÓ W TEO LO G ICZNYCH ". W i n c e n t y U r b a n , De testimoniis vitam Nankeri episcopi W ra- tislaviensis illustrantibus, Collectanea Theologica 27 (1956), 250— 286 i nadb. Biskup krakowski (1320— 26) i wrocławski (1326— 41) Nanker należy niewątpliwie do jednego z ciekawszych przedstawicieli średniowiecz nego episkopatu polskiego. Z jego imieniem związane są statuty syno- • Autor połączył ten przekaz z faktem przeniesienia zwłok Pięc Męczenników do Gniezna, wynikającego z wiadomości Kosmasa, poda nej pod r. 1039. 10 Ks. U m i ń s k i , jak o tym była już mowa wyżej, z faktu, że zwłoki Pięciu Braci Męczenników przeniesiono do Gniezna, wnosi iż biskupstwo poznańskie wówczas nie istniało, w przeciwnym bowiem razie zwłoki męczenników umieszczono by w Poznaniu. Trzeba tu jed nak mieć na uwadze fakt, że nie znamy okoliczności towarzyszących przeniesieniu zwłok spod Międzyrzecza do Gniezna. Należy również uwzględnić możliwość sprowadzenia zwłok do Gniezna podczas fak tycznej (po uwięzieniu na terenie Niemiec Ungera) sedyswakancji bi skupstwa poznańskiego. 11 Teza o utworzeniu biskupstwa poznańskiego w r. 1000 wydaje się najbardziej prawdopodobna, poza nią może wchodzić w rachubę jedynie pogląd ks. N o w a c k i e g o o powstaniu niezależnego biskupstwa po znańskiego w r. 968. RE C E N ZJE i S P R A W O Z D A N IA R E C E N ZJE i S P R A W O Z D A N IA 298 299 ■dslne diecezji krakowskiej z 1320 r., uważane za jeden z najwspanial szych pomników tego rodzaju u nas. W pamięci potomnych utkwił przekazany obszernie przez Kronikę Książąt Polsk.ch cbraz jego zmagań w ostatnich latach życia ze zdobywcą Śląska, królem Janem Luksem burskim. W grę wchodzi także zagadnienie kultu religijnego Nankera, w y s u w a n e w X V III w., a następnie po odzyskaniu przsz Polskę Śląska od 1945 r. styl. Sama myśl pisania tego typu dzieł i to właśnie przez specjalistów zasługuje jak najbardziej na poparcie. W ydaje się jednak, że nawet -w takich pracach nie powinniśmy unikać wprowadzenia czytelnika w warsztat badawczy, w źródła i literaturę, że powinniśmy pokazywać mu w sposób możliwie prosty drogi naszego rozumowania, wskazywać na trudności i możliwości różnych rozwiązań, dbać pilnie o precyzję sformułowań i dostarczanie czytelnikowi przsde wszystkim argumentów, które same niejako prowadzić go będą do właściwych wniosków. Tymczasem praca prof. 1 wyd. w Księdze Pamiątkowej Bolesława Orzechowicza, Lw ów 1916. t. I. * wyd. PAU , Kraków 1939. T R Z Y TO M Y „STU D IÓ W TEO LO G ICZNYCH ". Urban zdaje się tych wszystkich niebezpieczeństw nie dostrzegać postępując dokładnie tak, jak wyżej opisaliśmy. Pierwsze i jedyne z czasów życia Nankera świadectwa o wewnętrznym, religijnym życiu biskupa znaj duje w bullach papieskich. Jan X X II w bulli z 1 października 1326 r. przenoszącej Nankera z Krakowa do Wrocławia pisze m. in.: „Con- sideratis tuarum multiplicibus virtutum meritis nobis et eisdem fra- tribus non innotis, ąubus personam tuam dominus illustravit“ (cytat u Urbana, s. 253). Czyż historyk może przyjąć tego rodzaju świadectwa bez gruntownego rozpatrzenia czy nie chodzi tu o zwykłą formułę, czy też istotnie kryje się za tym jakaś treść? I tu, i w e wszystkich na stępnych przypadkach Autor nie stawia sobie nawet tego pytania. W szczególności szkoda, że nie rozpatrzono dokładnie relacji Kroniki Książąt Polskich. Ogólne podkreślenie prawdziwości Kroniki nie w y starczy, wiadomo bowiem, że powstała ona w otoczeniu wrażliwym na niezależność Śląska, co sprzyjało przychylnej pamięci tak o Nanke- rze, jak i o związanym z nim inkwizytorze dominikańskim Janie Svenkenfelt. Wiadomości Kroniki i Długosza stanowią podstawę, do której nawiązują następni pisarze wspominający o Nankerze i doda ją cy od siebie nowe elementy. Ciekawszy jest ustęp artykułu Autora, w którym w oparciu o archiwalia wrocławskie podaje wiadomości •o wszczęciu przez kapitułę wrocławską w drugim dziesiątku lat X V III w. pierwszych kroków w celu beatyfikacji Nankera. Poglądy nowożytnych historyków przytoczone zostały w - sposób równie schematyczny jak i średniowiecznych. Uwagi o pracy Silnickiego ograniczyliśmy do kilku tylko, najistot niejszych zresztą aspektów metodyczno-konstrukcyjnych, gdyż ustosun kowanie się do wielu poruszonych w niej problemów historycznych musiałoby z konieczności znacznie rozszerzyć wąskie ramy obecnej recenzji. Zauważmy jedynie, że J.'B aszkiew icz w sw ym - omówieniu pracy Silnickiego3 kwestionuje, nieraz zbyt ostro i zwłaszcza jedno stronnie, szereg poglądów Autora dotyczących zwłaszcza oceny działal ności kościelno-politycznej Nankera. Nie ulega wątpliwości, że i te sprawy obok wielu innych muszą się znaleźć na nowo w nowoczesnym warsztacie historyka. Z omówienia obu prac o Nankerza widzim y więc, że zarówno życie biskupa jak i jego kult czekają jeszcze na historyków, którzy dać nam mogą w dzisiejszym stanie w iedzy obraz znacznie bardziej po głębiony od tego, którym dysponujemy obecnie. Z drobiazgów, dorzućmy do uwag Baszkiewicza zauważone przeze mnie pomyłki: s. 43 — archidiakonat radomski połączony był z kole giatą kielecką a nie wiślicką; Kinga osiadła u klarysek w Sączu, a nie w Zawichoście; s. T R Z Y TO M Y „STU D IÓ W TEO LO G ICZNYCH ". Nanker Silnickiego jest zbyt wyabstrahowany ze współczesnej mu rzeczywistości społecznej. Sprowadzanie najgłęb szych przyczyn wydarzeń związanych z Nankerem, jak przykładowo- przeniesienie z Krakowa do Wrocławia, do jego charakteru, musi być uznane w tej sytuacji za mało uzasadnione. W dodatku rozbicie opisu życia biskupa na kilkanaście dość odrębnych fragmentów nie sprzyja wyrobieniu w pełni uzasadnionego poglądu na badanego człowieka i jego rolę historyczną. Stąd i główna, przewijająca się od pierwszej strony myśl Silnickiego, uznająca Nankera za człowieka o cechach z innej, poprzedniej epoki, który nie chce i nie może dostosować się do nowych warunków czy to w Krakowie, czy w e Wrocławiu, nie może być uważana za udowodnioną przez Autora, co nie przesądza oczywiście- kwestii jej słuszności względnie niesłuszności. grafii. Ma ona przecież ukazać żywego człowieka w możliwie pełnym kontekście życia, w konkretnym środowisku, pokoleniu. Można wskazafr we współczesnej historiografii europejskiej <na znakomite przykłady rozwiązania tego trudnego zadania, ale nie zapominajmy, że i w Polsce jeszcze w X IX w., w klasycznej już dziś w pewnym sensie naszej hi storiografii, znajdziemy duże na tym polu osiągnięcia widoczne cho ciażby w ujęciu Mieszka Starego przez Smolkę. Z tego stanowiska można zauważyć, że prof. Silnicki zbyt silnie rozbudowuje tradycyjnie pojęte tło kosztem dokładniejszego przedstawienia konkretnych sytuacji, warunków, sił, środowisk, jednym słowem całego „m ilieu“ , w którym wypadło żyć Nankerowi. Nanker Silnickiego jest zbyt wyabstrahowany ze współczesnej mu rzeczywistości społecznej. Sprowadzanie najgłęb szych przyczyn wydarzeń związanych z Nankerem, jak przykładowo- przeniesienie z Krakowa do Wrocławia, do jego charakteru, musi być uznane w tej sytuacji za mało uzasadnione. W dodatku rozbicie opisu życia biskupa na kilkanaście dość odrębnych fragmentów nie sprzyja wyrobieniu w pełni uzasadnionego poglądu na badanego człowieka i jego rolę historyczną. Stąd i główna, przewijająca się od pierwszej strony myśl Silnickiego, uznająca Nankera za człowieka o cechach z innej, poprzedniej epoki, który nie chce i nie może dostosować się do nowych warunków czy to w Krakowie, czy w e Wrocławiu, nie może być uważana za udowodnioną przez Autora, co nie przesądza oczywiście- kwestii jej słuszności względnie niesłuszności. wania jakby na równi „świadectw11 pochodzących z różnych okresów -czasu bez przeprowadzenia dostatecznej analizy, bez zbadania wza jem nej zależności pisarzy, zajęcia się powodami ich zainteresowań Nankerem itd., niebezpieczeństwo pomieszania źródeł do Nankera z dziejami Nankerowej legendy. Samo streszczenie czy nawet przyto czenie w całości poglądów pisarzy, zwłaszcza gdy chodzi o dzieła dru kowane, nie ma większego naukowego znaczenia. Tymczasem ks. 3 Kwartalnik Historyczny, L X I, nr 4, s. 264-—270. T R Z Y TO M Y „STU D IÓ W TEO LO G ICZNYCH ". T. Silnickiego i ks. W. Urbana, jakie poświęcono w ostatnim czasie Nankerowi. Chęć zbliżenia Nankera do czytelnika czy też nieuzasadnione w pracy przekonanie o niezmienności osobowości tego człowieka prowadzi Autora do sformułowań mało ostrożnych, jak np. to, żs „Nanker — to od po czątku człowiek z jednej bryły i to z najszlachetniejszego kruszcu...“ (s. 25). Mało też przekonywuje nas stosowanie tego założenia do po szczególnych sytuacji życiowych Nankera i wiązanie np. objęcia archi diakonatu sandomierskiego ze skromnością i obojętnością przyszłego biskupa na w zględy ambicjonalno-materialne. Zdaje się, że w szcze gólności w opracowaniach tego typu co „Biskup Nanker“ trzeba się wystrzegać wszystkiego, co tchnie jakim kolwiek panegiryzmem, właśnie ze względu na tak łatwo grożące tu odejście od prawdy i ścisłości historycznej. T. Silnicki oparł się w zasadzie o istniejącą literaturę przedmiotu i jej ustalenia wykorzystując w szczególności Przeszłość Nankera F i jałka 1 i własne Dzieje i ustrój Kościoła na Śląsku.1 P o raz pierwszy uzyskaliśmy w dziele Autora obszerniejsze przedstawienie całości ży cia Nankera. W ykład podzielono na 20 kilkustronicowych rozdziałków, z których każdy poświęcony jest jakiemuś fragmentowi czy zagadnieniu związanemu z życiem Nankera. Autor bardzo rozbudował partie opi sowe malując obszernie tło wydarzeń. Ułatwia to czytelnikowi nie- historykowi zapoznanie się z Nankerem. Dzieło Silnickiego ma charak ter popularno-naukowy, którego walory znacznie podnosi dobry, jasny Zasygnalizujmy także innego rodzaju zastrzeżenia budzące się w związku z ujęciem postaci Nankera przez Autora, z konstrukcją jego dzieła. N ie zapominajmy przy tym, że biografia — tak jak ją dziś poj mujemy — należy niewątpliwie do najtrudniejszych działów historio R E C EN ZJE i S P R A W O Z D A N IA R E C EN ZJE i S P R A W O Z D A N IA 301 300 grafii. Ma ona przecież ukazać żywego człowieka w możliwie pełnym kontekście życia, w konkretnym środowisku, pokoleniu. Można wskazafr we współczesnej historiografii europejskiej <na znakomite przykłady rozwiązania tego trudnego zadania, ale nie zapominajmy, że i w Polsce jeszcze w X IX w., w klasycznej już dziś w pewnym sensie naszej hi storiografii, znajdziemy duże na tym polu osiągnięcia widoczne cho ciażby w ujęciu Mieszka Starego przez Smolkę. Z tego stanowiska można zauważyć, że prof. Silnicki zbyt silnie rozbudowuje tradycyjnie pojęte tło kosztem dokładniejszego przedstawienia konkretnych sytuacji, warunków, sił, środowisk, jednym słowem całego „m ilieu“ , w którym wypadło żyć Nankerowi. T R Z Y TO M Y „STU D IÓ W TEO LO G ICZNYCH ". 72 — lepiej nie używać określenia augustianie dla kanoników regularnych na Piasku wrocławskim, aby nie mylono ich z augustianami — eremitami. Jerzy Kloczowski A l e k s a n d e r R o g a l s k i , Kościół katolicki na Śląsku. Studia nad dziejami diecezji wrocławskiej, Warszawa 1955, PA X , s. 288, map 3. Ks. Wincenty Urban stawia za zadanie swego artykułu zabranie- wszelkich „świadectw11 dotyczących wewnętrznego życia biskupa Nan kera począwszy od czasów współczesnych biskupowi aż po dzień dzi siejszy. Już w samym założeniu tkwi tu niebezpieczeństwo potrakto Historiografia wrocławskiej diecezji znajduje się w dość szczęśliwym położeniu i stanie, ponieważ ma w swym dorobku nie tylko cały szereg pozycji źródłowych, ale bardzo w iele opracowań monograficznych, czy też drobnych prac szczegółowych. Żadna z polskich diecezji nie może się z nią równać w tym względzie. Wprawdzie niektóre z tego całego RE C E N ZJE i S P R A W O Z D A N IA 302 RE C E N ZJE I S P R A W O Z D A N IA 303 sienie obrazu, cytując całe partie z innych autorów, np. z „Historii Śląska w zarysie-* Piwarskiego, por. stronice 30— 31, lub z Silnickiego, s. 16. zasobu prac nie odznaczają się historyczną obiektywnością, gdyż za ciążył na nich bardzo silnie duch tendencyjności, nie mniej jednak przynoszą one cały szereg spostrzeżeń słusznych i pożytecznych, a dla naukowych badań nieodzownych. Również i syntetyczne ujęcia dziejów wrocławskiej diecezji nie przedstawiają się najgorzej w porównaniu z innymi discezjami Polski. Praca autora dzieli się na pięć części oraz zawiera dokumenty, zwłaszcza nowsze, Uczy 288 stronic i 3 orientacyjne mapki. Pierwsza część pt. „Dzieje diecezji wrocławskiej od założenia do dni dzisiejszych11 składa się z 10 rozdziałów, druga „Słudzy ołtarza i tronu" (ideologia i postawa społeczna władz kościelnych w diecezji wrocławskiej w w. X IX i X X ) z 7 rozdziałów, trzecia „Arka przymierza między dawnymi a nowym i laty" (walka o polskość i z polskością na terenie Kościoła w diecezji wrocławskiej w X IX i X X w.) ma 12 rozdziałów, czwarta „Protestantyzm-katolicyzm-polskość (procesy relig jne w łonie katoli cyzmu diecezji wrocławskiej w X IX i X X w.) liczy 11 rozdziałów, piąta „Duchowni — przywódcy ludu śląskiego w wieku X IX /X X ) ma tylko 6 rozdziałów. Zaznaczyć też trzeba, że niektóre rozdziały są bar dzo krótkie, bo liczą zaledwie dwie stronice (np. T R Z Y TO M Y „STU D IÓ W TEO LO G ICZNYCH ". Dla przejrzystości na leżałoby osobno wyliczyć źródła rękopiśmienne, drukowane i opraco wania, a wśród materiałów archiwalnych trzeba było podać tytuły wykorzystanych jednostek lub zespołów, bp podanie źródeł rękopiśmien nych ogólnie: „Akta Archiwum Państwowego we Wrocławiu1* nie w iele nam mówi i nie ułatwia bynajmniej dalszych badań i kontroli. Przy aktach Archiwum Archidiecezjalnego autor podał sygnatury I A 17— 24. Tutaj należało wymienić tytuły poszczególnych archiwaliów i wówczas autor nie musiałby cytować dopiero pod literą D jednostki „Dioecesan- Berichte an den Heiligen Vater“. W polskiej literaturze historycznej stanowi niewątpliwie najlepszą pozycję praca prof. Tadeusza S i l n i c k i e g o pt. „Dzieje i ustrój K o ścioła na Śląsku do końca w. X IV . Historia Śląska od najdawniejszych czasów do roku 1400", t. II, z. 1, Kraków 1939. Wznowienie tej pracy ukazało się nakładem „P a x“ w Warszawie w 1953 r. pt. „Dzieje i ustrój Kościoła katolickiego na Śląsku do końca w. X IV “ . Praca profesora Silnickiego sięga tylko do roku 1400. Stąd też w nauce polskiej, zw ła szcza po roku 1945, odczuwano dotkliwie brak opracowania całości dziejów wrocławskiej diecezji, niezmiernie potrzebnego w związku ze zmianami, jakie się dokonały na Śląsku. Tego pięknego zadania podjął się Aleksander R o g a l s k i , ale podszedł do niego już z pewnym na stawieniem, jak wyznaje w przedmowie: „celem niniejszej książki jest przedstawienie i zanalizowanie zasadniczych zjawisk i procesów, jakie miały miejsce na terenie Kościoła w diecezji wrocławskiej od mo mentu zajęcia Śląska przez Prusy aż do pogromu Trzeciej Rzeszy, aż do jego powrotu do Polski". Inne zastrzeżenia dotyczą więcsj natury metodologicznej. W wyka zie źródeł i literatury podano około 160 pozycji. Dla przejrzystości na leżałoby osobno wyliczyć źródła rękopiśmienne, drukowane i opraco wania, a wśród materiałów archiwalnych trzeba było podać tytuły wykorzystanych jednostek lub zespołów, bp podanie źródeł rękopiśmien nych ogólnie: „Akta Archiwum Państwowego we Wrocławiu1* nie w iele nam mówi i nie ułatwia bynajmniej dalszych badań i kontroli. Przy aktach Archiwum Archidiecezjalnego autor podał sygnatury I A 17— 24. Tutaj należało wymienić tytuły poszczególnych archiwaliów i wówczas autor nie musiałby cytować dopiero pod literą D jednostki „Dioecesan- Berichte an den Heiligen Vater“. T R Z Y TO M Y „STU D IÓ W TEO LO G ICZNYCH ". str. 25— 27). Autor „Kościoła katolickiego na Śląsku1 musiał ogarnąć pod względem chronologicznym materiały bardzo szerokie, stąd miał liczne trudności do pokonania, dlatego też przy czytaniu niniejszej pracy nasuwają się- pewne zastrzeżenia. I tak ks. prof. Józef Ignacy R i t t e r w wydanej we W rocław ia w 1845 roku pracy pt. „Geschichte der Diocese Breslau. Erster Theil. Von der Pflanzung des Christenthums in Schlesien bis zum Jahre 1290“ , starał się torować pierwsze ślady. Po nim ks. Jan H e y n e, beneficjat przy kaplicy św. Elżbiety w katedrze wrocławskiej, bardzo pracowity, wydał w trzech tomach w latach 1860, 1864, 1868 pracę „Dokumen- tirte Geschichte des Bisthums und Hochstiftes Breslau". Praca ukazała się drukiem we Wrocławiu, dziś jest już nieco przestarzała, ale ze względu na zebrany w niej źródłowy materiał stanowi dla badacza ko ścielnej przeszłości na Śląsku rzecz nieodzowną. Zamiłowany w prze szłości Śląska i Kościoła ks. Jan C h r z ą s z c z , proboszcz w Pysko wicach, dał do ręki chętnych czytelników w popularniejszej form ie „Kirchengeschichte Schlesiens. Fur Schule und Haus“ , wydanej w e Wrociawiu w 1908 r. W związku z wyniesieniem wrocławskiego biskup stwa do godności arcybiskupstwa w 1929 r. ogłosił ks. prof. Franciszek Ksawery S e p p e l t w pięknej syntezie, wnikLwie, ale niekiedy z pew nym zabarwieniem tendencyjnym nakładem książęco-biskupiego ordyna riatu w e Wrocławiu „Geschichte des Bisthums Breslau. Najpierw sam tytuł nie pokrywa się z treścią i należałoby raczej poprzestać na podtytule „Studia nad dziejami diecezji wrocławskiej1' w X IX i X X wieku. Jedynie pierwsza część i to w zbyt krótkim rzucie, daje nam chronologiczny pogląd na całość dziejów biskupstwa wro cławskiego, a inne części są ściśle związane z tematyką X IX i X X w . Najpierw sam tytuł nie pokrywa się z treścią i należałoby raczej poprzestać na podtytule „Studia nad dziejami diecezji wrocławskiej1' w X IX i X X wieku. Jedynie pierwsza część i to w zbyt krótkim rzucie, daje nam chronologiczny pogląd na całość dziejów biskupstwa wro cławskiego, a inne części są ściśle związane z tematyką X IX i X X w . Inne zastrzeżenia dotyczą więcsj natury metodologicznej. W wyka zie źródeł i literatury podano około 160 pozycji. T R Z Y TO M Y „STU D IÓ W TEO LO G ICZNYCH ". Celowość podawanej w spisie literatury polega między innymi i na tym, że ułatwia on nam cytowanie w przypiskach, w których możemy korzystać już ze skróconego tytułu przy podaniu nazwiska autora, dla tego też w wykazie tytuł dzieła powinien być pełny. Autor nie prze strzega zawsze tego prawidła i zbyt skraca niekiedy tytuły dzieł, np- s. 279: E n g e l b e r t K , „Kasper von Logau“. W tym wypadku w spisie literatury należało podać „Kasper von Logau, Bischof von Breslau (1562— 1574)*, Breslau 1926, przy Bernardzie B u l o w i e , „D enk- j g p Ujmowanie historii Kościoła musi jednak dawać więcej. j j ę j Praca dra Aleksandra Rogalskiego ma charakter wybitnie popu larno-naukowy, autor włożył w nią niewątpliwie wiele wysiłku, zgro madził znaczny materiał, ale w wielu wypadkach ułatwia sobie kre- RE C E N ZJE i S P R A W O Z D A N IA R E C EN ZJE i S P R A W O Z D A N IA 304 305 wiirdigkeiten“ , Berlin 1930/31, przy F e l d m a n i e winien być również pełny tytuł „Problem polsko-fniemiecki w dziejach", Katowice 1946. Podobnie niepotrzebnie skrócony jest tytuł dzieła J a b ł o ń s k i e g o 0 książęco — biskupiej delegaturze w Berlinie. Na stronicy 284 czytamy: „Visitationsberichte Breslau1', t. I— III, W rocław 1902 i n. Ten skrót w tym miejscu jest stanowczo niewłaściwy. Publikacje w izytacji pa rafialnych z zasobów Archiwum Archidiecezjalnego w e Wrocławiu w y dawane przez zasłużonego i pracowitego badacza ks. Józefa J u n g - n i t z a ukazały się jako: „Veróffentlichungen aus dem Fiirstbischófli- chen Diozesan - Archive zu Breslau", jako tom I wyszły „Visita- tionsberichte der Diózese Breslau: Archidiakonat Breslau", Breslau 1902, drugi tom „Archidiakonat Oppeln“, Breslau 1904, trzeci „Archidiakonat Glogau“ , Breslau 1907, czwarty „Archidiakonat Liegnitz", Breslau 1908 Z przytoczonego skrótu można było korzystać najwyżej w przypisku- Szkoda też, że Wydawnictwo „Pax“ nie postarało się o sporządzenie indeksu do książki. podobne, że katedra nie uległa wtedy zniszczeniu. Na stronicy 22 na leżało podać lata rządów biskupstwem wrocławskim biskupa Nankiera i Przecława z Pogorzeli. Na stronicy 26 autor pisze, że „ośrodkiem niemczyzny stały się klasztory cysterskie na wsi, dominikanów zaś i franciszkanów w mieście". 1 Kronika Thietmara. tł. M. Z. J e d l i c k i Poznań 1953, s. 206— 208. - W o stand der romanische Dom des Breslauer Bischofs W a <1149— 1169)?, Archiv fur schlesiche Kirchengeschichte 2, 1937, s. 33—56. T R Z Y TO M Y „STU D IÓ W TEO LO G ICZNYCH ". Działalność kardynała Adolfa B e r t r a m a wymaga jeszcze sumiennego opracowania monograficznego, wydane bowiem do tychczas przyczynki, nie wyłączając pracy ks. Kurta E n g e l b e r t a 7, nie przedstawiają tej ciekawej postaci w e właściwym świetle, stąd też i wydane o nim sądy wydają się przedwczesne. Pod względem merytorycznym dużo zastrzeżeń budzą sformułowania treściowe drugiej części, zachodzą też w toku pracy i inne ujęcia, których nie da się utrzymać przy pełniejszym zbadaniu źródeł. Na stronicy 7 autor utrzymuje, że założono w 1000 r. „biskupstwa w K ra kowie i w e Wrocławiu, które wraz z biskupstwem poznańskim miały stanowić sufraganie arcybiskupstwa gnieźnieńskiego'1. Na stronicy 11 jest już w tej sprawie całkowicie poprawne sformułowanie, że w 1000 r. powstała metropolia w Gnieźnie, której poddano jako sufraganie utwo rzone biskupstwa w Krakowie, Wrocławiu i Kołobrzegu. Fakt założenia arcybiskupstwa w Gnieźnie i wszystkie okoliczności temu towarzyszące opisał świadek ówczesnych czasów, biskup merseburski Thietmar: „ar- cybiskupstwo to powierzył bratu wspomnianego męczennika Radzimowi 1 podporządkował mu z wyjątkiem biskupa poznańskiego Ungera na stępujących biskupów: kołobrzeskiego Reinberna, krakowskiego Pop- pona i wrocławskiego Jana'1 Na stronicy 10 korektor dopuścił się znacznego przeoczenia. Jest tu mowa o wyniesieniu wrocławskiego biskupstwa w 1929 r. do godności arcybiskupstwa i poddaniu mu jako sufraganii biskupstwa berlińskiego, warmińskiego oraz „polskiej pra- łatury w Pile". Nia idzie tu bowiem o taką prałaturę, tylko o prałaturę „nullius" w Pile. Na stronicy 16 zaszła pomyłka, że następcą biskupa Wojciecha został kanonik wrocławski Tomasz", poprawnie ma być „na stępcą biskupa Wawrzyńca..." Odnośnie kwestii pożaru katedry we Wrocławiu w 1241 r. w czasie najazdu Tatarów, zdania historyków są podzielone. W ydaje mi się, że argumenty ks. Ewalda W a l t e r a i Ludwika B u r g e m e i s t r a 3 w tej sprawie są bardziej prawdo Wymienione usterki nie osłabiają bynajmniej celowości i użytecz ności pracy dra Rogalskiego, która niewątpliwie stanowić będzie dla polskiego czytelnika pomoc w poznawaniu przeszłości ziemi śląskiej, jej kultury, jej zmagań, trudów i wzlotów. Autor zebrał też dużo ma teriału statystycznego dla zobrazowania zasięgu polskiego języka w na bożeństwach, duszpasterstwie i kaznodziejstwie, który będzie przydatny w przyszłych badaniach. Na pełniejszą historię Kościoła na Śląsku, która uwydatni wkład jego w życie duchowe i materialne społeczeństwa będziemy musieli jeszcze poczekać. Ks. Wincenty Urban y Ks. 3 Die Kunstdenkmaler der Stadt Breslau, Breslau 1930, s. 57. 4 Sprawy narodowościowe u Franciszkanów śląskich w X III w., Warszawa 1954, s. 51— 56 i nn. 5 Por. Ks. Wincenty U r b a n , Język polski w duszpasterstwie wroc ławskiej diecezji. Wrocław 1956 (maszynopis). 8 Dominikanie polscy na Śląsku w X III— X IV wieku, Lublin 1956, s. 171 i nn. ' A dolf Kardinal Bertram-Fiirsttrzbischof von Breslau (1914— 1945), Archiv fur schlesische Kirchengeschichte 7, 1949, s. 7— 37. ' A dolf Kardinal Bertram-Fiirsttrzbischof von Breslau (1914— 1945), Archiv fur schlesische Kirchengeschichte 7, 1949, s. 7— 37. T R Z Y TO M Y „STU D IÓ W TEO LO G ICZNYCH ". Jest prawdą, że- klasztorycysterskie i fran ciszkańskie uległy dość wcześnie silnie niemieckim wpływom, chociaż niedawno wydana przez o. Czesława Cezara B a r a n a * praca o fran ciszkanach wykazała jeszcze znaczną ilość zakonników polskiej na rodowości. Nie można się jednak zgodzić n a -te n -zaraut* pod adresem dominikanów. Szerzej wykazuję to podtrzymywanie polskiej kultury językowej przez wrocławskich dominikanów na innym miejscu.5 Pod kreślił to również Jerzy K l o c z o w s k i * , że łączność śląskich domi nikanów z polską macierzą miała w pływ na utrzymanie się polskości w nadodrzańskim kraju. Zresztą i sam autor przyjmuje (str. 107) zda nie J. S. B a n d t k i e g o o wybornych kazaniach polskich w kościele św. Wojciecha we Wrocławiu. Na stronicy 44 jest nietrafne wyrażenie o gitarzystach". Działalność kardynała Adolfa B e r t r a m a wymaga jeszcze sumiennego opracowania monograficznego, wydane bowiem do tychczas przyczynki, nie wyłączając pracy ks. Kurta E n g e l b e r t a 7, nie przedstawiają tej ciekawej postaci w e właściwym świetle, stąd też i wydane o nim sądy wydają się przedwczesne. podobne, że katedra nie uległa wtedy zniszczeniu. Na stronicy 22 na leżało podać lata rządów biskupstwem wrocławskim biskupa Nankiera i Przecława z Pogorzeli. Na stronicy 26 autor pisze, że „ośrodkiem niemczyzny stały się klasztory cysterskie na wsi, dominikanów zaś i franciszkanów w mieście". Jest prawdą, że- klasztorycysterskie i fran ciszkańskie uległy dość wcześnie silnie niemieckim wpływom, chociaż niedawno wydana przez o. Czesława Cezara B a r a n a * praca o fran ciszkanach wykazała jeszcze znaczną ilość zakonników polskiej na rodowości. Nie można się jednak zgodzić n a -te n -zaraut* pod adresem dominikanów. Szerzej wykazuję to podtrzymywanie polskiej kultury językowej przez wrocławskich dominikanów na innym miejscu.5 Pod kreślił to również Jerzy K l o c z o w s k i * , że łączność śląskich domi nikanów z polską macierzą miała w pływ na utrzymanie się polskości w nadodrzańskim kraju. Zresztą i sam autor przyjmuje (str. 107) zda nie J. S. B a n d t k i e g o o wybornych kazaniach polskich w kościele św. Wojciecha we Wrocławiu. Na stronicy 44 jest nietrafne wyrażenie o gitarzystach". 8 Dominikanie polscy na Śląsku w X III— X IV wieku, Lublin 1956, s. 171 i nn. 5 Por. Ks. Wincenty U r b a n , Język polski w duszpasterstwie wroc ławskiej diecezji. Wrocław 1956 (maszynopis). Ś 3 Die Kunstdenkmaler der Stadt Breslau, Breslau 1930, s. 57. 4 Sprawy narodowościowe u Franciszkanów śląskich w X III w., Warszawa 1954, s. 51— 56 i nn. T R Z Y TO M Y „STU D IÓ W TEO LO G ICZNYCH ". Artykuł ks. Schletza, który dał się już poznać w szeregu prac dotyczących przede wszystkim własnego Zgromadzenia, a który z przyczyn od siebie niezależnych był przez kilka lat skazany na m il czenie, daje czytelnikom Annałów, więc Francuzom i innym nie Polakom, skondensowaną syntezę części działalności synów św. W in centego w Polsce. Celem swoim, zasięgiem czasu i działalności misjo narzy różni się ten artykuł znacznie od wydanej przed 30 laty „Księgi pamiątkowej trzechsetlecia Zgromadzenia Księży Misjonarzy 1625— 1925“ , Kraków 1925. Artykuł przeznaczony jest dla licznych czytelników poza Polską, jest i ma być jedynie treściwym artykułem, dotyczy jedynie misjonarzy w Polsce, jedynie od sprowadzenia ich do Polski przez Marię Ludwikę do czasu kasaty głównych ich ośrodków pod berłem carów. A ogranicza się wyraźnie do trzech zadań, które misjonarze spełniali w tym czasie w Polsce i krająch sąsiednich w dziedzinie cyw ilizacji i kultury umysłu i ducha, a mianowicie: praca wśród włościan, działal ność pedagogiczna, praca naukowa i wydawnictwa. Artykuł ten, choć pomyślany jako podstawowa orientacja dla nie Polaków w tym przed miocie, przyda się także historiografii polskiej; nie tylko dzięki skrzęt nemu zebraniu ważniejszych faktów i osób w każdym z wymienionych trzech punktów, ale także dzięki wcale obfitej bibliografii przedmiotu, także archiwalnej, przytoczonej w dokumentacji poruszanych zagadnień. Krasław Białystok Śmiłowicze Bydgoszcz Łysków Wilno Zasław Oświej Kraków Siemiatycze 3. Seminaria duchowne diecezjalne, prowadzone przez misjonar wylicza Autor tylko w liczbie 19 (ss. 119 nn). Było ich łącznie 25: Białystok Brzozów Chełmno Gniezno Iłłukszta Krasław Krasnostaw Lublin 2 Lw ów Warszawa Łowicz 2 Wilno Mińsk Wornie Mohylów n. Dnieprem Żytomierz Płock --------- Poznań Wiedeń Przemyśl Vacs Tykocin Tyrnawa Białystok 2 Lw ów Warszawa Brzozów 2 Wilno Łowicz Chełmno Mińsk Wornie Gniezno Mohylów n. Dnieprem Ż Mohylów n. Dnieprem Żytomierz Żytomierz Płock Poznań Już dokładniej podaje Autor liczby niektórych misyj ludowych, odprawianych przez misjonarzy polskiej prowincji. Jedynie niektórych, bo inne dane nie zachowały się, ale i te są bardzo wymowne (ss. 114 nn). Np. misjonarze ze Stradomia w Krakowie przeprowadzili w latach 1682— 1782, a więc w okresie stu lat, 459 misyj; w latach 1686— 1763 misjonarze wileńscy 240 misyj, w latach 1741— 1844 misjonarze warszaw scy 336 misyj. Do bezstronnej oceny tej pracy trzeba jednak brać pod uwagę szereg czynników i okoliczności, wpływających dodatnio lub ujemnie na natężenie działalności misjonarzy. Więc przebieg poszcze gólnych misyj, które wraz z nauczaniem katechizmu trwały w wiekach 17 i 18 od 2 do 6 tygodni. T R Z Y TO M Y „STU D IÓ W TEO LO G ICZNYCH ". Wincenty Urban A l p h o n s e S c h l e t z C. M., L ’Activite culturelle des Pretres de la Mission en Pologne (1651— 1864), Annales de la Congregation de la Mis- sion, t. 119— 120, Paris 1954, ss. 113— 134 i nadb. Paris 1954. Recenzje o artykułach nie weszły dotąd w ogólny zwyczaj. Ma to 20 20 Kasza Przeszłość Kasza Przeszłość RE C EN ZJE i S P R A W O Z D A N IA 306 RE C E N ZJE i S P R A W O Z D A N IA 307 swoje strony dodatnie, jeśli się ograniczamy do omawiania krytycz nego prac obszerniejszych, ale ma też swoje strony ujemne. Dotyczy to zwłaszcza artykułów ogłaszanych w periodykach i wydawnictwach seryjnych poza Polską, które docierają do szerszych kół naukowców dopiero w późniejszych, często bardzo późnych wykazach bibliogra ficznych. Artykuł ks. Schletza, który dał się już poznać w szeregu prac dotyczących przede wszystkim własnego Zgromadzenia, a który z przyczyn od siebie niezależnych był przez kilka lat skazany na m il czenie, daje czytelnikom Annałów, więc Francuzom i innym nie Polakom, skondensowaną syntezę części działalności synów św. W in centego w Polsce. Celem swoim, zasięgiem czasu i działalności misjo narzy różni się ten artykuł znacznie od wydanej przed 30 laty „Księgi pamiątkowej trzechsetlecia Zgromadzenia Księży Misjonarzy 1625— 1925“ , Kraków 1925. Artykuł przeznaczony jest dla licznych czytelników poza Polską, jest i ma być jedynie treściwym artykułem, dotyczy jedynie misjonarzy w Polsce, jedynie od sprowadzenia ich do Polski przez Marię Ludwikę do czasu kasaty głównych ich ośrodków pod berłem carów. A ogranicza się wyraźnie do trzech zadań, które misjonarze spełniali w tym czasie w Polsce i krająch sąsiednich w dziedzinie cyw ilizacji i kultury umysłu i ducha, a mianowicie: praca wśród włościan, działal ność pedagogiczna, praca naukowa i wydawnictwa. Artykuł ten, choć pomyślany jako podstawowa orientacja dla nie Polaków w tym przed miocie, przyda się także historiografii polskiej; nie tylko dzięki skrzęt nemu zebraniu ważniejszych faktów i osób w każdym z wymienionych trzech punktów, ale także dzięki wcale obfitej bibliografii przedmiotu, także archiwalnej, przytoczonej w dokumentacji poruszanych zagadnień. swoje strony dodatnie, jeśli się ograniczamy do omawiania krytycz nego prac obszerniejszych, ale ma też swoje strony ujemne. Dotyczy to zwłaszcza artykułów ogłaszanych w periodykach i wydawnictwach seryjnych poza Polską, które docierają do szerszych kół naukowców dopiero w późniejszych, często bardzo późnych wykazach bibliogra ficznych. T R Z Y TO M Y „STU D IÓ W TEO LO G ICZNYCH ". Konarskiego. Stu dia klasyczne pijarów polskich, Lublin 1953, str. 72. W ciągu ostatnich lat ukazał się cały szereg publikacji dotyczących polskiego Oświecenia. Nie wchodząc w tej chwili w ocenę wartości naukowej tych publikacji — wartość ta jest bardzo różna — możnaby je z grubsza podzielić na dwie grupy. Pierwsza obejmuje publikacje o charakterze syntetycznym, publikacje, które chcą dać syntetyczny obraz całokształtu polskiego życia umysłowego w tym okresie np.: S u c h o d o l s k i B o h d a n : Nauka polska w okresie Oświecenia. 1953 (wstęp); L e ś n o d o r s k i B o g u s ł a w , O p a ł e k K a z i m i e r z : Nauka polskiego Oświecenia w walce o postęp. 1951; lub też jednego tylko, ale szerokiego jego wycinka np. O p a ł e k K a z i m i e r z : Prawo na tury u polskich fizjokratów. Warszawa, 1953. Druga obejmuje publi kacje o charakterze raczej monograficznym; nie zawsze są to mono grafie w ścisłym tego słowa znaczeniu, niemniej jednak charaktery styczne jest dla nich, że nie chcą syntetyzować, ale stawiają sobie za zadanie zbadanie, i to m ożliwie szczegółowe jednego, mniej lub w ię cej obszernego wycinka polskiego życia umysłowego w okresie Oświe cenia. Jako przykład takich publikacji mogą służyć: K o l a s a J a n : Prawo narodów w szkołach polskich wieku Oświecenia. Warszawa, 1954. R z a d k o w s k a E w a : Encyklopedia i Diderot w polskim Oświeceniu. Wrocław, 1955; O p a ł e k K a z i m i e r z : Hugona Kołłątaja poglądy na państwo i prawo. Warszawa, 1952 i in. Kilka szczegółów, które podaje Autor, można by zakwestionować. Np.: Czy ks. 0'Fogerti jest istotnie Francuzem (114), czy też Irlandczy kiem należącym do francuskiej prowincji ? Czy ks. A. Zygmunt jest istotnie wynalazcą alfabetu dla głucho niemych (124), czy też tylko przystosował go i rozbudował zgodnie z po trzebami języka, lub co najwyżej wynalazł nowy system. Wszak w y nalazcą właściwym był Hiszpan Pedro Ponce w 16 wieku. T R Z Y TO M Y „STU D IÓ W TEO LO G ICZNYCH ". a stałyby się znakomitą pomocą dla naszej historiografii już nie tylko kościelnej, ale i świeckiej. Gotowym do druku jest o ile mi wiadomo jedynie katalog jezuitów-artystów, który opracował zmarły przedwcze śnie o. Poplatek TJ. Również nieżyjąca już Hanna Pohoska pracowała nad katalogiem pijarów-pisarzy. Nad katalogiem polskich franciszka nów konwentualnych pracuje o. Cezar Baran. Zrobiono więc w tej dziedzinie bardzo mało, inne zakony wogóle się jeszcze do tego nie zabrały. Życzyć można, aby ks. Schletz nie dał się od tej pracy odciąg nąć współpracą nad ogólnym słownikiem biograficznym misjonarzy, na co się podobno zanosi. a stałyby się znakomitą pomocą dla naszej historiografii już nie tylko kościelnej, ale i świeckiej. Gotowym do druku jest o ile mi wiadomo jedynie katalog jezuitów-artystów, który opracował zmarły przedwcze śnie o. Poplatek TJ. Również nieżyjąca już Hanna Pohoska pracowała nad katalogiem pijarów-pisarzy. Nad katalogiem polskich franciszka nów konwentualnych pracuje o. Cezar Baran. Zrobiono więc w tej dziedzinie bardzo mało, inne zakony wogóle się jeszcze do tego nie zabrały. Życzyć można, aby ks. Schletz nie dał się od tej pracy odciąg nąć współpracą nad ogólnym słownikiem biograficznym misjonarzy, na co się podobno zanosi. Podsumowując, trzeba stwierdzić, że zestawienie jakie dał ks. Schletz dla czytelników nie znających polskiego języka, jest niewątpliwie bar dzo pożyteczne dla ich zainteresowań dziejami misjonarzy w Polsce. 1 jest dla nich podstawowe, bo chyba pierwsze tego rodzaju, jakie im dano. Innym postulatem, którego spełnienie naświetliłoby lepiej pozy tywny wkład misjonarzy w dzieło oświaty i nauki w Polsce, byłby kata log dzieł napisanych przez nich oraz druków, wydanych w ich drukar niach. Analogicznie czekamy na podobne wykazy prac i druków przy najmniej pijarów, jezuitów, bazylianów. Ks. Zdzisław Obertyński Za ortografię i korektę Autor nie odpowiada. Jednakowoż jest rzeczą przykrą, że pozostaną na zawsze w Annałach usterki, jak np. Lukawszewicz (114), Glemme (124), Chodzyński (127). Niestety takie A n - nales nie są pod tym względem u Francuzów wyjątkiem : ostatnio w y dane tomy w ielkiej poważnej Histoire de 1’Eglise, redagowanej obecnie przez profesorów Duroselle i ks. Jarry, roją się od podobnych i gorszych zniekształceń, za które — i za sporo innych, także rzeczowych — odpo wiadają autorowie i redakcje. M a r i a n P l e z i a , Dokoła reformy szkolnej St. Konarskiego. Stu dia klasyczne pijarów polskich, Lublin 1953, str. 72. M a r i a n P l e z i a , Dokoła reformy szkolnej St. T R Z Y TO M Y „STU D IÓ W TEO LO G ICZNYCH ". Uwzględnić trzeba dalej ilość misjonowanych parafij, ich zagęszczenie ludnościowe, zmieszanie z ludnością niekato licką, liczbę pracujących misjonarzy, stosunek do nich duchowieństwa parafialnego, klęski elementarne, wydarzenia polityczne, jak rozbiory Polski, w ojny napoleońskie, powstanie listopadowe i jego skutki. Jednakowoż dla użytku czytelników i badaczy niepolskich, były by się przydały dane nieco bogatsze, pomimo koniecznego zacieśniania się do ram artykułu. Wkład bowiem polskiej prowincji misjonarzy w du chowe życie Polski i krajów ościennych jest tak poważny, że należało moim zdaniem podać jako dokumentację kilka suchych a wymownych cyfr. Na przykład: Na nieco dokładniejsze omówienie zasługiwałyby dla obcokrajowców moim zdaniem Gorzkie Żale; nabożeństwo wprowadzone pierwotnie w kościele św. Krzyża w Warszawie przez wizytatora ks. Bartłomieja Tarłę, późniejszego biskupa poznańskiego, a w następstwie rozpow szechnione w całej Polsce i Litw ie (s. 132). Nabożeństwo tak charakte rystyczne obecnie dla polskich zwyczajów wielkopostnych. 1. Misjonarskie kościoły parafialne i nieparafialne: Sokółka Białystok Lw ów Lw ów Łysków Chełmno Subocza Głogów Mikulińce Tykocin Horodenka Mław Warszawa Autor podaje czytelnikowi szereg wybitnych polskich misjonarzy, zasłużonych w dziedzinie organizacji, duszpasterstwa, kaznodziejstwa, nauki. Krótkie te z konieczności wzmianki nasuwają myśl o postulacie wykończenia m ożliwie rychło i wydania posuniętej już daleko przez Autora pracy nad Słownikiem Biograficznym polskich misjonarzy. Nawet dobrze się do tego nadaje 300-lecie przybycia do Polski misjonarzy, które obchodzono w r. 1951. Prace nad takimi słownikami zakonów i zgro madzeń zakonnych w Polsce są u nas ogółem dopiero w kołyskach, 2 Wilno Ihumen Orsz Wojciech Oświe Iłłukszta Gdańska Kraków Sambo Zasław Siemiatycze Krasław Lublin Śmiłowicze 2. Misjonarskie szkoły parafialne i średnie, których Autor nie podaje w całości, a działające w liczbie 11: RE C E N ZJE i S P R A W O Z D A N IA R E C E N ZJE i S P R A W O Z D A N IA 309 308 zjalnych seminariów przez misjonarzy dlatego, że Szczuka jest opatem paradyskim. Ten wniosek jest jednak chyba mylny; zapewne chodzi tu 0 darowiznę bpa chełmińskiego Jana Małachowskiego z r. 1677 na rzecz seminarium chełmińskiego przez misjonarzy, wzgl. dla samych misjo narzy, darowiznę, którą sejm zatwierdził w r. 1678, a zapewne także Innocenty X I. Zaś Kazim ierz Szczuka był w Paradyżu jedynie opatem komendataryjnym — zresztą bardzo dobroczynnym, znacznie później dopiero i na krótko bpem chełmińskim. A teraz musiał się w jakiś sposób zajmować sprawą osiedlenia misjonarzy w Chełmie. T R Z Y TO M Y „STU D IÓ W TEO LO G ICZNYCH ". Wątpliwym się wydaje, czy późniejszy pierwszy biskup wigierski Karpowicz sam ustalał dla swych kazań jubileuszowych w W ilnie te maty, czy też zostały one z góry wytyczone przez księcia biskupa Mas salskiego, również fizjokratę (125). Czy słusznym jest używanie w języku francuskim nazwy „Bernar- dins“ ? (114, przyp. 4). Wszak poza granicami Polski nazwa ta jest nie zrozumiała, a w e Francji nazywają się oni poprawnie ..Franciscains observants“, a popularnie _Recollets“ . Nie sądzę, że wolno tłumaczyć dosłownie na język obcy polskie nazwy miejscowości, jeżeli nie są już zdawna ustalone. Na próżno po szukiwaliby więc Francuzi — jak wiadomo ogółem słabi w geografii — koło Gdańska miejscowości „Saint Adalbert1, zamiast święty Wojciech (117). Przy obecnym stanie badań nad polskim Oświecaniem, który mimo przeciwnych pozorów, wywołanych stosunkowo licznymi publikacjami, dotyczącymi tego okresu, pozostawia jeszcze bardzo w iele do życzenia, najpożyteczniejszymi zdają się być rozprawy, należące do drugiej grupy. ( ) Z krótkiej wzmianki (118) wynikałoby, że Innocenty X I pisze do Kazimierza Szczuki, zalecając polskim biskupom prowadzenie diece R E C EN ZJE i S P R A W O Z D A N IA R E C EN ZJE i S P R A W O Z D A N IA 310 311 Na nich dopiero mogą się oprzeć prawdziwie naukowo owocne roz prawy, należące do grupy pierwszej. Na nich dopiero mogą się oprzeć prawdziwie naukowo owocne roz prawy, należące do grupy pierwszej. rżenia autora, niemniej jednak w wykonaniu ramy te ulegają zacie śnieniu i ostatecznie rozprawa, z wyjątkiem rozdziału pierwszego, dotyczy głównie okresu, nazywanego zazwyczaj polskim Oświeceniem. rżenia autora, niemniej jednak w wykonaniu ramy te ulegają zacie śnieniu i ostatecznie rozprawa, z wyjątkiem rozdziału pierwszego, dotyczy głównie okresu, nazywanego zazwyczaj polskim Oświeceniem. p y g py p j Rozprawę Mariana Plezi pt.: Dokoła reform y szkolnej St. Konarskiego. Studia klasyczne pijarów polskich. Lublin, 1953 chciałoby się na pierw szy rzut oka zaliczyć do grupy drugiej. Przy tak szeroko zakreślanych ramach chronologicznych należało się spodziewać raczej wąskich ram tematycznych. Tymczasem w e wstępie, na tej samej str. 6 daje Plezia swojej rozprawie bardzo szerokie ramy tematyczne: „..Tematycznie interesować nas będzie najpierw dorobek poprzedników, współczesnych i następców Konarskiego na polu pi śmiennictwa polsko-łacińskiego, następnie reforma nauczania łaciny dokonana przez niego samego, a w łączności z nią próby wprowa dzenia nauki języka i literatury greckiej do szkół pijarskich, dalej pod ręczniki do nauczania przedmiotów klasycznych wydane przez pijarów polskich... T R Z Y TO M Y „STU D IÓ W TEO LO G ICZNYCH ". a wreszcie ich przekłady z literatur klasycznych na język polski..." Problematyka rozprawy ma więc być bardzo bogata i różno raka; częściowo należy ona do historii naue^ania (reforma nauczania łaciny, próby wprowadzenia nauki języka i literatury greckiej), czę ściowo do historii nauki (podręczniki), a częściowo wreszcie do historii piśmiennictwa polsko-łacińskiego. Tytuł jej został tak sformułowany, że w niewielkim tylko stopniu poucza nas o tym, czego możemy się po rozprawie spodziewać. Tytuł składa się z dwóch części: właściwego tytułu oraz podtytułu. Tytuł właściwy: Dokoła reform y szkolnej St. Konarskiego, nic nam prawie 0 rozprawie nie mówi. Właściwe jej oblicze, ale również tylko w przy bliżeniu, określa dodany przez autora podtytuł: Studia klasyczne pi jarów polskich. Podtytuł ten należałoby chyba uznać za właściwy tytuł rozprawy. Jego sformułowanie nasuwa jednak możliwości w ielo rakiego jego rozumienia. Mówiąc o studiach klasycznych pijarów polskich, można mieć na myśli wykształcenie klasyczne (jego program, organizację), jakie otrzy m ywali w Polsce członkowie zgromadzenia pijarskiego. Takie ujęcie sprawy nasuwałoby się chyba przede wszystkim przy zetknięciu się z podobnie sformułowanym tytułem. Stosując szersze jego rozumienie, możnaby również spodziewać się, że opatrzona nim rozprawa, będzie rozpatrywać zagadnienie wykształcenia klasycznego, jakie polscy pijarzy dawali swoim wychowankom. W obu wypadkach rozprawa wchodzi łaby w zakres historii nauczania. Obok tego możliwe jest jeszcze trzecie rozumienie tytułu rozprawy Plezi, a mianowicie: mówiąc o stu diach klasycznych pijarów polskich, możnaby mieć na myśli ich do robek naukowy w zakresie filologii klasycznej. W tym wypadku roz prawa wchodziłaby w zakres historii nauki. Zapowiedź tak bogatej, tak różnorakiej problematyki budzi poważny niepokój, a to z dwóch względów: przede wszystkim nasuwa się py tanie, co stanowi właściwy problem pracy, która ma poruszać tak wiele zagadnień? Czy nadmiar problemów nie doprowadzi ostatecznie do tego, że będzie to rozprawa bezproblemowa? Obok tego niemniej niepokojącym jest pytanie, jak zdoła się autor uporać z tymi wszyst kimi zagadnieniami na 72 stronach, zwłaszcza przy tak szeroko zamie rzonych ramach chronologicznych? Wyczerpujące, wszechstronne, praw dziwie naukowe opracowanie wszystkich tych zagadnień, musiałoby chyba zapełnić kilka tomów. Tak więc przystępując do lektury rozprawy Plezi po zapoznaniu się z jej tytułem i podtytułem, nie wiem y jakie zagadnienia będą w niej poruszone, nie w iem y też dokładnie, jakich czasów będzie ona dotyczyć. Podtytuł nie daje nam w tej mierze żadnych wskazówek, dla odmiany granice chronologiczne zakreśla tytuł, ale są to granice bardzo płynne 1 mało określone. T R Z Y TO M Y „STU D IÓ W TEO LO G ICZNYCH ". Tytuł wskazuje jedynie, że rozprawa będzie doty czyć czasów zbliżonych do reform y szkolnej Konarskiego, Bliższe ramy zarówno chronologiczne, jak i tematyczne zostały zakreślone dopiero we wstępie, a trzeba stwierdzić, że zakreślono je bardzo szeroko. Spróbujmy najpierw znaleźć odpowiedź na pierwsze z wysuniętych pytań, a mianowicie czy rozprawa Plezi posiada problem i co ten pro blem stanowi? Plezia we Wstępie i w Zakończeniu wysuwa aż kilka wersji problemu rozprawy. Na str. 5 pisze: „...w niniejszym studium podjęto dla próby zadanie u w y p u k l e n i a w p ł y w ó w a n t y c z n y c h w r e f o r m i e s z k o l n e j St. K o n a r s k i e g o (podkreśle nia recenzenta), na str. 6 formułuje już jednak problem odmiennie: „...postanowiono zatem p r z e d s t a w i ć w niniejszej pracy d z i a ł a l n o ś ć K o n a r s k i e g o n a t l e p e ł n i e j s z e g o o b r a z u s t u d i ó w k l a s y c z n y c h , jakim oddawano się przed nim i po nim w prowincji polskiej..."; nie koniec na tym, bo na str. 7 spotykamy jeszcze jedną wersję problsmu, którym teraz ma być p o p r o s t u t y l k o r e f o r m a n a u c z a n i a ł a c i n y p r z e p r o w a d z o n a p r z e z K o n a r s k i e g o . W Zakończeniu spotykamy jeszcze czwartą wersję problemu — tutaj kładzie Plezia nacisk na w p ł y w , j a k i w y w a r ł a d o k o n a n a p r z e z K o n a r s k i e g o r e f o r m a n a u- Ramy chronologiczne ustala Plezia na str. T R Z Y TO M Y „STU D IÓ W TEO LO G ICZNYCH ". Nawet reforma Konarskiego w zakresie nauczania łaciny nie jest jej punktem centralnym, ale raczej zajmuje w niej nie wiele miejsca. Jest ona tylko jednym z wielu poruszonych w rozprawie zagadnień. Dlaczego tak się stało? Czy jest to rzeczą przypadku, czy może ja kiegoś nieporozumienia? Nie — jest tylko następstwem tego, jak osta tecznie Plezia określił zadania swej pracy. Chociaż bowiem wysuwa aż cztery wersje rzekomego problemu pracy, ostatecznie nie koncentruje swej uwagi na żadnym z nich, ale zadania swej pracy określa zupełnie odmiennie. W tym samym Wstępie, w którym wysunął aż trzy zagad nienia, z których każde osobno miało stanowić' problem pracy, osta tecznie, po wskazaniu tematów, jakie mają być w pracy poruszone, tak określa stawiane przed nią zadania : „...wypadło zadowolić się zesta wieniem i najogólniejszym uporządkowaniem OFafc interpretacją ma teriału drukowanego (dotyczącego wysuniętych przez autora tematów — wyjaśnienie recenzenta) przy świadomej rezygnacji z dociekań szcza- gółowych...“ (str. 7). Takie określenia zadań pracy przesądza o jej charakterze — będzie to praca rejestracyjno-informująca, a nie rozpra wa monograficzna, usiłująca rozstrzygnąć taki czy inny problem nau kowy. Zresztą sam Plezia wyraźnie rezygnuje z opracowania mono graficznego (str. 7), motywując swoją decyzję, zresztą zupełnie trafnie, niedostatecznym stanem badań nad zagadnieniami, którymi ma się za jąć jego praca. W ydaje się jednak, że wobec tego wysuwanie we Wstę pie i w Zakończeniu zagadnień merytorycznych, i to jeszcze z wyraź nym zaznaczeniem, że to właśnie zagadnienie ma być problemem roz prawy, jest niepotrzebne. Czytelnik może czuć żal do autora, że zapo wiada rozważania, na które w pracy rejestracyjno-informującej nie może być miejsca. Nasuwa się tutaj jeszcze jedno pytanie. Zestawienie, uporządkowanie i interpretacja niektórych z pośród wskazanych przez Plezię materia łów drukowanych, jest nie tylko możliwe do wykonania, ale podjęcie takiej pracy z punktu widzenia naukowego jest celowe, co więcej na w et -bardzo pożądane. Dotyczy to materiałów, wchodzących w zakres łacińskiego dorobku piśmienniczego pijarów polskich, ich dorobku w dziedzinie podręczników do nauczana języków łacińskiego i greckiego, a wreszcie ich dorobku w dziedzinie przekładów z tych języków. Czy jednak równie celowym będzie zestawienie i interpretacja materiałów drukowanych, dotyczących przeprowadzonej przez Konarskiego reformy nauczania łaciny? Co do tego można żywić wątpliwości. Reforma nauczania łaciny stanowi tylko jeden fragment jego ogólnej reformy szkolnictwa i nauczania. Materiały, zwłaszcza drukowane, dotyczące tej reform y dawno już zostały zestawione, zarejestrowane, uporządkowane, zinterpretowane, i to pod różnymi kątami widzenia. T R Z Y TO M Y „STU D IÓ W TEO LO G ICZNYCH ". 6 w następujący sposób: „...Chronologicznie zatem interesujący nas przedmiot zamyka się gra nicami lat 1642, kiedy pijarów sprowadzono do Polski, oraz 1832, kiedy po powstaniu listopadowym uległy likwidacji nieomal wszystkie ich kolegia..." Plezia pragnie więc swymi badaniami objąć dwa wieki, niezmiernie bogate w wydarzenia zarówno, gdy bierzemy pod uwagę historię wewnętrzną zakonu oo. Pijarów, jak i jego działalność zew nętrzną, jego udział w ówczesnym życiu umysłowym. Takie są zamie- R E C EN ZJE i S P R A W O Z D A N IA RECENZJF. i S P R A W O Z D A N IA 312 313 c z a n i a ł a c i n y n a w s z y s t k i e d z i e d z i n y t w ó r c z o ś c i ł a c i ń s k i e j p i j a r ó w p o l s k i c h . Mamy więc ostatecznie aż cztery wersje problemu, wersje dcść rozbieżne, poza tym jednym po dobieństwem, że każda z nich w jakiś sposób dotyka reformy naucza nia łaciny, przeprowadzonej przez Konarskiego. nych informacji, która może być osiągnięta tylko dzięki ogromnej sta ranności przy gromadzeniu danych bibliograficznych, dzięki wyczer paniu m ożliwie wszystkich źródeł, które tych danych mogą dostarczyć i dzięki ich starannemu i systematycznemu opracowaniu. nych informacji, która może być osiągnięta tylko dzięki ogromnej sta ranności przy gromadzeniu danych bibliograficznych, dzięki wyczer paniu m ożliwie wszystkich źródeł, które tych danych mogą dostarczyć i dzięki ich starannemu i systematycznemu opracowaniu. Zacieśnienie przez Plezię zadań pracy do rejestracji i interpretacji źródeł, i to tylko drukowanych, dotyczących wskazanych przez niego zagadnień, mniej niepokojącym czyni fakt, że cała jego rozprawa liczy zaledwie 72 strony. Co prawda, jeżeli weźmie się pod uwagę, że obok rejestracji i opisu wskazanych źródeł, praca ma również zawierać ich interpretację (Plezia nie określa bliżej, o jaką interpretację mu chodzi — chyba wchodzi tu w grę tylko interpretacja filologiczna), jej rozmiary ciągle wydają się jeszcze nieco zbyt szczupłe w stosunku do stojących przed nią zadań. Każda rozprawa powinna być rozwiązaniem problemu, który został w niej wysunięty; niestety rozprawa Plezi nie tylko nie rozstrzyga żadnego z wymienionych problemów, ale nawet wyraźnie ich nie po rusza. T R Z Y TO M Y „STU D IÓ W TEO LO G ICZNYCH ". wiednich materiałów drukowanych, wręcz przeciwnie nosi on na sobie wyraźne cechy próby merytorycznego opracowania zagadnień, ale na takie opracowanie jest stanowczo zbyt szkicowy. Podobne łączenie różnych zagadnień w jednym, przy tym opa trzonym określonym tytułem, rozdziale znajdujemy w e wszystkich roz działach pracy Plezi, z wyjątkiem rozdz. V, który jeden tylko doty czy wyłącznie zagadnienia uwidocznionego w tytule. Praca Plezi miała być zestawieniem i interpretacją drukowanych m ateriałów pijarskich, dotyczących wybranych przez autora zagadnień. Czy praca jest istotnie interpretacją zestawionych materiałów? Nie wskazuje wprawdzie Plezia, o jakiego rodzaju interpretację tutaj jemu chodzi. Prawdopodobnie chodzi o interpretację filologiczną. Można je d nak mieć duże wątpliwości, czy omówienia, jakie dodaje Plezia do wskazanych pozycji bibliograficznych, są wyczerpującą interpretacją tego rodzaju. Są one bardzo szkicowe, zbyt szkicowe nawet na pracę o charakterze rejestracyjno-informacyjnym. Te zaś partie pracy, które są nieco obszerniejsze, są niestety w dużej części streszczeniem odpo wiednich fragmentów „Ordinationes". Zagadnieniem, do którego powraca Plezia w kilku rozdziałach, jest zagadnienie pi jarskich podręczników do nauczania języka łacińskiego i greckiego. Jakkolwiek rozdz. IV, zatytułowany: P o d r ę c z n i k i i w y d a n i a p i j a r s k i e , ma być temu zagadnieniu specjalnie po święcony, porusza je Plezia, i to dosyć obszernie również i w trzech pierwszych rozdziałach swojej pracy. W wyniku tego, w rozdz. IV znalazły się tylko te materiały, których nie zdążył Plezia wykorzystać w rozdziałach poprzedzających. Najlepszym przykładem gromadzenia w ramach jednego rozdziału szeregu różnych zagadnień jest rozdz. II, zatytułowany: K o n a r s k i e g o r e f o r m a n a u c z a n i a ł a c i n y . Rozdział ten rozpoczyna Plezia od krótkiej charakterystyki gramatyki łacińskiej Konarskiego, stwier dzając, że można ją w pełni zrozumieć tylko na tle reform y nauczania języka łacińskiego, przeprowadzonej przez Konarskiego w szkołach pi- jarskich, wobec czego daje... krótki rys nowych dążeń, jakie występują w tym samym czasie w nauczaniu łaciny w e Francji i w e Włoszech, aby następnie, po szkicowych uwagach o używanych ówcześnie w Pol sce gramatykach łacińskich, powrócić znowu do gramatyki Konarskiego. T R Z Y TO M Y „STU D IÓ W TEO LO G ICZNYCH ". Po rozstrzygnięciu kwestii jej autorstwa i ogólnikowym omówieniu jej zawartości, przechodzi Plezia do zagadnienia metody nauczania łaciny, wprowadzonej przez Konarskiego, następnie omawia program jej nau czania, zawarty w „Ordinationes visitationis apostolicae“ , szczególnie starannie wyliczając wyznaczoną przez też „Ordinationes11 lekturę. Po tym następują uwagi o dziele Konarskiego „De emendandis eloąuentiae vitiis“, a wreszcie po wykazie lektury łacińskiej dla „professorium11, rozdział został zakończony kilku uwagami syntetycznymi, dotyzącym i roli reformy Konarskiego w nauczaniu przedmiotów klasycznych. I to wszystko na 20 stronach; nic dziwnego, że każde z tych zagadnień zo stało potraktowane bardzo szkicowo, a niektóre partie rozdz. II są tylko streszczeniem odpowiednich fragmentów „Ordinationes11. Pom ię dzy niektórymi z poruszanych zagadnień nie widać bliższego związku; może nawet taki związek istnieje, ale szkicowy charakter rozważań sprawia, że czytelnik tego związku nie dostrzega. Zresztą cały rozdz. II wyłam uje się z ram, jakie ostatecznie zakreślił Plezia swej pracy — w żadnym wypadku nie jest on zestawieniem i interpretacją odpo Wspomniano już, że nawet prace, ograniczające się do samego tylko zestawienia źródeł, mogą posiadać dużą wartość naukową, pod warun kiem jednak, że zawarte w nich zestawienia są m ożliwie kompletne. W jakim stopniu zestawienia materiałów zawarte w pracy Plezi speł niają warunek zupełności? Najwięcej zastrzeżeń pod tym względem budzi rozdz. I. Otrzymał on wprawdzie mało sprecyzowany tytuł : W k r ę g u s z k o l n e j r e t o r y k i , ale odnosi się wrażenie, że chyba właśnie w tym rozdziale miało się znaleźć, zapowiedziane w e Wstępie, zestawienie łacińskiego dorobku piśmienniczego pijarów polskich. Jeżeli jednak zapowiadał Plezia zestawienie p i ś m i e n n i c t w a łacińskiego pijarów polskich, powinien był uwzględnć tutaj dorobek pijarów w tej dziedzinie w ca łej pełni. Należało więc uwzględnić zarówno poezję, jak i prozę, a w ra mach prozy należało uwzględnić pijarską literaturę piękną, popu- lamo-kaznodziejską i naukową, w tej mierze, w jakiej została ona napisana w języku łacińskim. Tymczasem Plezia daje w rozdz. I prze gląd łacińskiej poezji pijarskiej, głównie przy tym poezji panegirycz- nej, w zakresie prozy uwzględnia tylko kazania i m owy panegiryczne oraz daje niezmiernie krótką wzmiankę o Gdańskim i Dmochowskim. Zresztą nawet zestawienia poezji i kaznodziejstwa panegirycznego są bardzo niekompletne. Wymienieni przez Plezię autorowie stanowią tylko część panegirystów pijarskich. T R Z Y TO M Y „STU D IÓ W TEO LO G ICZNYCH ". Uwzględniano na wet sprawę reformy nauczania łaciny, choć może nie tak wyczerpująco, jak to interesuje filologa klasycznego (np. C h r z a n o w s k i I g n a c y : W ielka reforma szkolna Konarskiego, Warszawa 1908; K o n o p c z y ń s k i W ł a d y s ł a w : Stanisław Konarski, Warszawa 1926; K o t S.: Reforma szkolna Konarskiego, Kraków 1923; Ł a g o w s k i : Konarski jako reformator szkól. Przegląd Pedagogiczny 18R4; tenże: Collegium Nobilium, ibid. 1887— 88; R o s e W i l i a m S.: Stanislas Konarski Rs- form er of Education in X V IIIth Century Poland, London 1929; S a r g A.: Die Piaristenschulen im ehemaligen Polen und ihre Reform durch Konarski, 1864). Dlatego chcąc dzisiaj naukowo opracować to zagad nienie, nie wystarczy już zestawienie materiałów i ich interpretacja; celową z punktu widzenia naukowego zdaje się być tutaj tylko ob szerna, szczegółowa, bardzo wszechstronnie opracowana monografia. Z niej jednak Plezia z góry zrezygnował. Skoro praca Plezi ma posiadać charakter rejestracyjno-informacyjny, nie można jej zaliczyć do drugiej, ze wspomnianych na początku, grup rozpraw o polskim Oświeceniu. Grupa ta bowiem obejmuje prace for mułująca i rozwiązujące pewien problem naukowy. Nie oznacza to jed nak, aby w założeniu swym rozprawa Plezi nie miała posiadać w ar tości naukowej. Wręcz przeciwnie prace rejestracyjno - informacyjne jeśli tylko spełniają określona warunki, posiadają pewną wartość nau kową. służąc za niezbędny punkt wyjścia do wszelkich dalszych badań. Jakie są to warunki? Przede wszystkim kompletrość i ścisłość podawa Zobaczmy teraz w jakim stopniu praca Plezi zrealizowała ustalone we Wstępie zamierzenia. RE C E N ZJE i S P R A W O Z D A N IA R E C EN ZJE i S P R A W O Z D A N IA 315 314 Bogactwo zagadnień, jakie mają być w pracy poruszone, stwarza konieczność bardzo starannego jej podziału rzeczowego. Najlepszym rozwiązaniem byłoby poświęcenie każdemu z tych zagadnień osobnego rozdziału. Na tym stanowisku stanął też Plezia, dzieląc swoją roz prawę na 5 rozdziałów. Niestety podział na rozdziały jest czysto po wierzchowny; niektóre zagadnienia są omawiane w ramach kilku rozdziałów, a jednocześnie większość rozdziałów nie ogranicza się do jednego, zapowiedzianego w tytule, zagadnienia, lecz porusza ich cały szereg. T R Z Y TO M Y „STU D IÓ W TEO LO G ICZNYCH ". W brzmieniu tytułów nic nie wskazuje na to, że chodzi tu wyłącznie o podręczniki do nauki języków klasycz nych, o tłumaczenia z tychże języków i o wydania tekstów do lektury łacińskiej. Być może Plezia zamierzał dać tylko wybór panegirystów pijar- skich. Dawanie jednak wyboru autorów w pracy tego typu, zwłaszcza bez wskazania, że jest to wybór, oraz bez wskazania kryterium, na podstawie którego wybór został dokonany, m ija się z celem pracy. Zwłaszcza, że zestawienie bardziej kompletnego wykazu panegirystów pijarskich nie nastręcza większych trudności. Wystarczy pełniejsze w y korzystanie źródeł, z których Plezia i tak już korzystał, a więc Estrei chera i Bielskiego i uwzględnienie dwóch jeszcze źródeł, o których Plezia nie wspomina, a które mogą oddać dużs usługi, a mianowicie: H o r a n y i A .: Scriptóres Piarum Scholarum, Budae 1809; V i n a s T h o m a s : Index Bio-bibliographicus C. C. R. R. P. P. Matris Dei Scholarum Piarum qui in universo ordine pietatem, litteras ac scien- tias scriptis suis... omaverunt, Romae 1908. Druga uwaga dotyczy sposobu korzystania przez autora z „Ordina- tiones“ ; autor korzysta nie z łacińskiego oryginału „Ordinationes“ , ale z ich polskiego przekładu z r. 1925. Zasadniczo nie jest przyjętym korzystanie w opracowaniach naukowych z przekładu źródła o ile jego oryginał istnieje i jest dla badaczy dostępny, a już szczególnie nie jest przyjęte, jeżeli źródłem tym jest pomnik prawny, jakim są nie wątpliw ie „Ordinationes". N ie stanowią one rzadkości bibliotekarskiej, są łatwo dostępne w każdym prawie większym księgozbiorze, posiada jącym druki polskie z w. X V III. Trudno więc ustalić, czemu autor, 1 to właśnie jeszcze filolog klasyczny, korzysta z przekładu polskiego zamiast łacińskiego oryginału. Reasumując, należy stwierdzić, że praca Plezi posiada wiele braków, czy oznacza to jednak, że nie posiada ona wartości. Bynajmniej. Za w arty w niej materiał inform acyjny może oddać usługi badaczom polskiego życia umysłowego w okresie Oświecenia. Zwłaszcza materiały z zakresu podręczników pijarskich do nauczania łaciny i greki, po dobnie jak i dotyczące pijarskich przekładów z języków klasycznych mogą stanowić punkt wyjścia do dalszych, tym razem już merytorycz nych badań. T R Z Y TO M Y „STU D IÓ W TEO LO G ICZNYCH ". Pominął Plezia takich, jak np.: B i e n - k i e w i c z R e g i n a l d u s S. P.: Caelestum iustitiae vice-regia nu- minum Poloniae maiestate excelsum... Jasna Góra 1739; B ł e s z y ń s k i H i e r o n i m S. P.: Oecumenicum ecclesiae universalis concilium D. Thomas Aąuinas declaratus oratio, Kraków 1743; tenże: Oratio de R E C EN ZJE i S P R A W O Z D A N IA R E C E N ZJE i S P R A W O Z D A N IA 316 317 laudibus D. Joseph, b. m. 1748; D o g i e l M a c i e j S. P.: Arcus trium- phalis in... domus Oginscianae... Vilnae 1740; J a s t r z ę b s k i J o s e - p h u s S. P.: Oratio in laudem S. Sebastiani, Varsaviae 1727; K r a s n o - d ę b s k i K l a u d i u s z S. P. Oratio de laudibus S. Bonaventurae, Varsavias 1717; tenże: Arbor triumphalis ecclesiae ad aąuinatam flu- men radicata... Jasna Góra 1721; M a d e j s k i A e g i d i u s S. P.: Rex libertatis in absoluto scientiarum regno D. Thomas Aąuina... Varsaviae 1720; P a l f f y T h o m a s S. P.: Oratio in laudibus D. Thomae Aqui- natis, Varsaviae 1719 i wielu innych, których już tutaj z braku miejsca nie będziemy podawać. jej rozdziałów. Tytuł rozdz. I: W k r ę g u s z k o l n e j r e t o r y k i nie wskazuje, czego możemy się po tym rozdziale spodziewać. Tytuły rozdz. IV i V: P o d r ę c z n i k i i w y d a n i a p i j a r s k i e oraz T ł u m a c z e p i j a r s c y , mogą nasuwać przypuszczenia, że chodzi tu 0 wszystkie podręczniki i wszystkie przekłady, jakie wyszły z pod pióra pijarów polskich, jak również wszystkie dzieła, które zostały wydane w ich drukamii. T R Z Y TO M Y „STU D IÓ W TEO LO G ICZNYCH ". Jak widać z powyższych pozycji tematyka tego rodzaju nie jest obcą naszym historykom. narzy, a po jego kasacie w r. 1864 przez kler diecezjalny. Tego rodzaju praca o nauce i profesorach biblistyki jest nie tylko ciekawa, ale nadto pożyteczna. Przynosi bowiem nowe naświetlenie i nieznane wiadomości z tej tak mało znanej, a nietkniętej prawie przez historyków oświaty nauki w polskich zakładach teologicznych. Stąd też witać należy z uznaniem każdą rozprawę, która do nauki teologicznej wnosi w ia domości nowe, dotąd nieznane. Objektywne i na źródłach oparte na świetlenie i publikowanie krótkich chociażby studiów sprostuje nie jeden fałsz historyczny; Seminaria duchowne w Polsce wym agają w dalszym ciągu opracowania, a rzetelna w tym względzie praca rzuci nowy snop światła na kulturalną i oświatową rolę Kościoła w Polsce, zwłaszcza, kiedy weźm ie się pod uwagę tendencyjne i jednostronne na świetlenie problemów historyczno-kościelnych, chociażby ubocznie tylko traktowanych, naszych historyków epoki stalinowskiej. Szczegółowiej zajmę się omówieniem rozprawy ks. Gryglewicza. Co stanowi jej wartość, a jakie posiada usterki. Praca ks. Gryglewicza po siada niewątpliwie swoją wartość. Zarówno tematyka, jak i jej prze prowadzenie stanowi w zapoznanej dziedzinie badań nad dziejami teo logii polskiej pozycję dodatnią. Autor zarejestrował nazwiska profeso rów Pisma św. w lubelskim seminarium duchownym, zwłaszcza na przestrzeni X IX stulecia, podał krótką ich biografię i omówił treść w y kładów z zakresu biblistyki. W e form ie uzupełnień podajemy tu na stępujące dane. Po krótkim wstępie, który stanowi wprowadzenie do całej rozprawki, autor omawia wykłady Pisma św. w studium OO. Dominikanów w Lu blinie (s. 100— 112). Pismo św. nie było osobnym przedmiotem, ale trak towano je łącznie z dogmatyką. Tak było u dominikanów od r. 1643 z przerwą do r. 1847. Ks. Gryglewicz zajmuje się wykładami Pisma św. w latach 1844— 48. Przytacza kilka nazwisk i autorów podręczników. To wszystko. A szkoda, że nie wykorzystał lepiej rękopisu: L.ber studii formalis in conrentu regali lublinensi Sacri Ordinis Praedicatorum (rkps w Arch. Sem. Duch. -w Lublinie), na który powołuje się. Znalazły by się zapewne i inne jeszcze materiały w archiwach dominikańskich. Wiemy na ogół mało o dominikanach polskich X V III stulecia, o ich studiach zakonnych. Polska prowincja znajdowała się w tym okresie w stadium wewnętrznego rozluźnienia, piękną natomiast kartę posiadają domini kanie w tym czasie na Litw ie i Rusi. Szczegóły, dotyczące studiów w X V III w. rzuciłyby niewątpliwie jakieś nowe światło na poziom naukowy dominikanów lubelskich. , 7 Nasza Przeszłość 4 (1948) 209— 224 Roczniki Teologiczno-kan niczne 2 (1955) 61— 107; 3 (1956) 119— 160. 1 Nasza Przeszłość 3 (1947) 162— 190. 2 Józef Jakubowski, żołnierz i kapłan, Kraków 1945. 3 Nasza Przeszłość 2 (1947) 245— 253. 4 Ruch B iblijny i Liturgiczny 1949 i odb. s. 19. 5 Homo Dei 19 (1950) 527— 534. Bolletino Ceciliano 1952 nr 2, s. 24. 7 Nasza Przeszłość 4 (1948) 209— 224 Roczniki Teologiczno-kan niczne 2 (1955) 61— 107; 3 (1956) 119— 160. ( ) 2 Józef Jakubowski, żołnierz i kapłan, Kraków 1945. T R Z Y TO M Y „STU D IÓ W TEO LO G ICZNYCH ". Należy jednak wyrazić żal, że rozprawa, która na pierwszy rzut oka tak sympatycznie zapowiadała się jako merytoryczny wkład do badań nad życiem umysłowym polskiego Oświecenia, przy bliższym z a p o z n a n iu się z nią, okazała się szkicowym przyczynkiem, a nawet raczej zbiorem kilku szkicowych przyczynków o charakterze rejestra- cyjno-informacyjnym. Pełne wykorzystanie wszystkich czterech wspomnianych źródeł roz szerzyłoby być może również i zasoby materiałów, wymienionych w po zostałych rozdziałach, chociaż trzeba przyznać, że są one znacznie kompletniejsze od materiałów, zgrupowanych w rozdz. I. Zastrzeżenia, tym razem nie co do kompletności zestawienia, ile raczej co do sposobu ich podania, budzą zamieszczone w rozdz. IV in formacje o pijarskich wydaniach tekstów do lektury łacińskiej. Infor macje te mają charakter bibliograficzny. Niestety w dużej części nie spełniają one wymagań, stawianych wszelkim informacjom tego ro dzaju. Innego jeszcze typu zastrzeżenia budzi rozdz. V, dotyczący pijarskich przekładów z języków klasycznych. Każdą wskazaną tam pozycję, zaopatruje Plezia uwagami, w których stara się określić jej wartość jako przekładu Szkoda tylko, że uwagi te są tak sumaryczne i że z re guły prawie nie zawierają własnej opinii Plezi, lecz referują tylko opinie Ogrodzińskiego lub Sinki. Hanna Waśkiewicz Ks. F e l i k s G r y g l e w i c z , Studium Pisma św. w Lubelskim Seminarium Duchownym. Roczniki Teologiczno-kanoniczne 2 (1955), 109— 157 i nadb. Lublin 1955. Na zakończenie dwie drobniejsze uwagi. Pierwsza dotyczy sposobu formułowania tytułów poszczególnych rozdziałów. Plezia wykazuje ten dencję do bardzo ogólnego formułowania tytułów, co może prowadzić do niejasności lub nieporozumień. Wystąpiło to w tytule całej roz prawy, powtarza się również w odniesieniu do tytułów poszczególnych Rozprawa ks. Feliksa Gryglewicza stanowi przyczynek do historii nauk biblijnych w seminariach polskich, w szczególności w zakładach teologicznych lubelskich: w studium OO. Dominikanów, seminarium OO. Jezuitów, do którego uczęszczali też alumni diecezjalni, seminarium diecezjalnym, kierowanym przez kapłanów Zgromadzenia Księży M isjo RE C E N ZJE i S P R A W O Z D A N IA RE C E N ZJE i S P R A W O Z D A N IA 319 318 10— 11). Ta ostatnia nie w iele szczegółów dorzuca do dawniejszej roz prawy J. Bielińskiego. Jak widać z powyższych pozycji tematyka tego rodzaju nie jest obcą naszym historykom. 10— 11). Ta ostatnia nie w iele szczegółów dorzuca do dawniejszej roz prawy J. Bielińskiego. 1 Nasza Przeszłość 3 (1947) 162— 190. 5 Homo Dei 19 (1950) 527— 534. f p 3 Nasza Przeszłość 2 (1947) 245— 253. * Bolletino Ceciliano 1952 nr 2, s. 24. ( ) 4 Ruch B iblijny i Liturgiczny 1949 i odb. s. 19. T R Z Y TO M Y „STU D IÓ W TEO LO G ICZNYCH ". Zasady nauczania misjonarskiego zostały RE C E N ZJE i S P R A W O Z D A N IA RE C E N Z JE i S P R A W O Z D A N IA 320 321 zebrane w osobnym dyrektorium, wydanym ostatecznie w Paryżu w r. 1895 pt.: D irectoire des grands seminaires confies aux Pretres de la Mission. - Zawiera ono w oparciu o uchwały trydenckie metody w y chowania seminaryjnego w myśl wskazań św. Wincentego a Paulo, a uzu pełnione w ciągu w ieków przez konwenty generalne Zgromadzenia Misji. Obok ogólnego dyrektorium istniały reguły dla poszczególnych urzędów, a także dla profesorów, wykładających w seminariach diece zjalnych. Reguły te były oparte na starej tradycji, sięgającej również czasów św. Wincentego a Paulo, o dostosowane w późniejszych czasach do nowych potrzeb Kościoła, ukazały się również drukiem kilkakrotnie (bez karty tytułowej) pt.: Regulae communes professoribus tum theolo- giae, tum. philosophiae (b. r. w.). Wśród nich były reguły i przepisy dla profesorów Pisma św. a dodać trzeba, że wszystkie te zasady naucza nia obowiązywały profesorów w seminariach duchownych, a więc także polskich. Znajomość wspomnianego dyrektorium i reguł dawałaby roz prawie ks. Gryglewicza możność lepszego i szerszego poznania przed miotu. wanych w seminariach X V III wieku, jak Calmeta, Gravesona, Brictiusa. Nie wspomina natomiast podręcznika polskiego biblisty, profesora w kilku kolejno seminariach duchownych, a najdłużej wykładającego w seminarium zamkowym w Krakowie, ks. J a k u b a J a c h i m o w - s k i e g o . Wydał on w Krakowie w r. 1789 swoje wykłady Pisma św. SŁ Testamentu pt. Praelectiones Theologicae in Sacram Scripturam Veteris Testamenti, ad usum Seminariorum intra Regnum Poloniae existentium concinnatae (s. 372). Ocena tego podręcznika od strony bi blijnej należy oczywiście do biblistów. Jedno jest pewne, że dzieło Ja- chimowskiego było owocem długoletnich wykładów i naukowych do ciekań, oparte jednak w dużej m ierze na pracach Calmeta. Przygoto wywał je autor podręcznika do druku w czasie długotrwałej a przykrej choroby. Jachimowski wyznał to w przedmowie do czytelnika: „Lucru- bationem istam, quam fieri curavi. feci, tum per otium diutumae ac molestae infirmitatis meae; ąuatenus molestias aegntudinis jucun- diores iacerem, tum utilitatem intendens Cleri junioris educatio- nis; cui fere totum tempus vocationis et status mei vacavi“ 8. T R Z Y TO M Y „STU D IÓ W TEO LO G ICZNYCH ". Trudno dziś z całą pewnością stwierdzić, w których seminariach wykładano z książki Jachimowskiego, w każdym wypadku wykładowcom-misjo- narzom musiała być znana i wykorzystana przy wykładach . Przejdźm y do szczegółów. Na s. 112 jest mowa o erekcji seminarium lubelskiego i krasnostawskiego. Ks. Gryglewicz powołuje się jako na źródło na notatki ks. J. A. Wadowskiego: Seminarium duchowne (w L u blinie), przechowywane w tece Kurii Biskupiej w Lublinie, Rep. 60. V III. 6. Możemy wskazać autorowi starsze i pewniejsze źródła, dotyczące po czątku fundacji misjonarzy i seminarium lubelskiego. U śp. ks. L. Za lewskiego w Lublinie znajdował się rkps: Liber jurium fundationis do- mus lublinensis Congregationis Missionis, który według relacji ks. St. Młynarczyka otrzymała po śmierci właściciela Biblioteka Narodowa w Warszawie. Nadto w Archiwum Księży Misjonarzy w Krakowie na Stradomiu przechowuje się duży rękopis łaciński pt.: Historia Congre gationis Missionis in Polonia, w którym znajduje się 1) opis fundacji lubelskiej (Initia, progressus et status praesens quoad temporalia D o- mus Lublinensis Congregationis Missionis conscriptus anno 1734) i 2) opis fundacji Zgromadzenia M isji w Krasnymstawie (Initia, progressus et praesens status Crasnostaviensis Congr. Miss. anno 1775 conscriptus). W r. 1778 wydali misjonarze (prawdopodobnie wileńscy) streszczenie Pisma św. w form ie wierszowanej (heksametrem). Streszczenie to po dawało studentom krótką wiadomość o autorach ksiąg świętych, na stępnie relację wierszowaną Pisma św. (Biblia Sacra carminicis m em o- ricis comprehensa). Autor podpisał się inicjałami: J. C. C. M .10 Dziełko kursowało po seminariach misjonarskich i dotarło zapewne i do Lublina. Ks. Ignacy Laws (t 1843), o którym pisze ks. Gryglewicz na s. 122, pochodził z Brunsbergi (dzisiejsze Braniewo). Z nowicjatu OO. K ar m elitów przeszedł do Zgromadzenia M isji w Warszawie (1779). Warto było wspomnieć, że swoją karierę profesorską rozpoczął on właśnie w lubelskim seminarium duchownym, a wiadomość ta znajduje się w cytowanym przez autora rękopisie lubelskim. Czytamy w nim: „RD. Ignatius Laws d. 10 Augusti 1785 venit ex domo Varsaviensi ad docen- O seminarium duchownym w Lublinie, kierowanym przez kapłanó Zgromadzenia M isji od r. 1714 — do końca X V III wieku, a ściśle mó wiąc do r. 1797 i o nauce Pisma św. z tychże lat ks. Gryglewicz pisze mało, i nie wymienia z tego okresu nazwisk profesorów tego przedmiotu. Prawda, że w X V III w. łączono często -wykłady Pisma św. z dogmatyką. Pismo św. stanowiło podstawy dla teologii dogmatycznej. 8 Posługiwanie się podręcznikami przez profesorów misjonarski było powszechnie stosowane. Jeśli nawet jakiś profesor posługiwał się skryptem, był on tylko streszczeniem podręcznika. Konwent generalny Zgromadzenia Misji, odbyty w Paryżu w r. 1717 pod kier. generała wspomnianej kongregacji, ks. Jana Bonnet, zabronił ks. Cyboniemu z Polski na wydanie podręcznika z własnymi objaśnieniami. Por. Recueil des principales circulaires des superieurs generaux de la Congregation de la Mission, t. I. Paris 1877, 309, przyp. 2. 10 Por. ks. F r. B ą c z k o w i c z , Z dziejów domu stradomskieg Roczniki ob. Zgromadzeń św. Wincentego a Paulo 27 (1925' 32. p yp • Praelectiones Theologicae, Author ad lectorem, przy końcu książ ss. nlb. T R Z Y TO M Y „STU D IÓ W TEO LO G ICZNYCH ". y y y p j W ostatnim 10-leciu pojawił się szereg prac z historii polskich za kładów teologicznych. Warto wyliczyć ważniejsze pozycje. Ks. W ła dysław Wicher zajął się nauką teologii moralnej w dawnych semina riach mi s j o n a r s k i c h O seminariach duchownych na przełomie X V III i X IX w. pisał autor niniejszej recenzji w swej monografii o ks. Jó zefie Jakubowskim2. Nadto publikował tenże wiadomość „O studiach kard. Ledóchowskiego w seminarium świętokrzyskim w Warszawie" i „O niektórych podręcznikach liturgicznych w dawnych seminariach duchownych w Polsce“ 4. Ks. Leon Świerczek C. M. opublikował cie kawy artykuł o muzyce liturgicznej w seminariachs, który znalazł od dźwięk i za granicą, a dotyczy on programu na czasy obecne8. Sprawą W ielkiego Seminarium Misyjnego zajął się ks. Marian Rechowicz (K ra ków 1948). Ten sam autor opracował w szeregu artykułach źródłowo dzieje Seminarium Generalnego w e L w o w ie 7. Nie mniej posunęły się hadania nad historią wydziałów teologicznych. Prof. JBarycz ogłosił na łamach Naszej Przeszłości metrykę promowanych Wydziału Teolo gicznego U. J. Cenną pozycją jest niewątpliwie sumienna rozprawa ks. Tadeusza Glemmy o W ydziale Teologicznym Uniwersytetu Jagielloń skiego w latach 1795— 1847 (Kraków 1949). Sprawami wyższych uczelni warszawskich zajęli się: ks. abp Michał Godlewski (Epizod z dziejów Seminarium Głównego w Warszawie w 1831 roku, Collectanea Theolo- gica 21 (1949), 16— 41), oraz ks. W ładysław Kwiatkowski (Początki W y działu Teologicznego U. W., Wiadomości Archidiec. Warsz. 1952, nry Kilka zaledwie wierszy poświęcił ks. Gryglewicz studiom teologicz nym jezuitów lubelskich. To trochę za mało, a brak odpowiedniej pro porcji jest w rozprawie rażący. Wychowywaniem i kształceniem kleru na terenie Lublina zajmowali się w przeszłości: dominikanie, jezuici, misjonarze i kapłani diecezjalni. Musiała istnieć wśród nich jakaś rywalizacja, a drogą porównania można było ustalić, które spośród studiów lubelskich odpowiadało swemu za daniu. Ponieważ jednak ks. G ryglewicz za cel pracy wziął seminarium diecezjalne, prowadzone przez kapłanów Zgromadzenia Misji, a po ich kasacie przez kapłanów diecezjalnych, poświęcił temu zakładowi teolo gicznemu najwięcej troski. Do cech ujemnych części rozprawy z okresu misjonarskiego (s. 112— — 133) należy brak znajomości bibliografii, dotyczącej misjonarzy pol skich, zarówno starszej, jak i powojennej. Autor nie jest obeznany z dziejami misjonarzy, nie orientuje się w zasadach tej kongregacji, do tyczącej dyrekcji seminariów. 14 S c h l e t z , Słownik Biograficzny Zgrom. Księży Misjonarzy w P o l sce, t. II (“w maszynopisie) i tamże podane archiwalia. 15 T a m ż e, t. I. T R Z Y TO M Y „STU D IÓ W TEO LO G ICZNYCH ". Nie mniej spotykamy w X V III stuleciu szereg autorów z dziedziny nauk biblij nych. Podręczniki tych pisarzy teologicznych były używane współcześnie w innych seminariach, prowadzonych przez misjonarzy. A ponieważ istniała „uniformitas“ , przypuszczać należy, że i w lubelskim seminarium 7. nich wykładano. Ks. Gryglewicz w ylicza ważniejszych autorów, uży Nasza Przeszłość Nasza Przeszłość 21 21 R E C EN ZJE i S P R A W O Z D A N IA RE C E N ZJE i S P R A W O Z D A N IA 322 323 dos clericos. Discessit hinc Culmam d. 22 7-bris 1788 anno“ Z w y czajem ówczesnym każdy z profesorów uczył kilku przedmiotów teolo gicznych. Nie ma w rękopisie wzmianki o Lawsie jako o wykładowcy Pisma św. Możemy natomiast wnioskować, że w okresie pobytu w Lu blinie uczył skrypturystyki, skoro wydał popularną egzegezę do pery kop Ewangelii pod dziwacznym tytułem: Porozdzielane i objaśnione niedzielne i świąteczne Ewangelie, służące kapłanom do używania (W ar szawa 1787) 12. Ks. Laws wykładał przez długie lata w różnych semi nariach: w Chełmnie, Łowiczu i w seminarium św. Krzyża w Warsza wie. Ks. Gryglewicz cytuje na s. 122 dziełko ks. Lawsa: Hebdomadale sive sacrarum scientiarum synopsis per..., Varsaviae ad S. Crucem, 1805. Dzieło Lawsa o takim tytule jest m i nieznane. Być może, że ks. G ry glewicz miał takie dziełko z taką właśnie kartą tytułową, doklejoną może później. Praca powyższa znana jest jednak w literaturze biblijnej pt.: Hebdomadale studiorum seu rudimenta theologica loco exercitiorum hebdomadalium ad usum Seminarii Varsaviensis S. Crucis concinnata, Varsaviae die 11 Februarii 1805. Celowo przytoczyłem dokładny tytuł. O ks. Lawsie jako o autorze książki nie ma wzmianki na karcie ty tułowej, podpisał się dopiero po „praefatio authoris'1 pełnym tytułem: ,.sexvus obsequentiasimus, Professor historiae ecclesiasticae, herme- neuticae et pastoralis J. A. Laws, Congr. Miss. Domus Varsaviensis“ . Treść tej książki podał ks. Gryglewicz w swej p racy1. kapłańskich (1828) został w sierpniu t. r. wysłany do Lublina. Wykładał kolejno historię Kościoła (od 1828), teologię dogmatyczną (od r. 1829) i hermeneutykę (od r. 1830), oraz uczył śpiewu przez trzy lata. Wiado mość powyższą mógł ks. Gryglewicz zaczerpnąć nie tylko z rękopisu misjonarskiego w Krakowie: Catalogus seminaristarum Seminarii In- tem i d. Varsaviensis, ale także z akt znajdujących się w arch. Kurii Biskupiej w Lublinie, a mianowicie: Seminarium Lubelskie, Rep. 60, V III. nr 22. Z Lublina wyjechał ks. ss. 188. 13 Ks. L a w s jest postacią dość znaną w naszej literaturze teolo gicznej. Pisali o nim: ks. S. Ch(odyński) w Encykl. Kość. ks. Nowo dworskiego, t.: X II, 16— 17; ks. F r. B ą c z k o w i c z , Z dziejów domu stradomskiego, Roczniki, j. w. 21 (1915) 138— 139 (błędnie go pisze Laws); ks. A. S c h l e t z , Działalność naukowo-oświatowa ks. Józefa Jakubow skiego, Lw ów 1939, 25; T e n ż e , Józef Jakubowski, żołnierz i kapłan, Kraków 1945, 142; T e n ż e , O niektórych podręcznikach liturgicznych w dawnych seminariach duchownych w Polsce, Kraków 1949, 8—'10 (odb. z Ruchu Bibl. i Liturg.); T e n ż e , Słownik Biograficzny Zgrom a dzenia Misjonarzy w Polsce (1651— 1951), t. II (w maszynopisie); ks. H. F e i c h t , X. M chał Mare n Mioduszewski 1787— 1868, Księga Pam. ku czci prof. A. Chybińskiego, Kraków 1930, 70 (pisze go także błędnie Laws). Ks. Gryglewicz powyższych pozycji nie zna. 11 Cathalogus Missionariorum in domo Lublinensi commorantium, pod r. 1785, w fasc. Memoriale defunctorum fratrum nostrae Congrega- tionis, (mój odpis lubelskiego rękopisu). 11 Cathalogus Missionariorum in domo Lublinensi commorantium, pod r. 1785, w fasc. Memoriale defunctorum fratrum nostrae Congrega- tionis, (mój odpis lubelskiego rękopisu). 12 Drugie wydanie ukazało się pod zmienionym nieco tytułem: Ewan gelie porozdzielane i objaśnione niedzielne i świąteczne służące kate- chistom do używania w cesarsko-królewskich państwach, Lw ów 1820, ss. 188. 13 Ks. L a w s jest postacią dość znaną w naszej literaturze teolo gicznej. Pisali o nim: ks. S. Ch(odyński) w Encykl. Kość. ks. Nowo dworskiego, t.: X II, 16— 17; ks. F r. B ą c z k o w i c z , Z dziejów domu stradomskiego, Roczniki, j. w. 21 (1915) 138— 139 (błędnie go pisze Laws); ks. A. S c h l e t z , Działalność naukowo-oświatowa ks. Józefa Jakubow skiego, Lw ów 1939, 25; T e n ż e , Józef Jakubowski, żołnierz i kapłan, Kraków 1945, 142; T e n ż e , O niektórych podręcznikach liturgicznych w dawnych seminariach duchownych w Polsce, Kraków 1949, 8—'10 (odb. z Ruchu Bibl. i Liturg.); T e n ż e , Słownik Biograficzny Zgrom a dzenia Misjonarzy w Polsce (1651— 1951), t. II (w maszynopisie); ks. H. F e i c h t , X. M chał Mare n Mioduszewski 1787— 1868, Księga Pam. ku czci prof. A. Chybińskiego, Kraków 1930, 70 (pisze go także błędnie Laws). Ks. Gryglewicz powyższych pozycji nie zna. , ( j p g ę p ) 12 Drugie wydanie ukazało się pod zmienionym nieco tytułem: Ewan gelie porozdzielane i objaśnione niedzielne i świąteczne służące kate- chistom do używania w cesarsko-królewskich państwach, Lw ów 1820, ss. 188. T R Z Y TO M Y „STU D IÓ W TEO LO G ICZNYCH ". Piotra Damiana (Wiktora) Ożarowskiego, ani innych mniej lub zupełnie nie znanych ze swego życia wewnętrznego duchownych. Misjonarzy ówczesnych pomawia Autorka o brak ubóstwa (179), ale może nie uwzględnia, że każdy z nich ma zgodnie z konstytucjami Zgro madzenia prawo do własności prywatnej, a tylko dowolne rozporządza nie nią jest ograniczone. Krytycznie też patrzy Autorka na krakowskich kapucynów za to, że nie chcieli pod pozorem braku miejsca przyjąć do swego klasztoru starca, który się podawał za ich brata Grzegorza, sybiraka, który po 40 latach zesłania przyszedł do Krakowa pieszo, 0 żebranym Chlebie (101). Nie podaje nam Autorka, czy kapucyni istotnie rozpoznali w nim swego współbrata zakonnego, przypuszcza, że może był nie łatwy w pożyciu, że stracił ducha zakonnego, ale że jed nak... M ówi też dalej (tamże, przyp. 2), że albertyni pisząc do niej o ka pucynach są bardzo wstrzemięźliwi w wiadomościach, aby „nie zranić świętej miłości" bliźniego. A jednak rezerwa kapucynów i franciszkanów konwentualnych — o reformatach nic nie słyszymy — wobec br. A l berta i jego poczynań jest zupełnie zrozumiała. Rezerwa i nieufność. To samo nastawienie cechowało niegdyś w ciągu wieków i musiało cechować franciszkanów konwentualnych wobec nowych bernardynów, bernardynów wobec nowych reformatów, reformatów wobec nowych kapucynów. Dodajmy do tego jeszcze powtarzające się w dziejach tylu zakonów pojawienie się zelantów pojedynczych lub też całych ich grup, chcących gwałtownie ulepszać i poprawiać własny zakon, a kończących często niechlubnie, a zrozumiemy stanowisko kapucynów i franciszka nów wobec początków albertynów i twierdzenie tychże, że dla kapu cynów byli „solą w oku“. Twierdzenie zresztą, moim zdaniem, przesadne 1 niesłuszne, bo wyraża także uznanie dla swojej własnej, wyższej do skonałości. A le nawet wśród tak życzliwych albertynom bernardynów Na książkę tą składa się 25 rozdziałów, które można by sklasyfi kować w następujący sposób: 1— 7, przed „nawróceniem"; 8— 12 szuka nie drogi; 13— 25, „ojciec zadżumionych“ . Rozdziały są bez tytułów, ale podają szczegółowo swą zawartość. Co prawda, zawartość ta podana jest często według mody francuskiej dość enigmatycznie, choć poetycznie i pięknie. Wcale obfita jest wykorzystana bibliografia (s. 287— 290), na którą składają się nie wydane rękopisy samego Alberta, inne o nim, dalej książki, broszury, dzienniki. W ydaje się jednak, że Autorka nie prze prowadziła podziału tej bibliografii według ich wartości, ani krytyki źródeł. Obok firm bardzo solidnych i znanych, spotykamy tam jednak pozycje, które dają do myślenia. W idzimy tam nieprzeciętne powagi, czy to stanowiskiem czy charakterem: rektor ks. T R Z Y TO M Y „STU D IÓ W TEO LO G ICZNYCH ". P o święceniach R E C E N ZJE i S P R A W O Z D A N IA R E C E N ZJE i S P R A W O Z D A N IA 324 325 Pom ijam inne jeszcze drobne braki, bo wszystkie prawie sylwety profesorów lubelskich wymagają małych retuszów i uzupełnień. daleko idąca ostrożność i czujność i nie wolno mu ufać a priori każdemu zwierzeniu penitenta o jego niezwykłych przeżyciach. daleko idąca ostrożność i czujność i nie wolno mu ufać a priori każdemu zwierzeniu penitenta o jego niezwykłych przeżyciach. p y g j y p Dobrze by było, gdyby ks. Gryglewicz zajął się i opracował historię nauki Pisma św. w dawnych seminariach duchownych w Polsce. Żmudna co prawda, ale jakże wdzięczna okazała by się tego rodzaju praca, która byłaby wkładem poważnym do dziejów teologii polskiej. Nie w ydaje się także słusznym twierdzenie Autorki, że „od czasów św. Franciszka z Asyżu rzadcy są święci, którzy osiągnęli taki stopień równowagi" (267), jak właśnie br. Albert. Czyżby wolno było odmówić wysokiego stopnia równowagi setkom kanonizowanych i niekanonizo- wanych świętych, którzy wśród przeciętnych społeczeństw uchodzą za niezrównoważonych właśnie dla tego, że są szaleńcami bożymi? T o za leży od tego, co się rozumie w treści rzeczownika „równowaga". Wszak nie brakło ludzi i to nie głupich, którzy przynajmniej początkowo w i dzieli brak równowagi właśnie u br. Alberta. Ks. Alfons Schletz M a r i a W i n o w s k a , Frere Albert ou la Face aux outrages, Paryż, ćd. La Colombe 1953, ss. 294, w tym 1 mapka + 15 ilustracji poza tekstem. Autorka wiana jest chlubnie i nie potrzebuje reklamy. Można się cieszyć, że duchowa sylwetka br. Alberta Chmielowskiego została skre ślona właśnie jej piórem. Skreślona z pietyzmem i miłością, która nie rzadko przechodzi wręcz w entuzjazm. Praca jej należy do dziedziny zarówno i przede wszystkim hagiografii, ale także i historii. Autorka dobrze się orientuje w dziejach zewnętrznych życia Adama Chmielow skiego. Porusza się swobodnie na terenie dogmatyki i mistyki i umie jętnie wnika w jego życie wewnętrzne. Autorka jest ogółem surowa dla ówczesnego duchowieństwa krakow skiego. Może poniekąd ma słuszność twierdzić, że ok. r. 1900 było tam niewielu kapłanów, naprawdę uduchowionych (217). A le przecież bywał tam o. Rafał Kalinowski, o. Marian Morawski, przejściowo bywał o. Henryk Jackowski, nie licząc zmarłego wcześniej o. T R Z Y TO M Y „STU D IÓ W TEO LO G ICZNYCH ". Sopuszyński do Tykocina, zajm o wał się tam pracą duszpasterską i uczył prawdopodobnie w seminarium tykocanskim (1832— 33). W r. 1833 objął stanowisko profesora semina rium duchownego w Poznaniu, gdzie po roku (18 V III 1834) zakończył życie u. O profesorze Pisma św. z lat 1846— 1848, ks. Andrzeju Dorobi podał ks. Gryglewicz (s. 129— 130) uwagi, oparte na notatach jednego z słuchaczy. W ten sposób odtworzył autor w przybliżeniu wykłady Dorobisa, autorów biblijnych, z których czerpał i metodę nauczania. Ks. Dorobis należał wogóle obok księży Chmielewskiego, Malinowskiego i Bołdoka do bardziej znanych profesorów lubelskich. Dorobis był dość znaną postacią Kościoła polskiego w X IX stuleciu. Choć cenne i trafne są uwagi o nim ks. Gryglewicza, można by jednak wspomnieć jeszcze o innych placówkach i pracach ks. Dorobisa. W Lublinie wykładał Do robis dwukrotnie: 1838— 1841 historię Kościoła i j. łaciński; 1844— 1847 Pismo św., teologię moralną i rubryki. Piastował nadto urząd dyrektora kleryków. Potem został profesorem seminarium duchownego w W ło cławku (1847— 1851), a stamtąd powołano go do Warszawy, gdzie wkrótce objął urząd wizytatora misjonarzy polskich (1851— 1860). Jako w izy tator wydał drukiem szereg listów okólnych do misjonarzy i sióstr m i łosierdzia. Przygotował również do druku i dwukrotnie powiększył dzieło ks. Pawła Rzymskiego: Wykład obrzędów kościelnych (Warszawa 1857) i Cantionale Ecclesiasticum (Warszawa 1856). W rękopisie pozo stały Dzieje Kościoła unickiego, wykorzystane przez ks. Likowskiego przy pisaniu dzieła o unii. Dorobis wręczył też powracającemu z Rosji przez Warszawę M gr Chigiemu memoriał o stanie zakonów w Polsce (De institutis regularibus in Polonia). W latach 1860—63 spełniał Dorobis funkcje asystenta domu św. Krzyża w Warszawie, a później wykładał do r. 1867 w seminarium duchownym w Płocku. Po kasacie zgroma dzenia poświęcił się pracy duszpasterskiej w diecezji płockiej, gdzie w Zadusznikach rozpoczął budowę nowego kościoła. Ostatnie lata swego życia przepędził w diecezji sejneńskiej, gdzie zmarł jako oficjał i prałat archidiakon katedry w Sejnach w r. 1883 15. Na s. 124 pisze ks. Gryglewicz: „O ks. Janie Sopuszyńskim brak w ia domości, poza podaną datą, kiedy wykładał Pismo św. i uczył śpiewu w Seminarium Duchownym". Możemy skądinąd biograficzne dane o ks. Sopuszyńskim uzupełnić. Ur. 1805 r. w Raszynach, pow. Mława, wstąpił w r. 1824 w Warszawie do Zgrom. Księży Misjonarzy. T R Z Y TO M Y „STU D IÓ W TEO LO G ICZNYCH ". Jednakowoż wiadomo każ demu obeznanemu z życiem modlitwy, że u dusz nim żyjących, zakorze nionych już w życiu modlitewnym, trwającym w modlitewnym zjed noczeniu z Bogiem, wyrabia się i Irwale pozostaje szczególne, nowe in tensywne nabożeństwo, skierowane formalnie do osoby Boga Ojca. Tego Autorka nie uwypukliła ani w życiu samego br. Alberta, ani w trzech rozdziałach (20— 22), poświęconych s. Bernardynie, pierwszej przełożo nej generalnej ss. albertynek, a zawierających liczne jej wypowiedzi, charakterystyczne dla jej życia wewnętrznego. Opierając się więc tylko na wywodach Autorki, wnioskować by należało o braku tego nabożeń stwa zarówno u br. Alberta, jak u s. Bernardyny. Autorka przyznaje, że do tragedii Chmielowskiego w Starej Wsi mamy bardzo mało źródeł z pierwszej ręki. Pomimo tego w ydaje się, że po trzebna by tu była diagnoza dokładniejsza, przynajmniej hipotetyczna. A mianowicie: 1. Przypuśćmy, że lekarze istotnie nie poznali się na jego chorobie. To jednak nie wyklucza faktu, że casus Chmielowski — zawsze według szczupłych danych, jakie posiadamy — doskonale m iejci się w ramach psychiastenii i połączonych z nią potwornie męczących skrupułów. 2. W Starej Wsi widzim y już dalszy rozwój choroby, która zapowiada się przynajmniej na rok przedtem. Jest to melancholia, któ rej symptomem są niektóre obrazy z tych właśnie lat. Mam na myśli przede wszystkim dwa obrazy, oba z lat 1879— 1880, mianowicie „Szara godzina* oraz „W e Włoszech, zwany też „Mnich na cmentarzu. Oba te dzieła przypominają, żywo swym romantycznym smutkiem niektóre tw ory Bocklina, jak „Św ięty gaj“ , a zwłaszcza „W yspę umarłych w kilku jej odmianach. Ów symptom, „ekspresję marzycielskiej melan cholii... w okresie 1877— 1880“ zauważył trafnie świetny filozof i dobry psycholog ks. rektor Konstanty Michalski, który też stwierdza, że póź niej, tj. po wyzdrowieniu Chmielowskiego, znika w jego obrazach zu pełnie owa poprzednia melancholia. (Por. M i c h a l s k i . K. ks., Brat Albert, Kraków 1946, 127 oraz cytat za nim. 117 i 126, Katalog dra J. S i e n k i e w i c z a , nr 15 i 16). Do jezuitów przychodzi więc Adam Chmielowski w jesieni 1880 r. już w stanie równowagi niedoskonałej. 3. Dalszym czynnikiem, który tę niestałą, i niepewną równowagę silnie podrywa, jest fizyczny skutek nagłego zaprzestania palenia i całkowi tego wyrzeczenia się nikotyny. Wygląda to na poryw wielce szlachetny, ale nieprzemyślany i nieroztropny. Gorzej, bo wygląda to na postano wienie, zrobione bez zgody przełożonych. T R Z Y TO M Y „STU D IÓ W TEO LO G ICZNYCH ". Konstanty Michalska, metropolita Szeptycki, kard. Dunajewski, biskup Nowak, o. Jan Paweł - ski, ks. Czesław Lewandowski, Helena Modrzejewska, Zdzisław Dębicki, brat Leon. Ale znajdziemy tam również wypowiedzi osób, których w ia- rogodność winna być dopiero udowodniona a nie budzi zaufania. Z dru giej jednak strony, nawet goście br. Alberta z Ogrzewalni mogą być cennymi i wiarogodnymi świadkami, choć ich charakter ogółem zaufania nie budzi. Przy całej wartości tej biografii br. Alberta nasuwają się jednak przy jej czytaniu niektóre wątpliwości. Np. nie w ydaje się słusznym potępienie tego, że spotykały się z nieufnością w Polsce pod koniec ubiegłego stulecia drogi „niezwykłe" w życiu duchownym (80). Wszak w tej dziedzinie obowiązuje każdego roztropnego kierownika sumienia R E C E N ZJE i S P R A W O Z D A N IA RE C E N ZJE 1 S P R A W O Z D A N IA 326 327 notuje Autorka z krytyką, (206) niezadowolenie pewnego niewymienio- nego bernardyna, gdy mu na Kalatówkach podano śniadanie, jak jed nemu z „opuchlaków". Sądzę, że ów bernardyn miał w zasadzie słuszność; wszak odbył długą, nużącą wędrówkę na Kalatówki nie jako turysta, nie w odwiedziny, jak tylu pożądanych i niepożądanych gości, lecz po to, aby tam braciom czy siostrom odprawić mszę św., zapewne na ich w y raźne życzenie czy prośbę. wych skrupułów, pewności potępienia i, jak się wydaje, nie znajduje nikogo, któryby go zrozumiał i zastosował jedyne w tym stanie celowe lekarstwo duchowe: całkowitą, ślepą ufność w Bogu (79). Przeciwnie, po niewymienionych „ekstrawagancjach Chmielowskiego, jezuici „szyb- ko- uznają, że popadł on w rodzaj aberratio mentis, żegnają go i skie rowują nawet do domu zdrowia (80 n). Psychiatrzy nie umieją się poznać na jego chorobie, zabiera go do siebie jego brat Stanisław Chmielowski (81) i „uzdrowienie* ma miejsce u tamtejszego proboszcza, ks. Pogo rzelskiego w Szarogrodzie (85). Autorka wstawia uwagę, że casus Chmie lowski nie jest typem spotykanym w książkach duchownych (79). Obok twierdzeń, które uważam za wątpliwe, są jeszcze, nie liczne co prawda, ale poważniejsze niedomówienia, które trzeba uważać za braki. Możnaby je pominąć w dziele czysto historycznym, kronice czy syntezie, ale są one niewątpliwie brakami w dziele hagiograficznym. Autorka ogółem stara się wejść w miarę możności w sanctuarium su mienia br. Alberta i to z subtelnym umiarem, z delikatnością dykto waną zrozumiałym i należytym szacunkiem wobec zagadnień i przeżyć, rozgrywających się pomiędzy duszą i Bogiem. T R Z Y TO M Y „STU D IÓ W TEO LO G ICZNYCH ". Wszak jego przełożony, to „rozumny i dobry psycholog (73); w ięc wątpić wolno, czy ten dobry psycholog był by się zgodził bez zastrzeżeń na tak ryzykowne posta nowienie nowicjusza. Z drugiej zaś strony wiemy, że Adam miał bo lesne doświadczenia z kierownikami sumienia, „lękliw ym i lub ieno- rantami, którzy krępują duszę, zamiast pozostawić ją kierownictwu Ducha (121). Nasuwa się więc domniemanie, że Adam Chmielowski, nauczany ujemnym doświadczeniem, nie przedłożył przełożonemu do Drugim poważnym brakiem jest m. zd. tylko ułamkowe wyjaśnie nie wewnętrznej, a w swoich skutkach także zewnętrznej tragedii A da ma Chmielowskiego w nowicjacie jezuitów w Starej Wsi, 1880— 1881. Pociąga ono za sobą lekki, lecz wyraźnie dostrzegalny krytycyzm Autorki w stosunku do tamtejszych jezuitów. Oto krótki przebieg według opisu Autorki: Adam Chmielowski wstępuje 24 9 1880 do nowicjatu w Sta rej Wsi za rektoratu o. Kacpra Szczepkowskiego. Magister nowicjuszów, „rozumny i dobry psycholog" (73), o. Wojciech Baudiss nie zakazuje mu malarstwa. Adam rozpoczyna 30-dniowe rekolekcje i postanawia zerwać z paleniem papierosów, co jak wiadomo, może u namiętnych pa laczy wywołać zaburzenia nerwowe. Pewnego dnia znajduje niedopałek, nie umie się opanować i wypala go, a następnie popada w prawdziwą burzę wyrzutów sumienia, że nie dotrzymał postanowienia. Autorka zu pełnie słusznie podkreśla, że to cierpiała przede wszystkim jego miłość własna, że nie dotrzymał słowa, czego sam nie dostrzegł, a co powinien był dostrzec jego kierownik sumienia (78). Dołącza się do tego, zgodnie z programem pierwszego tygodnia ćwiczeń duchownych św. Ignacego, nakaz rozmyślania o grzechu, sprawiedliwości bożej, śmierci, „dla niego, który już nie potrafi rozmyślać** (79). Adam staje się ofiarą straszli R E C EN ZJE i S P R A W O Z D A N IA 328 RE C E N ZJE i S P R A W O Z D A N IA 329 Jeszcze inna refleksja: zakony pustelnicze, zwł. kartuzi, znają w y padki z nowicjuszami, które też ,.nie są notowane w książkach du chownych", mianowicie usposobienia, które psychicznie nie znoszą sa motności, życia pustelniczego. Charaktery bardzo szlachetne, ale któ rym ścisłe przestrzeganie reguły pustelniczej grozi chorobą umysłową i to pomimo własnego pociągu do tego życia. Czy dla tego Chmielowski nie wstąpił do kamedulów, swoich przyjaciół? Czy dlatego nie poszedł za pierwotną zachętą o. Rafała Kalinowskiego? Czy dla tego o. K a li nowski pisze mu później, że dusza żyw i się jedynie w samotności, więc doradza mu, aby przynajmniej brał „kąpiele pustelni"? (123). T R Z Y TO M Y „STU D IÓ W TEO LO G ICZNYCH ". Za tą radą br. Albert szedł sam, stosował ją wobec swych braci i sióstr, aby ich ustrzec od „największego niebezpieczeństwa apostolatu, jakim jest aktywizm " (188). Aby mogli się wzorować na św. Bernardzie, którego Autorka słusznie przytacza, który nauczył się „plus in silvis quam in libris" (238), ale i na nim samym: na artyście, którego oglądanie cudów przyrody prowadzi do kontemplacji Stwórcy. aprobaty swego postanowienia zerwania nagłego a zupełnego z niko tyną. 4. Wypalony łakomie niedopałek sprowadza burzę wyrzutów su mienia, a nie widać żadnego przełożonego, ani kierownika sumienia, który by wkroczył jako lekarz, chociażby za pomocą energicznego złajania miłości własnej nowicjusza. Znów się nasuwa podejrzenie, że Adam nie otworzył się całkiem szczerze swemu mistrzowi nowicjuszów czy też kierownikowi sumienia. 5. Szczytowe nasilenie i pokusy rozpaczy przychodzą u niego w pierwszym tygodniu wielkich rekolekcji, jako znany objaw na pewnym wyższym szczeblu życia wewnętrznego. Ze swymi skrupułami nie umie sobie poradzić, a pokusy rozpaczy dopro wadzają go do jakichś dziwactw, a następnie do choroby, którą jezuici ..pośpiesznie" zakwalifikowali jako rodzaj aberratio mentis. Dodać należy, że nie wiemy, czy Chmielowski pozwalał sobie samowolnie na dodat kowe umartwienia cielesne, które również mogły ujemnie wpływać na jego stan zdrowotny. 6. Że diagnoza jezuitów była słuszna, na to wska zuje fotografia Adama Chmielowskiego, po jego wyjściu ze Starej Wsi. Robi na niej wrażenie człowieka wycieńczonego fizycznie, ale i psy chicznie, a nawet niezupełnie normalnego umysłowo. 7. Że sam br. Albert uznawał tę diagnozę ale i terapię jezuitów za słuszną, tego daje sam dowód, a mianowicie: 1) darzy nadal stale jezuitów zaufaniem, a jezuici stale wspomagają swego eksnowicjusza (238). Jego zaufanie nie musi być jedynie przejawem tego, że nie ma do nich żadnego żalu (93). 2) Br Albert sam stosuje do s. Bernardyny, pierwszej przełożonej ss. albertynek, na jej skrupuły i pokusy potępienia analogiczną receptę, tj. ufność w Bogu ale i lekarza: „Zamiast zanudzać swego spowiednika idź zobaczyć się z dr. Sawickim; ten ci da trochę waleriany i każe ci dobrze się wyspać" (79). Zresztą to stara recepta, znana bez wym ienia nia waleriany od wieków i kartuzom, i św. Teresie i tylu innym. Tak mi wygląda założenie, rozwój i koniec tragedii br. Alberta w Starej Wsi; jest to oczywiście jedynie hipoteza, bo z powodu braku dokład niejszych źródeł, nie ma też zupełnie pewnych niektórych podstaw. Jeszcze jeden brak: Autorka nie uwypukliła stosunku br. Alberta do krakowskiego księcia biskupa, kardynała Puzyny. T R Z Y TO M Y „STU D IÓ W TEO LO G ICZNYCH ". RE C EN ZJE i S P R A W O Z D A N IA R E C E N ZJE i S P R A W O Z D A N IA 331 330 ku 1916 zwykłym pociągiem osobowym nie trzy (279), lecz przynajmniej S l/i godzin. . ku 1916 zwykłym pociągiem osobowym nie trzy (279), lecz przynajmniej S l/i godzin. . 142), lecz w r. 1889 otrzymał od Austrii dla siebie i swoich następców na stolicy krakowskiej tytuł książęcy. Przy wszystkich jego niezaprze czonych zaletach i zasługach trudno mu jednak przypisać „dobroć anielską" (156). Chociażby w świetle jego zatargu z krakowskimi m isjo narzami i decyzji odebrania im rzekomo prawnie fundacji bpa Sza niawskiego, a nadanie jej zmartwychwstańcom, którym kard. Dunajewski zawdzięczał w bardzo znacznej mierze swoje biskupstwo. Jak wiadomo, proces ten z misjonarzami kuria krakowska przegrała, ale przy tym zaginął oryginalny przywilej fundacyjny bpa Szaniawskiego dla m i sjonarzy... 3. Inne. — Cenzura wojenna austriacka (284) nie mogła być groź dla nekrologów br. Alberta w r. 1916; przeciwnie, dla byłego pow stańca przeciwko Rosji mogła być tylko bardziej względna i to jeszcze po ogłoszonym przez państwa centralne utworzeniu polskiego niepo dległego królestwa. — Nie jest ścisłym, że br. Albert „odgadł" społeczną rolę muzyki (141). To nie był jego wynalazek. Taką je] wartość znano i stosowano znacznie wcześniej, a w pracy misyjnej stosowali ją, jak wiadomo, ze świetnym wynikiem jezuici w Redukcjach paragwajskich. — W okolicach Ołomuńca nie mógł istnieć w r. 1863/4 proboszcz „cze- chosłowacki", lecz był to Morawianin (16). — Rysie nie istniały już wówczas w lasach Werchraty, a w ilki latem tam nie tylko nie napa dały ludzi (191), ale i nie bywały. j y — Książe biskup krakowski Sapieha nie był arcybiskupem w l tach 1914— 1926 (241, 275, 280). Narracja Autorki jest prawdziwa je dynie sensu diviso, nie zaś sensu composito. Ok. r. 1900 Kraków nie był jeszcze arcybiskupstwem (217), ani też późniejszy książę metropolita i kardynał Sapieha nie był tam wtedy jeszcze ordynariuszem, a objął diecezję osobiście dopiero w r. 1912. — Bp Nowak nie był krakowskim koadiutorem (275, 285), lecz biskupem pomocniczym, vulgo sufraganem, a później bpem przemyskim. Analogicznie bp Czesław Sokołowski nie był koadiutorem, lecz biskupem pomocniczym siedleckim (112, 290), a później nominatem sandomierskim. T R Z Y TO M Y „STU D IÓ W TEO LO G ICZNYCH ". — Siemieńscy Lucjan i jego córki Klementyna Chojecka, karmelitanka Małgorzata Teresa l Lucyna W il czyńska nie mają rezydencji w Chorostkowie (27n, mylnie pisane Chro- rostków). Mieszka tam wówczas Wilhelm Siemieński, który nie jest krewnym poprzednich. Dodać można, że do czasu pierwszej w ojny świa towej i jej zniszczeń istniał tam nieznany dzisiaj obraz pędzla Adama Chmielowskiego. — Nie nazwany szantażysta, który dokuczał br. A l bertowi, nazywał się Emil Doberschiitz, nie był ani włóczęgą, ani no torycznym bandytą (175), lecz „człowiekiem interesów". Nie był are sztowany, lecz na podstawie ówczesnych wojennych zarządzeń wysie dlony przymusowo z zaboru austriackiego, jako „lastiger Auslander", do swej rodzinnej Bydgoszczy, wówczas pod zaborem pruskim. Uzyskał to wysiedlenie nie nazwany w książce kapłan, mianowicie znany dzia łacz na Śląsku i w Brazylii, misjonarz ks. Jan Rzymełka, wówczas kie rownik zakładu ks. Siemaszki w Krakowie. Do niego zwracał się br. Albert z wyrzutem: „Zgubiłeś duszę!" Frere Albert jest biografią, nie zaś systematycznym wydaniem jego doktryny. A była by to rzecz pożyteczna, dla Polaka pociągająca, dla obcych pouczająca i budująca. Wiadomo zaś, jak mało wiedzą oni o pol skim dorobku świętości w Kościele. Np. jak szeroko znanym jest opie kun trędowatych o. Damian, jak mało nasz o. Jan Beyzym. Dlatego też książka Winowskiej ma taką wartość uświadamiającą. Pomimo chronologicznego układu strona ascezy i mistyki udała się doskonale. A le trzeba te zagadnienia wyłaniać, trzeba zapatrywania, nauki, prze życia br. Alberta wyszukiwać. A nie jest to rzeczą łatwą, bo spis rze- rzy jest dokładny, ale na modę francuską poetycki nie oddający treści. Temu zaradził by nieprzewidziany niestety przez wydawców dobry sko rowidz rzeczowy. A jest tam istotnie bardzo dużo zapatrywań, wska zówek, rad i nauk br. Alberta, mających trwałą wartość dla szerokich sfer katolickich, nie tylko dla jego własnego Zgromadzenia. Oto kilka przykładów. Codzienna komunia św. jako lekarstwo duszy, i to na tyle lat przed dekretem Piusa X (85n); uświęcenie człowieka przez pracę (145); klasyczne „porzucanie Boga dla Boga (215, 229), jeśli tego wymaga •obowiązek, tak zgodne z duchowością kartuzów, a za nimi Ignacego Loyoli. Klasyczne zachowanie się brata misjonarza Marcina Kudełki czy też ss. albertynek wobec ekstaz br. Alberta, czekających „aż mu to przejdzie" (159n), żywo) przypominających (tłumaczenie przerażonemu świadkowi lewitację m. M arceliny Darowskiej przez konwerskę s. T e resę Ciszkiewicz, jako „taki rodzaj m odlitwy". Klasyczna przestroga br. T R Z Y TO M Y „STU D IÓ W TEO LO G ICZNYCH ". Jego pontyfikat trwał 15 lat (1894— 1911). Puzyna nie był biskupem słomianym, a nadto był tercjarzem franciszkańskim, swoje obowiązki terjarskie brał bardzo poważnie, więc choćby z tych dwóch przyczyn musiał się interesować żywo albertynami. Ich cel i praca musiały go też bardzo obchodzić z pobudek duszpasterskich, które, jak wiadomo, były naczelną troską kardynała Puzyny. Oprócz niektórych braków istnieją w pracy Marii Winowskiej także nieścisłości, wzgl. pomyłki. Dla duchowej sylwetki br. Alberta są one obojętne. Tym bardziej są obojętne dla czytelnika nie Polaka i poza Polską. Jednakowoż żywot br. Alberta, choć należy przede wszystkim dc hagiografii, nie zwalnia od ścisłości historycznej, pomimo że licentia poetica może sobie dużo pozwolić. Oto kilka przykładów: 1. Osoby. — Ks. Czesław Lewandowski, cytowany bardzo częst otrzymuje chyba stale mylny tytuł „pere" = ojciec. Był on jednak m i sjonarzem (Lazarystą), a ci są Zgromadzeniem kapłanów świeckich i nie używają tytułu „ojciec", lecz „Monsieur". Możnaby poniekąd uspra wiedliwić tytułowanie go ojcem tym, że przez długie lata był ojcem du chownym alumnów krakowskiego seminarium diecezjalnego. — Zało życiel zrazu schroniska, później dużych zakładów wychowawczych w Krakowie i Czernej, misjonarz, nazywa się Siemaszko (Kazimierz, 1 1904), nie zaś Siemiaszko (97). — Jezuita, misjonarz wśród unitów na Podlasiu, przyjaciel i powiernik br. Alberta, nazywa się Pydynkow- ski, nie Pydynkiewicz (92). — Łucja Dębicka, której t>r. Albert jako dziecku przepowiedział godność królewską (265), siostra Celiny Bzow skiej, nie była nigdy karmelitanką, ale żyje dotąd jako niepokalanka. — Kard. Dunajewski nigdy nie był arcybiskupem ani metropolitą (120, 135, A le na marginesie tych ciężkich przeżyć br. Alberta nasuwają się pewne refleksje i analogie z przeżyciami św. Ignacego Loyoli. Wiadomo, że Ignacy również cierpiał od straszliwych skrupułów, na które żaden z jego ówczesnych spowiedników nie umiał znaleźć skutecznego lekar stwa. Że, nie wiedząc jeszcze wówczas, czym jest roztropność i umiar, nadużywał praktyk pokutnych fizycznych, co w sumie doprowadziło do ciężkiej choroby. Z drugiej zaś strony, właśnie przykład Ignacego, poświęcanie się tego hiszpańskiego arystokraty służbie nędzarzy, prze bywanie stale w ich odrażającym towarzystwie, przezwyciężanie natu ralnego wstrętu do brudu fizycznego i moralnego, do robactwa i cho rób, a nadprzyrodzona miłość do Chrystusa w osobie nędzarza, to mo gło stać się już wówczas w Starej Wsi podnietą d® późniejszego po wołania br. Alberta. T R Z Y TO M Y „STU D IÓ W TEO LO G ICZNYCH ". Nie odpowiada też za liczne błędy francuskie ortogra ficzne i opuszczenia, które nieraz zniekształcają znaczenie zdania. Jest to wina wydawnictwa La Colombe. Niemniej dzięki Autorce br. Albert przestaje być ukrytą wartością. Należy wyrazić życzenie, aby książka ta ukazała się niebawem również w naszym języku, dla naszych szero kich sfer. A le po uwzględnieniu wymienionych powyżej postulatów. Podział materiału maryjnego został ujęty według wskazań znanych i cenionych w świecie katolickim mariologów Roschini’ego i Be Szkoda tylko, że autorzy bibliografii, wprowadzając porządek i podział rzeczowy i bardziej teologiczny, nie wprowadzili wyraźnego oddzielenia materiału mariologicznego, tj. ściśle naukowego, od bogatego materiału naukowo-popularnego wzgl. całkowicie popularnego. Sądzę, że można było łatwo, przynajmniej odpowiednimi znakami wydzielić materiał dla teologów-mariologów czyli bibliografię mariologiczną, od materiału nie naukowego czyli od bibliografii maryjnej. Pewnie, że wygląda to może bardziej imponująco w tej formie, bo wydzielony materiał ściśle nau kowy, przedstawiałby się raczej skromnie. Nie posiadamy przecież roz praw i artykułów mariologicznych na miarę taką, jaką widzim y w in nych krajach, zwłaszcza w ostatnich latach. Ks. Zdzisław Obertyński Z. S z o s t k i e w i c z — S. W e s o ł y , Bibliographia Mariana Polo- norum ab anno 1903 ad annum 1955 — Polska Bibliografia Maryjna od roku 1903 do 1955, Ed. „Marianum“, Roma 1955, s. V, 76. W pracy naukowej i korzystaniu z wydanych druków i książek wiele pomaga dobrze opracowana w danym dziale nauki bibliografia. Tak jak inne gałęzie wiedzy, tak też i teologia a w teologii jej dział m a- riologia posiadają każda swoją bibliografię. W mariologii trzeba w y różnić opracowania w skali ogólnoświatowej od opracowań bibliogra ficznych krajowych wzgl. o bardziej zacieśnionym zakresie jak np. bibliografie zakonne. Myśl wydania takiej bibliografii nurtowała w wielu umysłach teolo gów polskich. Co więcej w ostatnich latach, zwłaszcza z okazji 100-lecia ogłoszenia dogmatu Niepokalanego Poczęcia przystąpiono do jej reali zacji '). Polska bibliografia maryjna winna być wydaną raczej w kraju, w Polsce, aby była łatwo dostępną dla ogółu wiernych a zwłaszcza dla kapłanów i teologów. Dlatego starania i wysiłki podjęte w kraju winny być podtrzymane i doprowadzone do pomyślnego końca, jakim jest w y danie kompletnej polskiej bibliografii maryjnej i m ariologicznej!), Do r. 1905 Polska nauka teologiczna nie posiadała swojej krajow ej bibliografii maryjnej. Dopiero rocznica dogmatu Niepokalanego Poczę cia (50-lecie) i z nią związane uroczystości w naszym kraju, przyniosły jakby w darze, wydanie polskiej bibliografii maryjnej. T R Z Y TO M Y „STU D IÓ W TEO LO G ICZNYCH ". Alberta dla dewotek, że w godzinę śmierci mogą się spostrzec, iż na leżały nie do Boga, lecz do spowiednika (191); przestroga tak świetnie idąca po linii nauk i rad św. Teresy W ielkiej, św. Franciszka Salezego, kardynała Lavigerie, o. Honorata, m. Marceliny Darowskiej. Klasyczne i niezawodne stwierdzanie przez mistyka prawdziwego uobecniania się Boga w jego duszy (221). Klasyczne stwierdzenie konieczności rozrywki ■czy pracy fizycznej jako psychicznego odprężenia w kontemplacji (229), 2. Miejscowości. — Narol nie leży na Podolu (209, 213), nie ma tam ani crarnoziemu (213), ani stepu (229), lecz gleba piaszczysta i lasy. — Horyniec nie jest słynnym miejscem pielgrzymkowym (210). Chociaż tamtejsza, zbudowana w lesie kapliczka św. Antoniego, wewnątrz której bije obfite źródło, otwarta dzień i noc, jest stale otoczona pieczołowitą czcią, miłością i troską ludności. — Poturzyce (27), nazywa się Potu- rzyca i nie leży na Podolu. — W Zarzeczu byli Dzieduszyccy, nie Sie mieńscy (46). — Podróż koleją z Zakopanego do Krakowa trwała w ro 2. Miejscowości. — Narol nie leży na Podolu (209, 213), nie ma ta ani crarnoziemu (213), ani stepu (229), lecz gleba piaszczysta i lasy. — Horyniec nie jest słynnym miejscem pielgrzymkowym (210). Chociaż tamtejsza, zbudowana w lesie kapliczka św. Antoniego, wewnątrz której bije obfite źródło, otwarta dzień i noc, jest stale otoczona pieczołowitą czcią, miłością i troską ludności. — Poturzyce (27), nazywa się Potu- rzyca i nie leży na Podolu. — W Zarzeczu byli Dzieduszyccy, nie Sie mieńscy (46). — Podróż koleją z Zakopanego do Krakowa trwała w ro R E C EN ZJE i S P R A W O Z D A N IA RE C E N ZJE i S P R A W O Z D A N IA 332 333 rzeczy bezwględnie wymaganej u kartuzów. Klasyczna zasada, że n ie wystarcza samo głoszenie ewangelii, lecz życie nią (258). rzeczy bezwględnie wymaganej u kartuzów. Klasyczna zasada, że n ie wystarcza samo głoszenie ewangelii, lecz życie nią (258). stawiony w porządku chronologiczno-alfabetycznym. Schemat podziału rzeczowego polskiej bibliografii maryjnej, wydanej w Rzymie, jest bar dziej przejrzysty i praktyczny. Pozycję tę należy witać z radością, jako ważny przyczynek dla polskiej mariologii. stawiony w porządku chronologiczno-alfabetycznym. Schemat podziału rzeczowego polskiej bibliografii maryjnej, wydanej w Rzymie, jest bar dziej przejrzysty i praktyczny. Pozycję tę należy witać z radością, jako ważny przyczynek dla polskiej mariologii. Autorka nie odpowiada za brzydkie wydanie, ani za ortografię pol ską i niemiecką. y ! Za pośrednictwem o. Przeora ze Skałki i dzięki życzliwości o. Syk stusa Szafrańca dowiedziałem się, że najwięcej materiałów do wydania bibliografii m aryjnej zgromadził p. B. Zynda. 1 Myśl opracowania bibliografii maryjnej zajęła umysły starszych i młodszych. Mam na myśli profesorów teologii i studentów. O ile mi wiadomo, zbieraniem materiału do wydania bibliografii maryjnej zajęli się: Seminarium krakowskie pod kierunkiem ks. K. Smoleńskiego, oo. paulini w Częstochowie, zwłaszcza o. Damian Nowak i o. Sykstus Sza- franiec (głównie bibliografią dotyczącą Jasnej Góry) i wreszcie p. Bo lesław Zynda. 1 Myśl opracowania bibliografii maryjnej zajęła umysły starszych i młodszych. Mam na myśli profesorów teologii i studentów. O ile mi wiadomo, zbieraniem materiału do wydania bibliografii maryjnej zajęli się: Seminarium krakowskie pod kierunkiem ks. K. Smoleńskiego, oo. paulini w Częstochowie, zwłaszcza o. Damian Nowak i o. Sykstus Sza- franiec (głównie bibliografią dotyczącą Jasnej Góry) i wreszcie p. Bo lesław Zynda. ! Za pośrednictwem o. Przeora ze Skałki i dzięki życzliwości o. Syk stusa Szafrańca dowiedziałem się, że najwięcej materiałów do wydania bibliografii m aryjnej zgromadził p. B. Zynda. T R Z Y TO M Y „STU D IÓ W TEO LO G ICZNYCH ". Autorzy zdają sobie z tego sprawę, że ich dzieło nie jest wyczerpującym zestawieniem wszystkich druków polskich o Maryi. Mimo braków, można jednak ta dziełko uważać za dość udaną i pożyteczną próbę bibliografii m aryjnej, po której czetkamy na coś doskonalszego w kraju. Dla uzupełnienia bi bliografii nie zamierzam podawać wszystkich pominiętych pozycji, które liczą się na setki. Przecież autorzy bibliografii, jak sami podają, oparli się tylko na niektórych źródłach im dostępnych, zwłaszcza na urzędo wych bibliografiach polskich i na rocznikach bibliograficznych, które okazały się mało dokładnymi oraz na dwóch czasopismach: Ateneum Kapłańskim i Przeglądzie Powszechnym. A to za mało. W iele artykułów jest rozrzuconych w różnych czasopismach. Są one nieraz drobne, ale wartościowe dla całości mariologii polskiej. Przy tym zaznaczyć należy, że i te dwa poważne czasopisma: Ateneum Kapłańskie i Przegląd P o wszechny nie są dokładnie wykorzystane. Oto niektóre braki i opusz czenia np. ks. Tad. Karyłowskiego, Stabat Mater, Przegl. Powsz. 159 (1923) 236— 251; ks. H. Cichowskiego, Nestorianizm i sob. efeski, Przegl. Powsz. 189 (1931) 155— 172; ks. Charszewskiego (recenzja maryjna), A te neum Kapł. 26 (.1930) 312— 322. Do opuszczonych druków wyżej wspomnianych można dołączyć sze reg dzieł maryjnych o charakterze raczej popularnym. N ie mniej jednak są to prace pożyteczne. Oto między innymi: ks. Wł. Kuc, Trium f M iry i Królow ej Różańca św., Bochnia 1934, s. 101; O. H. Petitot, Objaw en.e- Najśw. Maryi Pa/nny w Lourdes św. Bernadecie, Kraków 1935 s. 103; ks. M. Meschler, Najśw. Panna je j cnotliwe życie i błogosławiona śmierć. Tłum. z niem. Ad. Kuleszo, Wilno 1935, s. 152; ks. Wł. Smolarkiewicz, Sw.ęty rok Maryi. Nabożeństwo maryjne, b. m. w. 1937, s. 224; ks. J. Cyrek, Ślubowanie jasnogórskie, Kraków 1936, s. 32; ks. J. Szczepański, Zjaw ienie się M atki Bożej na Górze Sałetyńskiej, Dębowiec 1936, s 59; ks. St. Kuczyński, Najśw. Panna z Lourdes, Warszawa 1905, s. 99. Na ten sam temat opuszczono dziełko pt.: Lourdes, Kraków-Kleparz 1925, s. 242; A licja Byszewska, Cudowne Objawienie Matki Boskiej w Lourdes, b. m. w. 1933, s. 30. Do liczby pominiętych w bibliografii a tylko popularnych broszur i dzieł o M aryi należy dołączyć: Ed. Paszkowskiego, Doi osa, Warszawa 1905, s. 136; O. St. Cz. Mączki, Co nam mówią obrazy M ryi, Lw ów 1934, s. 41; Wł. Bełzy, Królow a Korony Polskiej. Żywot Najśw- M aryi Panny, Matki Chrystusa Pana, Lw ów 1906, s. 88; K. T R Z Y TO M Y „STU D IÓ W TEO LO G ICZNYCH ". Dla upamiętnie nia mianowicie uroczystego jubileuszu wydano (Lw ów 1905) dwuto mową „Księgę Mariańską", która oprócz szeregu różnych artykułów, po święconych czci N. M. Panny w Polsce, przyniosła nam w drugiej swej części polską bibliografię maryjną pt.: Polska bibliografia maryjna od wynalezienia sztuki drukarskiej do roku 1902, w opracowaniu prof. W. Bruchnalskiego. Bibliografia maryjna Szostkiewicza-Wesołego jest wartościowym wkładem w literaturę teologiczno-maryjną polską. Nie mniej jednak trzeba przy wielkim uznaniu dla pracy i trudu, jakie włożono w jej "wydanie, zaznaczyć, że posiada braki i że nie jest pełnym wyrazem m y śli mariologicznej a tym bardziej myśli maryjnej w ogóle. Zebrano dużo, bo aż 1210 pozycji maryjnych. Autorzy zebrali mnóstwo pozycji •w postaci książek, broszur, artykułów, recenzji, wydawanych tak w kraju, jak i za granicą. Co więcej, bibliografia ta obejmuje liczne druki wydane przez Polaków za granicą w obcych językach. Od r. 1903 wydawano jednak i pisano w dalszym ciągu różne rozr- prawy, dzieła, broszury, artykuły o Matce Bożej. Jednym słowem litera tura maryjna nieustannie wzbogacała się nowymi pozycjami. A kiedy Polska doczekała 100-lecia dogmatu Niepokalanego Poczęcia, powstała znów piękna myśl zebrania w jednym dziełku bibliograficznym dorobku maryjnego, minionego, ostatniego 50-lecia, aby kontynuować rozpoczętą przez prof. Bruchnalskiego pracę. Myśl taka powstała i została zamie niona w czyn przez członków Sodalicji Mariańskiej, istniejącej przy Papieskim Kolegium Polskim w Rzymie. I oto posiadamy nowowydaną za granicą polską bibliografię maryjną, która aczkolwiek jest konty nuacją poprzedniej, jest jednak odmiennym sposobem ułożona. Materiał bowiem bibliograficzny został tu zebrany i ułożony według zagadnień rzeczowo uporządkowanych, a nie, jak u Bruchnalskiego, gdzie jest ze- Bibliografia o której piszemy, nie jest jednak pełnym wyrazem tego, RE C E N Z JE i S P R A W O Z D A N IA RE C EN ZJE i S P R A W O Z D A N IA 334 kult na tle życia religijnego w dawnej Bochni, Bochnia 1934 s. 128; ks. Ign. Charszewski, Kościół i obraz M atki Boskiej w Żurom inie, Kielce 1904, s. 36; ks. J. Midura, Dzieje obrazu M atki Boskiej w Zawadzie k Dę bicy, Kraków 1935 s. 202; ks. M. Jastrzębiec, W obronie czci Jasn^góryy Poznań 1911 i 1912 s. 6 nlb. 191; ks. Fr. Smagliński, Cudowny obraz M atki Boskiej Byszewskiej, Toruń 1937, s. 75; Fr. Mączyński, K ściół Najśw. M aryi Panny w Krakowie, Kraków 1938, s. 127; ks. A. J. T R Z Y TO M Y „STU D IÓ W TEO LO G ICZNYCH ". Zło towski, F.gura łaskami słynąca M atki Boskiej i kościół sejneński, b. m. w. 1938, s. 90; Tadeusz Dobrowolski i Edward Szramek, Obraz M atki Boskiej Piekarskiej w Opolu. Roczniki Tow. Przyj. Nauk. na Śląsku. T. VI. Katowice 1938, s. 522; Wł. Horbacki, Kościół Mariacki, Kraków 1948, s. 111, 1 nlb. kult na tle życia religijnego w dawnej Bochni, Bochnia 1934 s. 128; ks. Ign. Charszewski, Kościół i obraz M atki Boskiej w Żurom inie, Kielce 1904, s. 36; ks. J. Midura, Dzieje obrazu M atki Boskiej w Zawadzie k Dę bicy, Kraków 1935 s. 202; ks. M. Jastrzębiec, W obronie czci Jasn^góryy Poznań 1911 i 1912 s. 6 nlb. 191; ks. Fr. Smagliński, Cudowny obraz M atki Boskiej Byszewskiej, Toruń 1937, s. 75; Fr. Mączyński, K ściół Najśw. M aryi Panny w Krakowie, Kraków 1938, s. 127; ks. A. J. Zło towski, F.gura łaskami słynąca M atki Boskiej i kościół sejneński, b. m. w. 1938, s. 90; Tadeusz Dobrowolski i Edward Szramek, Obraz M atki Boskiej Piekarskiej w Opolu. Roczniki Tow. Przyj. Nauk. na Śląsku. T. VI. Katowice 1938, s. 522; Wł. Horbacki, Kościół Mariacki, Kraków 1948, s. 111, 1 nlb. co wydrukowano o Maryi w ostatnim 50-leciu. Autorzy zdają sobie z tego sprawę, że ich dzieło nie jest wyczerpującym zestawieniem wszystkich druków polskich o Maryi. Mimo braków, można jednak ta dziełko uważać za dość udaną i pożyteczną próbę bibliografii m aryjnej, po której czetkamy na coś doskonalszego w kraju. Dla uzupełnienia bi bliografii nie zamierzam podawać wszystkich pominiętych pozycji, które liczą się na setki. Przecież autorzy bibliografii, jak sami podają, oparli się tylko na niektórych źródłach im dostępnych, zwłaszcza na urzędo wych bibliografiach polskich i na rocznikach bibliograficznych, które okazały się mało dokładnymi oraz na dwóch czasopismach: Ateneum Kapłańskim i Przeglądzie Powszechnym. A to za mało. W iele artykułów jest rozrzuconych w różnych czasopismach. Są one nieraz drobne, ale wartościowe dla całości mariologii polskiej. Przy tym zaznaczyć należy, że i te dwa poważne czasopisma: Ateneum Kapłańskie i Przegląd P o wszechny nie są dokładnie wykorzystane. Oto niektóre braki i opusz czenia np. ks. Tad. Karyłowskiego, Stabat Mater, Przegl. Powsz. 159 (1923) 236— 251; ks. H. Cichowskiego, Nestorianizm i sob. efeski, Przegl. Powsz. 189 (1931) 155— 172; ks. Charszewskiego (recenzja maryjna), A te neum Kapł. 26 (.1930) 312— 322. co wydrukowano o Maryi w ostatnim 50-leciu. T R Z Y TO M Y „STU D IÓ W TEO LO G ICZNYCH ". Żulińskiej, Królow a Korony Polskiej, Lw ów 1906, s. 78; o. Lud. Perroy S. J., Ifajśw. Panna wzorem pokory i cichości. Przekł. z franc. Kraków 1934, s. 230. Z książek maryjnych ks. Majewskiego opuszczono trzy pożyteczne, mia nowicie: Orędowniczko nasza w życiu i przy śmierci, Warszawa 1935, s. 128; Matka łaski Bożej, Warszawa 1936, s. 177; Przyczyna naszej ra dości, Warszawa 1938, s. 145. Nie ma też wzmianki o wydanym dziele o teologu i poecie: St. Sarbiewski M. C. S. J. Poloni poemata omnia. Sta- raviesiae 1911 s. L X IV + 624. Opuszczono niektóre druki wydawnictw dokumentów papieskich, do tyczące N. M. Panny, ogłoszone w języku polskim, w ostatnim 50-leciu, a szczególnie ważne dla polskiej mariologii i jej rozwoju. Pod nr 61— 64 w Bibliografii podano jeden tylko drukowany w Polsce dokument ma ryjny, mianowicie P. Popiela Leon XI I I i jego Encykliki o Różańcu M ariań skim, Przegl. Pol. 176, (1910) 21— 44,191— 209. Trzy inne podane dokumenty maryjne są wydane poza granicami Polski. A tymczasem i w kraju ma my dawniej i w ostatnich latach powojennych drukowane tłumaczenia dokumentów papieskich, dotyczące czci N. M. Panny. Np. encyklikę pap. Piusa X I-go o Różańcu św. przetłumaczył ks. bp St. Okoniewski (Pel plin 1937, s. 8). Posiadamy tłumaczenie Bulli: „Ineffabilis Deus“ pap.f Piusa IX -go, (Maryawita I — 1904, s. 313—315, 346— 347, 455— 458, 458— 489) jak i również polski przekład encykliki pap. Piusa X-go: „Ad diem illum laetissimum“ ks. A. Szaniawskiego (Przegl. Kat. 1904, s. 225— 227, 241— 244, 257— 260, 273— 276, 289— 291, 301— 308). Ten sam dokument ma ryjny, ukazał się po polsku na łamach „M aryaw ity" (I — 1904, 360— 364, 391— 394, 423— 426). Bulla dogmatyczna o Wniebowzięciu N. M. P. „M u - nificentissimus Deus“ pap. Piusa X II-go, została wydrukowana w H o mo Dei 20 (1951) 26— 40. W ydrukowały ten ważny dokument m aryjny i inne czasopisma np. Tyg. Powsz. 1950, nr 299. Wszystkie ważniejsze dokumenty papieskie, dotyczące Najśw. Dziewicy, były żywo i bezpo średnio omawiane i drukowane w kraju. Dobrze, że uwzględniono w bibliografii czasopisma i kalendarze ma riańskie (nr 940— 1027) i objęto również stowarzyszenia mariańskie oraz bractwa różańcowe (nr 1028— 1129), lecz nie podano jednak i tu pełnego ich zestawienia. Opuszczono cały szereg kalendarzy maryjnych m. i. K a lendarze Cudownego Medalika, które ukazały się w Krakow ie w la- tarh 1930— 1938. W bibliografii pominięto liczne druki maryjne, dotyczące historii kultu maryjnego w Polsce. W yliczym y tylko ważniejsze: O. B. Łubień ski, H'storia Cudownego Obrazu M atki Boskiej Nieustającej Pomo~y, Kraków 1916, s. V I + 335; St. Fischer, Matka Boska Bocheńska i Jej T R Z Y TO M Y „STU D IÓ W TEO LO G ICZNYCH ". Panny na soborze w Efezie, Głos Kar. 5 (1931) 345— 349; O. J. Puchalik C. SS. R., Dogmatyczna podstawa pośred nictwa M. Bożej, Przegl. Kat. (1924) 724— 726; St. Windakiewicz, Trady cja Mariańska w Polsce, Sodalis Marianus 30 (1931) 113— 116; K. Gołba, Było to w Efezie, Sodal. Mar. 30 (1931) 138— 145; ks. Al. Zychliński, Nasz udział w życiu Bożym. II. O Chrystusie historycznym i o Maryi Matce Kościoła, Głos Karmelu 16 (1947) nr 3, s. 4— 5; Ed. Ostachowski, 300-lecie Karm elu w Czernej i jego znaczenie dla Polski, Głos Karm elu 7 (1981) 182— 192; Tenże, Ojciec Rafał (Kalinow ski) czciciel Najśw. Maryi Panny, Głos Karmelu 8 (1934) 387— 390; ks. Fr. Dziasek, Najśw. Maryja Panna w Ciele Mistycznym Chrystusa, Wiad. Dusz. 8 (1948) 133— 144; ks. J. K o walski, Serce Ciała mistycznego, Meteor 31 (1939) 101— 112; ks. Lech Kaczmarek, Pełnia łaski w życiu Bogarodzicy, Wiad. Duszp. 8 (1952) 241— 245; Encykliki maryjne, Wiad. Duszp. 7 (1951) 106— 108; ks. J. P i wowarczyk, Przed Dogmatyzacją Wniebowzięcia, Tyg. Powsz. 6 (1950) nr 36, s. 2; ks. St. Franki, Wniebowzięcie Bogarodzicy, Gazeta Kość. 45 (1938) 514— 516, 532— 534, 548— 550; ks. A. Kwieciński, Wniebowzięta K ró lowa w sztuce, Królowa Apost. 37 (1951) 64— 66; ks. A. Słomkowski, K ult Serca Maryi — rec. dzieła O. A. Krupy: K ult Serca Maryi, Tyg. Powsz. 4 (1948) nr 45, s. 11; O. Neuman, Z dziejów kultu N. Serca Maryi, Głos Karmelu 20 (1951) 56— 62; ks. Al. Prumbs, Dogmatyczne naświetlenie kultu Niepokalanego Serca Maryi. Wiad. Duszp. 3 (1947) 181— 185; Rozwój kultu Najśw. M. Panny, Wiad. z Diec. Łódz. 20 (1946) 22— 38; ks. J. W oj- tukiewicz, Najśw. Marya Panna, Ku Szczytom, Wilno I. grudz. 1938 — styczeń 1939, s. 9— 17; J. D. — Papież Niepokalanej (Pius X ) błogosła wionym, Rycerz Niep. 30 (1951) 171— 173; ks. Fr. Myszka CM., Niepokalane Poczęcie a Kościół Wschodni, Meteor 30 (1938) 198—205; Jan Dobraczyń ski, Matka Boża w literaturze współczesnej, Rycerz Niep. 31 (1952) 44— 45; ks. K. Szweda, Bł. Pius X o wszechpośrednictwie N. M. Panny, Rycerz Niep. 31 (1952) 262— 264; ks. St. Franki, Współodkupicielka — z zagadnień M ariologii współczesnej, Gazeta Kość. 46 (1939) 322— 324; O. R. Kostecki, Matka Odkupiciela i Matka Nasza, Królow a Apost. 3 7 (1951) 63, 67; Pośrednictwo Matki Boskiej, Homiletyka 32 (1914) 557; ks. T R Z Y TO M Y „STU D IÓ W TEO LO G ICZNYCH ". Dział o stowarzyszeniach mariańskich jest niezu pełny. Pominięto np. Stowarzyszenie Dzieci Maryi, które posiadało w Polsce swoje własne czasopismo m aryjne pt.: Rocznik Mariański, po Dobrze, że uwzględniono w bibliografii czasopisma i kalendarze ma riańskie (nr 940— 1027) i objęto również stowarzyszenia mariańskie oraz bractwa różańcowe (nr 1028— 1129), lecz nie podano jednak i tu pełnego ich zestawienia. Opuszczono cały szereg kalendarzy maryjnych m. i. K a lendarze Cudownego Medalika, które ukazały się w Krakow ie w la- tarh 1930— 1938. Dział o stowarzyszeniach mariańskich jest niezu pełny. Pominięto np. Stowarzyszenie Dzieci Maryi, które posiadało w Polsce swoje własne czasopismo m aryjne pt.: Rocznik Mariański, po 336 R E C EN ZJE i S P R A W O Z D A N IA R E C E N Z JE i S P R A W O Z D A N IA 337 święcony szerzeniu czci Niepokalanie Poczętej oraz sprawom stowarzy szeń Dzieci Maryi i Cudownego Medalika (miesięcznik), redagowany przez Zgr. X X Misj. Kraków Stradom 4. Rocznik mariański wychodził od końca r. 1924— 1939. Wśród artykułów są godne wzmianki: ks. Fr. Smidody, Kościół i klasztor OO Karm elitów na Piasku w Kraków.e i jego cud. obraz M. Boskiej (RM. 3 (1927) 367— 375); ks. Wl. Stawinogi, K ult Matki Bożej w literaturze polskiej, szczególnie w poezji (RM. 3 (1927) 164— 168, 208— 211, 256— 261); ks. Tad. Petrzyka, Początek czci dla Matki Bożej Lourdeńskiej w Polsce (RM. 1 (1925) 18— 23. Rocznik Ma riański był tym dla Dzieci Maryi, czym dla Sodalicji Mariańskich Wiara i Zycie oraz Sodalis Marianus. przeniósł się i przesunął do czasopism. Był czas, kiedy można było je dynie wypowiedzieć swoją myśl w czasopismach. Dobrze się więc stało, że włączono do bibliografii maryjnej małe artykuły i krótkie recenzje, a zwłaszcza pisane przez fachowych teologów. Przyszła bibliografia ma riologiczna i maryjna musi jak najszerzej uwzględnić tego rodzaju lite raturę maryjną. Z drobnych artykułów i recenzji podajemy również nie które ważniejsze uzupełnienia: ks. F. Gryglewicz, Jerozolimska tradycja Wniebowzięcia N. M. Panny, Homo Dei 20 (1951) 40— 45; ks. St. Abt., Nc 1500-lecie Soboru Efeskiego, Przegl. Kat. 69 (1931) 17— 18; Celina Stoń- ska, Ruch O xfordzki a nabożeństwo do M atki Najśw. Przegl. Kat. 69 (1931) 22— 24; ks. L. Maciejewski, O cnotach Maryi, Maryawita (I — 1903) 25— 26, (I — 1904) 75— 77, 172— 173, 426— 428; O. Eugeniusz, Dogmat Bo skiego Macierzyństwa Najśw. T R Z Y TO M Y „STU D IÓ W TEO LO G ICZNYCH ". Bączek, Krytyczna recenzja o jednym z wielkich cudów współczesnych Piotra Rudera, Homiletyka 25 (1910) 305— 313; ks. Fr. Błotnicki, H isto ria nabożeństwa majowego. Gazeta Kość. 45 (1938) 274— 275); O. R. K o stecki, Najśw. M aryja Panna na tle dogmatów, Królowa Apost. 35 (1949) 67— 68, 84—86; ks. Z. Golian, Wykład Pozdrowienia Anielskiego, Homi letyka 20 (1908) 330— 336; O. R. Kostecki, Matka Najśliczniejsza, Róża Duch. 7 (1950) 178— 180; ks. Fr. Dziasek, Matka Jezusa Maryja, Królowa Apost. 37 (1951) 43— 45; O. R. Kostecki, Znak życia na wzór Maryi, Głos Karmelu 20 (1951) 196— 199; ks. A. Fiat (Generał Zgrom. XX. Misj. z Paryża), Konferencja o Niep. Pocz. Maryi, wygłoszona i wydana w Krakowie nakł. SS. Mił. 1904, 4-a s. 10; O. R. Kostecki, Poświęcenie się Niepokalanemu Sercu Maryi, Róża Duch. 51 (1949) 243— 244, 341— 342; ks. bp Stefan Wyszyński, List pasterski na dzień poświęcenia Narodu Polskiego Niepokalanemu Sercu Maryi, Wiad. Diec. Lub. 23 (1946) 285— 293; Tenże, List pasterski o codziennym odmawianiu Różańca świętego, tamże 24 (1947) 290— 293; ks. kard. Stefan Wyszyński, Przemówienie R ó żańcowe w czasie uroczystości Różańca na Bielanach. 15 VI I I 1951 r. R y cerz Niep. 30 (1951) 230— 233. rowski, O czci N. M. Panny — praca dogmatyczna, Maryawita (I— 1903) 9— 12, (1— 1904) 104— 106, 135— 136, 200—201, 233— 235, 263— 268, 294— 296, 328— 330, 461— 463, 525— 526, 585— 586; ks. J. Matulewicz, O dogmacie N. M. P. Maryawita (1— 1903) 7— 9, (1— 1904) 56— 58, 168— 169, 248— 249, 782. W Bibliografii Szostkiewicza-Wesołego pod nr 229 i 230 są umiesz czone dwa manuskrypty, jeden z podaniem nazwiska autora, a drugi bez podania nazwiska. Nie ulega wątpliwości, że rękopisy nie należą do bibliografii w ścisłym tego słowa znaczeniu. Takich manuskryptów w ostatnich czasach wiele czeka na wydrukowanie. Należy także wspomnieć o błędach drukarskich, które się wkradły, do samej książki wzgl. do spisu nazwisk np. zamiast właściwego nazwi ska Smoroński czytamy Smorkoński, (s. 1) albo zamiast Skimborowicz jest błędnie Skimbrowicz (s. 24), a zamiast G. Augustynik jest G. Augu styniak (s. 25 i 45) itd. Podobnie i w spisie nazwisk „Index analiticus'1 notuje na s. 68: Fudalewski Wł., 470 gdy tymczasem jest ta pozycja pod nr 490. Żaczek nazywa się Żaczek itd. Są to rzeczy nieuniknione w tak drobiazgowej pracy i nie przedstawiają braków istotnych. T R Z Y TO M Y „STU D IÓ W TEO LO G ICZNYCH ". Podo bnie jak i nieścisłości w podawaniu dokładnym liczby stron itp. powinny być w przyszłości uzupełnione. Najważniejszy zarzut dotyczy dużej ilości opuszczonych pozycji maryjnych. Powstaje więc pytanie, jak należy uzupełnić bibliografię Szostkiewicza-Wesołego jak również i W. Bruchnalskiego? Czy należy podjąć się opracowania obszernej cało ści: Bibliografia mariologiczna i maryjna polska od wynalezienia druku do naszych czasów? Czy też może tylko wydać trzecią bibliografię pt.: Uzupełnienia do bibliografii W. Bruchnalskiego i Szostkiewicza-Wesołego? Czy wreszcie wydać tylko bibliografię mariologiczną ściśle naukową X X -go wieku? 3) W zór ostatniej bibliografii, którą omawiamy, z pew nymi zmianami, należy przy następnych wydaniach zatrzymać i udo skonalić. Na doskonałą bibliografię mariologiczną, jako na dobry prze wodnik po literaturze mariologicznej w Polsce, czekają polscy teologo wie, a na bibliografię maryjną wydaną w kraju czekają zarówno ka płani jak i czciciele Maryi w Polsce. Oto krótki szereg niektórych tylko tzw. „małych artykułów1', w któ rych są nieraz wartościowe wypowiedzi dla mariologii polskiej. Warto wiedzieć i o tych małych drukach, zwłaszcza gdy pisali je ludzie o du żej wiedzy teologicznej i maryjnej. Do powyższych braków dołączmy _ pominięte jeszcze druki maryjne o większych rozmiarach, czy to popularno-naukowe czy też tylko po pularne, czy wreszcie ściśle naukowe. Podajemy niektóre z ważniej szych opuszczeń: ks. Cz. Pacuszka, Matka Boska i matka ludzi, Płock 1946, s. 96; ks. M. Nassalski, Upomnienie Duchowne, Włocławek 1903, s. 424; ks. P. Mańkowski, Ecce Mater tua, Kraków 1916 s. XI, 239; ks. Fr. Jarniński, Wykład Litanii Loretańskiej, Płock 1903, s. 364; ks. P. Kurtyka CM., Fatima. Sławne Objawienie Niepokalanej z r. 1917. Z dodatkiem dziewięciu kazań i modlitw na nowennę do N. M. P. Fa timskiej oraz Niepokalanego Serca Maryi. Erie Pa 1950, s. 230; ks. G. Augustynik, Pielgrzymka Jubileuszowa do miejsc wsławionych cudami Najświętszej M aryi Panny czyli Nabożeństwo majowe na uczczenie 50- letniego Jubileuszu Dogmatu Niepokalanego Poczęcia N. M. Panny, Kielce 1904, s. 389; O. B. Smyrak, Życie mariańskie Karmelu, Kraków 1938, s. 127; ks. W. Bogacki, Chwała Maryi, Łomża 1938, s. 238; ks. Ski biński, Miesiąc Maryi Saletyńskiej (wyd. Il-ie), Dębowiec 1937, s. 170; ks. Fr. Kuligowski, Czytania majowe na temat pieśni polskich o N. M. Pannie w zastosowaniu do czasów obecnych (wyd. Il-ie), Warszawa 1948, s. 239 + 1 nlb. Wł. Szafrański, Niepokalana Tw oim wzorem, Włocławek 1946, s. 199; ks. W. Bogacki, Marja wzór młodzieży, Łomża 1927, s. 160; ks. Br. Obuchowicz, Sposób odmawiania i śpiewania Różańca św. wyd. V. T R Z Y TO M Y „STU D IÓ W TEO LO G ICZNYCH ". Gdy jest mowa o zasłużonym i wartościowym czasopiśmie Sodalis Marianus, to trzeba zaznaczyć, że i tu są opuszczone liczne artykuły maryjne, jak np. M. Bartynowskiego, Leon XI I I Papież, czciciel Matki Bożej, Sodal. Mar. 3 (1904) 3, 75, 213; ks. J. Górki, Pius X jako czciciel Maryi, Sodal. Mar. 7 (1908) 247, 273 oraz liczne artykuły o. J. Godaczew- skiego. W iele materiału maryjnego można znaleść w miesięczniku Hom ile tyka. Biorę dla przykładu rocznik 1904-ty, gdzie mamy wartościowe artykuły maryjne, między innymi takie: Posłannictwo i godność Boga rodzicy, ks. abpa J. Bilczewskiego (s. 1— 17); Historya i wykład Pozdro wienia Anielskiego, ks. Wł. Chotkowskiego (s. 18— 37); Marya i Mara ks. bpa H. Kossowskiego (s. 38— 60); Różaniec. Przyczynek do jego hi- storyi i znaczenia (is. 61— 70); Z dania Ojców Kościoła i Świętych Pań skich o Najśw. Niepokalanej Bogarodzicy (s. 86— 96); Kazanie na u ro czystość Oczyszczenia N. M. P. (s. 333— 350); Jubileusz nadzwyczajny ku czci Niepokalanego Poczęcia N. M. Panny (s. 117— 130); Matka Dobrej Rady (s. 327— 337). O artykułach tych nie wspomina bibliografia ma ryjna. W iele pominiętego materiału maryjnego można znaleść w Przeglądzie Katolickim. Weźmy np. rocznik jubileuszowy 1904. Znajdujemy w nim dobry artykuł ks. Ł. Janczaka, O czci N. M. Panny, a osobliwie o czci Niepokalanie Poczętej N. Maryi Panny (s. 6— 7, 22— 24, 40—41, 56— 57, ''O— 71, 90— 91, 105— 106, 120— 122). Jest tu także ciekawy artykuł spra wozdawczy M ikołaja Krzystofowicza pt.: Kongres ku czci Matki Bożej (Przegl. Kat. (1904), s. 667— 670, 685— 688, 718— 719, 733— 735, 750— 752, 766, 799— 800, 811— 813, 828— 830). Trudno wyliczać wszystkie pozycje maryjne opuszczone, zwłaszcza, gdy się ma na myśli nie same tylko druki naukowe, ale także popularne broszury wzgl. dzieła popularno-naukowe, artykuły, iecenzje, kazania maryjne oraz małe artykuły, rozrzucone po różnych czasopismach. P o mysł bowiem, wykorzystania czasopism i zawartych w nich drobnych artykułów i recenzji maryjnych w bibliografii Szostkiewicza-Wesołego jest pożytecznym. W iemy przecież, że w obecnym stuleciu powstało w iele czasopism i cały ciężar podawania nauki maryjnej i nauczania 22 Nasza Przeszłość RE C E N ZJE i S P R A W O Z D A N IA RE C E N ZJE i S P R A W O Z D A N IA 338 339 J. 3 O. Joachim Bar, franciszkanin, jest Zdania, że należy wpierw w y dać bibliografię mariologiczną X X w. Jest ona najbardziej potrzebną dla teologów i mariologów polskich. O. H i e r o n i m Eug . W y c z a w s k i OFM., Wprowadzenie studiów w archiwach kościelnych, Warszawa 1956, str. 159. T R Z Y TO M Y „STU D IÓ W TEO LO G ICZNYCH ". W międzyczasie jednak prowadzona inten sywnie akcja odzyskania archiwaliów dała pozytywne wyniki w postaci rewindykacji „akt dawnych", o czym wspominam znowu w innym arty kule pt. Odnaleziony rękopis, drukowanym w „Orędowniku diecezji chełmińskiej" R. 6 (1950) N r 1 s. 29— 35. Dotyczy on najstarszej księgi tutejszego archiwum diecezjalnego, bo t. zw. „Codex epistolaris" bisku pów chełmińskich z lat 1574— 1606, i z tego tytułu mógłby być uwzględ niony w „wskazówkach bibliograficznych" tak na s. 8 jak 84. nizacji kościelnej (7— 45), 2) kancelarii (46— 70), 3) zawartości archiwów (71— 109) i 4) treści ksiąg archiwalnych (110— 152). nizacji kościelnej (7— 45), 2) kancelarii (46— 70), 3) zawartości archiwów (71— 109) i 4) treści ksiąg archiwalnych (110— 152). W rozdziale pierwszym zaczyna od zagadnienia terytorialnej organi zacji diecezjalnej, wskazując na poważne zmiany granic diecezji, jakie dokonały się w Polsce od chwili rozbiorów, co pociągnęło też za sobą przesunięcia w sieci archiwalnej. W przesunięciach tych winien orien tować się poszukujący źródeł w archiwach kościelnych, żeby mógł „tra fić do właściwych archiwów". Stara się Autor podać pod tym w zglę dem praktyczne wskazówki, ilustrując je szeregiem przykładów. Przy tym należało by jednak zwrócić uwagę i na to, że niektóre diecezje i ka pituły zachowały wprawdzie swoje archiwa, ale w niepełnym składzie zespołów, bo w niektórych wypadkach (mam na myśli diecezję cheł mińską) część archiwaliów zdeponowano ongiś w archiwach państwo wych, skąd nieraz wogóle nie wróciły. Stąd historyk w swoich poszu kiwaniach źródeł do danej diecezji winien pamiętać także i o archiwa liach kościelnych znajdujących się w zbiorach państwowych. Po omówieniu zagadnienia układu terytorialnego przechodzi Autor do „organizacji Kościoła również od strony kościelnych władz i urzę dów" (18), a więc: biskupa, administratora, sufragana, archidiakonatów, dekanatów, parafii, oficjalatów, kapituł katedralnych i kolegiackich, kolegiów niższego duchowieństwa i seminariów duchownych. Przy każ dym daje wyjaśnienie charakteru i zakresu ich działalności z częściowym uwzględnieniem ich historycznego rozwoju. Objaśnianie poszczególnych urządzeń kościelnych czy procedury sądowej nie należy zasadniczo do pojęcia heurystyki. Z uwagi jednak na znaną dziś powszechnie „słabą stronę" przygotowania studentów do pracy naukowej takie wyjaśnienia będą niewątpliwie z wielką korzyścią. W rozdziale drugim omawia Autor sposób urzędowania poszczególnych władz i instytucji oraz strukturę ich kancelarii. Najobszerniej traktuje 0 urządzeniu kancelarii biskupiej, podając jej genezę i czynniki, jakie wpływały na jej ukształtowanie, oraz zadania i czynności jej urzędników. W ramach kancelarii urzędów diecezjalnych omówił także krótko kan celarię oficjała stołecznego, oficjałów okręgowych i parafii. T R Z Y TO M Y „STU D IÓ W TEO LO G ICZNYCH ". Warszawa 1904 8-ka, s. 554; ks. Edm. Crapez, Chwalebna Kata rzyna Laboure. Tłum. z franc. Warszawa 1913, s. 222; ks. W. Niem ie- Ks. Franciszek Bracha C. M. O. H i e r o n i m Eug . W y c z a w s k i OFM., Wprowadzenie d studiów w archiwach kościelnych, Warszawa 1956, str. 159. Powyższa praca ukazała się jako N r 1 Skryptów Wydziału Prawa Kanonicznego Akademii Teologii Katolickiej w Warszawie. Zawartość jej ujął autor w cztery rozdziały, w których mówi kolejno o: 1) orga R E C E N Z JE i S P R A W O Z D A N IA R E C EN ZJE i S P R A W O Z D A N IA 341 340 ksiąg są zestawione następująco: Episcopalia, Officialia, Administra- torialia, Libri functionum pontificalium, Libri visitationum i na ko niec wszystkie inne" (s. 91). Przykładem odmiennego układu może być n. p. archiwum diecezji chełmińskiej, w którym „Acta dawne ułożone są według historycznego rozwoju diecezji (terytorialnego), na: Culmen- sia et Pomesaniensia, Gedanensia, Camienensia, a ponadto osobne działy tworzą: Monastica, Varia, Diplomata et epistolae", a w poszczególnych tych zespołach zachowany jest wewnętrzny układ nie według treści, a według lat = cfr. mój artykuł w Zapiskach Tow. Nauk. w Toruniu t. X I (1939— 45), Nr 5— 12, str. 125 nn. pt. Archiwa kościelne na P o morzu ich stan i potrzeby, w którym cmawiam nie tylko zawartość archiwów pelplińskich, ale informuję także o źródłach do historii diecezji chełmińskiej i w innych archiwach zawartych. Jest to najnowsza po zycja dotycząca tego archiwum, a której Autor nie uwzględnił w swoich zestawieniach bibliograficznych na str. 8, gdzie wyszczególnia przesta rzały już dawno artykuł K a u f m a n n a Die Archive in Pelplin (1917), a także nieaktualny już dzisiaj szkicowo tylko ujęty przegląd archiwów pelplińskich w dysertacji ks. Glemmy z r. 1926. Wspomniana zaś przez autora pozycja pt. Archiwa diecezji chełmińskiej jest jedynie notatką informacyjną z Pierwszego Ogólnopolskiego Zjazdu Historyków Pomo rza i Prus w Toruniu 1947 r., która nie zawiera nic więcej jak tylko „krótkie dzieje w o j e n n e " tychże archiwów, a mianowicie, że wszystkie archiwalia pelplińskie wywiezione zostały przez Niemców i że „dotąd (tzn. 1947 r.) nie wróciły". V II. Varia rescripta, petitiones, gravamina) litterae.... V II. Varia rescripta, petitiones, gravamina) litterae.... V II. Varia rescripta, petitiones, gravamina) litterae.... C. Bullae Summorum Pontificum, litterae Curiae Romanae, privi- legia regum et episcoporum, diversa (czyli odpowiada działowi „Diplo- mata et epistolae" w archiwum diecezjalnym). Z uwagi na to nie w y starcza znajomość powszechnie przyjętych podziałów, ale badacz chcący korzystać z pewnego określonego archiwum, powinien zapoznać się bli żej z jego odrębną organizacją. g j y ( ) Na str. 30 w bibliografii s z p i t a l n i c t w a pominięto ważną roz prawę R i n k J., Die christliche Liebestatigkeit im Ordenslande Preus- sen bis 1525, Freiburg im 3r. 1911. W rozdziale czwartym poświęconym omówieniu t r e ś c i ksiąg ar chiwalnych, Autor traktując łącznie księgi biskupie, administratorskie i oficjalskie, rozpatruje ich układ, wpisy dokumentów, protokóły praw nych czynności oraz przewód sądowy, przy czym najobszerniej omawia to ostatnie zagadnienie, objaśniając szczegółowo całą procedurę sądową (czy nie zbyt obszernie, jak na heurystykę?). Następnie oddzielnie opi suje księgi: funkcji pontyfikalnych, gospodarcze biskupie i kapitulne, beneficjów i retaksacji, wizytacyjne, protokółów sesji i czynności ka pitulnych, instalacji, a w końcu zbiory listów, księgi metrykalne i „inne", jak księgi testamentów, zbiory statutów kapitulnych 1 ustaw synodal nych. — Odnośnie do ksiąg metrykalnych, co do których zamieścił Autor słuszną uwagę, że „należy pamiętać, że w niektórych diecezjach zgro madzono sporo starszych ksiąg metrykalnych i innych rękopisów pa rafialnych w archiwach diecezjalnych" (s. 95), należałoby również za znaczyć, że z terenów „włączonych" ongiś przez hitlerowców do Rzeszy Niemieckiej zabrano prawie wszystkie starsze (do końca X V III w.) księgi metrykalne i wywieziono je w głąb Rzeszy, skąd po większej chyba części jeszcze nie wróciły. Jeśli chodzi o księgi metrykalne z te renu diecezji chełmińskiej, to pomimo stwierdzenia, gdzie się znajdują i pomimo kilkakrotnych starań nie udało się, jak dotąd, ich odzyskać. Poszczególne zagadnienia zaopatrzył Autor w „ w s k a z ó w k i bi - b l i o g r a f i c z n e“ , jakoże znajomość bibliografii jest nieodzowna w każdej dziedzinie naukowej, a więc i poszukiwaniach archiwalnych. Odnośnie do tych wskazówek pragnąłbym wnieść kilka poprawek i uzupełnień, mając na uwadze głównie sprawy diecezji chełmińskiej. Na str. 39 dodać trzeba dwa najnowsze opracowania odnośnie do K a p i t u ł : chełmińskiej P a n s k e P. T R Z Y TO M Y „STU D IÓ W TEO LO G ICZNYCH ". 21 przy k a t a l o g a c h b i s k u p ó w brak pracy W o e l k y C. P., Der Katalog der Bischófe von Culm (Zeitschrift fur die Gesch. u. Altertumskunde Ermlands t. VI, Leipzig 1878); oraz na str. 22 przy diec. Wrocławskiej: D. K a s t n e r a , Breslauer Bischófe Breslau (1929). Przy kwestii p r y m a s o s t w a wymienićby jeszcze można: O k o n i e w s k i Stanisław ks., K to jest Prymasem Polski, Poz nań (1919). Pozycji tej nie ma też i w Polskiej Bibliografii prawa ka nonicznego OO. Bara i Zm arza) gdzie obok poważnych artykułów w y mieniono szereg mniejszych znacznie broszur (N r 901— 916). I tak na str. 15 zestawienie m a p diecezjalnych uzupełnić należy po zycjami: P a w ł o w s k i J. N., Kartę der Diócesen Culm und Ermland, Graudenz 1890; K u j o t Stanisław ks., do dzieła „K to założył parafie..." (cytowanego przez Autora na s. 29) dodał dwie mapy historyczne; tak samo do pracy zbiorowej pt. Diecezja chełmińska (cyt. na s. 11) dołą czono mapę opracowaną w Instytucie Kartograficznym im. E. Romera we Lw ow ie (1928); S c h e l e r B., Album der Diócese Culm z r. 1856 (egz. archiwalny); oraz N. N., Album, diecezji chełmińskiej z r. 1818 (egz. arch.). Na str. 21 przy k a t a l o g a c h b i s k u p ó w brak pracy W o e l k y C. P., Der Katalog der Bischófe von Culm (Zeitschrift fur die Gesch. u. Altertumskunde Ermlands t. VI, Leipzig 1878); oraz na str. 22 przy diec. Wrocławskiej: D. K a s t n e r a , Breslauer Bischófe Breslau (1929). Przy kwestii p r y m a s o s t w a wymienićby jeszcze można: O k o n i e w s k i Stanisław ks., K to jest Prymasem Polski, Poz nań (1919). Pozycji tej nie ma też i w Polskiej Bibliografii prawa ka nonicznego OO. Bara i Zm arza) gdzie obok poważnych artykułów w y mieniono szereg mniejszych znacznie broszur (N r 901— 916). T R Z Y TO M Y „STU D IÓ W TEO LO G ICZNYCH ". Osobno scharakteryzował kancelarie kapituł katedralnej i kolegiackiej. Zasoby archiwów k a p i t u l n y c h podzielił Autor aż na 18 działów, a mianowicie: dyplomy pergaminowe, kopiarze dokumentów, protokóły sesji i czynności kapitulnych, księgi instalacji, statuty kapitulne, księgi obowiązków mszalnych i chórowych, księgi testamentów, księgi gospo darcze kapituły, inwentarze katedry i skarbca katedralnego, księgi gospodarcze stołu biskupiego, księgi beneficjów i retaksacji, statuty sy nodalne, księgi wizytacyjne, księgi czynności pontyfikalnych, zbiory li stów, opracowania archiwalne, księgi różnych fundacji, zbiory archi walne niższego duchowieństwa katedralnego. I tu słusznie Autor zazna czył, że „grupując w pewne zespoły rękopisy archiwów kapitulnych dla lepszej wśród nich orientacji, nie chcemy wcale twierdzić, że taki po dział istnieje bezwzględnie w e wszystkich zbiorach kapitulnych" (s. 105— 6). Przykładem na to może być tutejsze archiwum kapitulne, gdzie W rozdziale trzecim pisze o zawartości poszczególnych archiwów, a najobszerniej o diecezjalnych i kapitulnych, wskazując na rodzaj 1 układ akt, przy czym opiera się przede wszystkim na materiale kra kowskim. W zbiorach dawnych archiwów biskupich (dziś diecezjalnych) wyróżnia dziewięć działów-zespołów, a mianowicie: 1) Acta episcopalia, protocolla, terminarzej 2) Księgi czynności pontyfikalnych, 3) Księgi go spodarcze dóbr stołowych, 4) Księgi beneficjów i retaksacji, 5) Księgi wizytacyjne, 6) Zbiory listów, 7) Formularze, 8) Archiwalne opraco wania, 9) Fascykuły akt luźnych. Słuszna jest uwaga, że „układ ksiąg w diecezjalnych archiwach jest najrozmaitszy" i że „najczęściej zespoły RE C E N ZJE i S P R A W O Z D A N IA RE C E N ZJE i S P R A W O Z D A N IA 342 343 I tak na str. 15 zestawienie m a p diecezjalnych uzupełnić należy po zycjami: P a w ł o w s k i J. N., Kartę der Diócesen Culm und Ermland, Graudenz 1890; K u j o t Stanisław ks., do dzieła „K to założył parafie..." (cytowanego przez Autora na s. 29) dodał dwie mapy historyczne; tak samo do pracy zbiorowej pt. Diecezja chełmińska (cyt. na s. 11) dołą czono mapę opracowaną w Instytucie Kartograficznym im. E. Romera we Lw ow ie (1928); S c h e l e r B., Album der Diócese Culm z r. 1856 (egz. archiwalny); oraz N. N., Album, diecezji chełmińskiej z r. 1818 (egz. arch.). Na str. T R Z Y TO M Y „STU D IÓ W TEO LO G ICZNYCH ". w aktach dawnych utrzymał się dotychczasowy (niewątpliwie przesta rzały) układ datujący od X IX w., a dzielący całość na: A. Libri in- troligati (z wewnętrznym układem chronologicznym); B. Volumina ectorum „Acta“ z podziałem rzeczowym, na: I. Generalia, statuta, diversa... II. Cultus divinus, fundationes, facultates... II. Cultus divinus, fundationes, facultates... III. Proventus et emolumenta, possesio bonorum, diversa negotia pe- cuniaria... III. Proventus et emolumenta, possesio bonorum, diversa negotia pe- cuniaria... IV. Privilegia, tractatus, litterae pastorales, visitationes... V. Contractus, transactiones, iudicialia negotia... VI. Negotia singularium ecclesiarum... K S I Ą Ż K I N A D E S Ł A N E rzystanie z archiwów kościelnych" (6), i pod tym aspektem należy ją rozpatrzyć i ocenić. Nie objął Autor wszystkich zagadnień z tym zwią zanych, ale rozwinął „jeden tylko dział metodologii historii, a miano wicie heurystyki i to na ograniczonym odcinku, bo tylko w odniesieniu do archiwów kościelnych" (s. 153). Byłoby jednak bardzo pożądane, aby tym początkującym historykom podać nie tylko najkonieczniejsze w ia domości o sieci archiwów kościelnych i układzie ich zawartości, ale także i o s p o s o b i e k o r z y s t a n i a z ich zasobów, co również wchodzi przecież w zakres heurystyki. Pionierską swą pracą przysłużył sie Autor dobrze nauce historii, dając studentom do ręki obszerne i przystępne „vademecum“ studiów archiwalnych. Należy mieć nadzieję, że Autor wzbogaci w przyszłości naszą literaturę heurystyczną już nie „wprowadzeniem", ale pełnym podręcznikiem. rzystanie z archiwów kościelnych" (6), i pod tym aspektem należy ją rozpatrzyć i ocenić. Nie objął Autor wszystkich zagadnień z tym zwią zanych, ale rozwinął „jeden tylko dział metodologii historii, a miano wicie heurystyki i to na ograniczonym odcinku, bo tylko w odniesieniu do archiwów kościelnych" (s. 153). Byłoby jednak bardzo pożądane, aby tym początkującym historykom podać nie tylko najkonieczniejsze w ia domości o sieci archiwów kościelnych i układzie ich zawartości, ale także i o s p o s o b i e k o r z y s t a n i a z ich zasobów, co również wchodzi przecież w zakres heurystyki. Pionierską swą pracą przysłużył sie Autor dobrze nauce historii, dając studentom do ręki obszerne i przystępne „vademecum“ studiów archiwalnych. Należy mieć nadzieję, że Autor wzbogaci w przyszłości naszą literaturę heurystyczną już nie „wprowadzeniem", ale pełnym podręcznikiem. Album Studiosorum Universitatis Cracoviensis, T. V. (1720— 1780), ed. Carolus Lewicki, Cracoviae-Wratislaviae 1956, s. X II, 207. Ks. Antoni Liedtke Antemurale, T. I. Romae 1954, s. 155; T. II. ibid. 1955, s. 175; T. III. ibid. 1956, s. 167. Ateneum Kapłańskie, styczeń— luty 1957, s. 160. B a r a n C z e s ł a w C e z a r o., Sprawy narodowościowe u francisz kanów śląskich w X III w., Warszawa 1954, s. 170. F i a ł k o w s k i J ó z e f , Bp Andrzej S. Załuski, kanclerz wielki kor. na pierwszym etapie walki o polityczno-gospodarczą reformę pań stwa (1736— 1740), Warszawa 1955, nadb. V II. Varia rescripta, petitiones, gravamina) litterae.... P., Personalien der Mitglieder des Kulm er Domkapitels (Zeitschr. fur die Gesch. u. Altertumskunde Ermlands t. X X V , Braunsberg 1933— 34); i wrocławskiej: S a m u l s k i R., Untersuchungen iiber die persónliche Zusammensetzung des Breslauer Domkapitels im M ittelalter bis zum Tode des Bischofs Nanker (1341) Teil I, Weimar 1940; Na str. 76 przy i n w e n t a r z a c h brak najnowszej pozycji, miano wicie M i e n i c k i R., Inwentarze dóbr biskupstwa chełmińskiego t. I. (1646 i 1676), Fontes Tow. Nauk. w Toruniu 40 (1955); w r. 1956 ukazał się już t. II tychże inwentarzy (1723— 1747), Fontes 42. Na str. 95 przy a r c h i w a c h p a r a f i a l n y c h wartoby zaznaczyć, że na terenie diecezji chełmińskiej krótkie zestawienie ich zawartości podane zostało przy każdej parafii w pracy zbiorowej „Diecezja cheł mińska" (Pelplin 1928), wymienionej przez Autora na str. 11; Na str. 97 przy k o d e k s a c h d y p l o m a t y c z n y c h obok starego Codex Prussicus J. V o i g t ’a (1836— 61) należałoby przede wszystkim podać nowy Preussisches Urkundenbuch (Philippi-Seraphim-Hein) t. I— III, Kónigsberg 1882— 1944, a dla spraw Pomorza zachodniego w y dany najlepiej Pommersches Urkundenbuch (Klempin-Prumers-Frede- richs-Sandow), t. I— V II, Stettin 1886— 1940. Praca O. Wyczawskiego jest pierwszą tego rodzaju publikacją w na szej literaturze kościelnej, wprowadzającą do studiów archiwalnych. Autor postawił sobie jako cel: „ułatwić początkującemu historykowi ko R E C E N ZJE i S P R A W O Z D A N IA 344 Ks. ALFO N S SCHLETZ, Po dziewięciu la ta c h Roczniki Teologiczno-kanoniczne, T. III. Lublin 1956, s. 440. Słowo J. Em. Ks. Kard. S TE FA N A W YSZYŃSKIEG O , Prymasa Polski, do Redakcji „Naszej P rz e s z ło ś c i"................................ 6 T y r o w i c z M a r i a n , Drogi radykalizmu społecznego Aleksandra Puławskiego. Nadb. z Kwartalnika Historycznego 43 (1956), s. 128— 141. U r b a n W i n c e n t y , De testimoniis vitam Nankeri Episcopi W ra- tislaviensis illustrantibus, Collectanea Theologica 27 (1956), s. 250— 286 i nadb. — Leopold hr. Sedlnicki, książę biskup wrocławski (1836— 1840), Warszawa 1955, X I, 326. U r b a n W i n c e n t y , De testimoniis vitam Nankeri Episcopi W ra- tislaviensis illustrantibus, Collectanea Theologica 27 (1956), s. 250— 286 i nadb. — Leopold hr. Sedlnicki, książę biskup wrocławski (1836— 1840), Warszawa 1955, X I, 326. K A R O L GÓRSKI, Uwagi o źródłach budzisławskich . . . . 34 W a r s z a w s k i J ó z e f ks. T. J., Katolickość dzieł Mickiewicza. Cz. pierwsza kowieńsko-wileńska, Rzym 1956, s. 202. Nadb. z Sacrum Poloniae Millenium III. — Polonica z rzymskiego kodeksu nowicjuszy Towarzystwa Jezu sowego (1565— 1586), Rzym 1955, s. 205. W a r s z a w s k i J ó z e f ks. T. J., Katolickość dzieł Mickiewicza. Cz. pierwsza kowieńsko-wileńska, Rzym 1956, s. 202. Nadb. z Sacrum Poloniae Millenium III. B RO NISŁAW W ŁO D ARSKI, Salomea, królowa halicka . . . 61 TERESA KUJAW SKA-KOM ENDER, Wstęp do badań nad pismami Doroty z M ą t a w .................................................... 84 — Polonica z rzymskiego kodeksu nowicjuszy Towarzystwa Jezu sowego (1565— 1586), Rzym 1955, s. 205. Ks. W IN C EN TY URBAN, Konwersja Melecjusza Smotrzyskiego 133 Wiadomości Archidiecezjalne Warszawskie 1957, z. 1— 4. Ks. ALFO N S SCHLETZ, Ks. Jan Kanty Dąbrowski, biskup sufra- gan p o z n a ń s k i............................................................................217 W y c z a w s k i H i e r o n i m Eug . o. OFM., Wprowadzenia do stu diów w archiwach kościelnych, Warszawa 1956, s. 159. Ks. BRO NISŁAW N A TO N SK I T. J., Ks. Jan Poplatek T. J. (1903— 1 9 5 5 ).................................................................................... 238 K S I Ą Ż K I N A D E S Ł A N E z Collectanea Theologica 26 (1955), s. 46— 62. G r y g l e w i c z F e l i k s ks., Studium Pisma św. w Lubelskim Semi narium Duchownym, Lublin 1955, nadb. z Roczników Teologiczno- kanonicznych 2 (1955), s. 109— 157. J o b e r t A m b r o i s e , L ’£tat Polonais, la liberte religieuse et 1’Eglise orthodoxe au X V II-e siecle. Revue Internationale d’Histoire politiąue et constitutionnelle 1955, s. 236— 243 i nadb. K l e i n K a l i n a M a g d a l e n a , Podręczny słownik łacińsko-polski dla prawników, Warszawa 1956, s. X X X I, 224. K ł o c z o w s k i J e r z y , Dominikanie na Śląsku w X III— X IV wieku, Lublin 1956, s. X X V II, 356. L e w i c k i K a r o l , Habilitacja Ludwika Bernackiego w Uniwersytecie Jagiellońskim, Wrocław 1953, s. 22 (Odb. „Ze skarbca kultury" 1952, z. 2). Manuscriptum Gertrudae, filiae Mesconis II Regis Poloniae (ex Ante murale II), ed. Valerianus Meysztowicz, Romae 1955, s. 105— 157. M e y s z t o w i c z W a l e r i a n X., Koronacja pierwszych Piastów, Rzym 1956, s. 104 (Odb. z Sacrum Poloniae Millenium). M i c h u l k a W. ks., Ksiądz Bronisław Markiewicz, Londyn (1954), s. 341. K S IĄ 2 K I N A D E S Ł A N E 346 Pamiętniki spiskowców i więźniów galicyjskich w latach 1832— 1846, opr. Karol Lewicki, Wrocław 1954, s. X X X IX , 290. Pamiętniki spiskowców i więźniów galicyjskich w latach 1832— 1846, opr. Karol Lewicki, Wrocław 1954, s. X X X IX , 290. Pamiętniki spiskowców i więźniów galicyjskich w latach 1832— 1846, opr. Karol Lewicki, Wrocław 1954, s. X X X IX , 290. R e c h o w i c s M a r i a n , St. Jean Kanty a-t-il ete 1’auteur du com- mentaire conciliariste sur l’£vangile de St. Matthieu? Nadb. z Collectanea Theologica 26 (1955), s. 13— 45. R e c h o w i c s M a r i a n , St. Jean Kanty a-t-il ete 1’auteur du com- mentaire conciliariste sur l’£vangile de St. K S I Ą Ż K I N A D E S Ł A N E Matthieu? Nadb. z Collectanea Theologica 26 (1955), s. 13— 45. R e c h o w i c s M a r i a n , St. Jean Kanty a-t-il ete 1’auteur du com- mentaire conciliariste sur l’£vangile de St. Matthieu? Nadb. z Collectanea Theologica 26 (1955), s. 13— 45. — Studia alumnów w Galicyjskim Seminarium Generalnym (1790— — 1819), Lublin 1955, nadb. z Roczników Teologiczno-kanonicznych 2 (1955), s. 61— 107. — Karność alumnów w Galicyjskim Seminarium Generalnym 1790— 1813), Roczniki Teologiczno-kanoniczne 3 (1956), s. 119— 160. — Studia alumnów w Galicyjskim Seminarium Generalnym (1790— — 1819), Lublin 1955, nadb. z Roczników Teologiczno-kanonicznych 2 (1955), s. 61— 107. — Karność alumnów w Galicyjskim Seminarium Generalnym 1790— 1813), Roczniki Teologiczno-kanoniczne 3 (1956), s. 119— 160. S P I S T R E Ś C I S P I S T R E Ś C I R e w s k i Z b i g n i e w , Majstersztyki krakowskiego cechu murarzy i kamieniarzy X V I— X IX wieku, W rocław 1954, s. 108, 132 ilustr. — Str. Ks. ALFO N S SCHLETZ, Po dziewięciu la ta c h ................................ 5 Słowo J. Em. Ks. Kard. S TE FA N A W YSZYŃSKIEG O , Prymasa Polski, do Redakcji „Naszej P rz e s z ło ś c i"................................ 6 J. Eksc. Ks. Arcybiskup EUGENIUSZ B A Z IA K do Redakcji „Naszej Przeszłości" ............................................................... ? List J. Eksc. Ks. Biskupa CZESŁAW A FALKO W SKIEG O do Re daktora ..Naszej P r z e s z ło ś c i" ................................................... 3 Ks. JÓZEF U M IŃSKI, Plan „Dziejów Kościoła w Polsce" . . 9 K A R O L GÓRSKI, Uwagi o źródłach budzisławskich . . . . 34 B RO NISŁAW W ŁO D ARSKI, Salomea, królowa halicka . . . 61 TERESA KUJAW SKA-KOM ENDER, Wstęp do badań nad pismami Doroty z M ą t a w .................................................... 84 Ks. W IN C EN TY URBAN, Konwersja Melecjusza Smotrzyskiego 133 Ks. ALFO N S SCHLETZ, Ks. Jan Kanty Dąbrowski, biskup sufra- gan p o z n a ń s k i............................................................................217 Ks. BRO NISŁAW N A TO N SK I T. J., Ks. Jan Poplatek T. J. (1903— 1 9 5 5 ).................................................................................... 238 M I S C E L L A N E A Ks. A N TO N I LIEDTKE, W poszukiwaniu grobu błog. Juty. Przy czynek do dziejów jej k u lt u ...................................................271 Bp FRANCISZEK JOP, Przemówienie żałobne w Bazylice Wawelskiej d. 24 maja 1956 r. podczas pogrzebu śp. Ks. A rcy biskupa Michała G o d le w s k ie g o ............................................ 282 M I S C E L L A N E A Ks. A N TO N I LIEDTKE, W poszukiwaniu grobu błog. Juty. Przy czynek do dziejów jej k u lt u ...................................................271 Bp FRANCISZEK JOP, Przemówienie żałobne w Bazylice Wawelskiej d. 24 maja 1956 r. podczas pogrzebu śp. Ks. A rcy biskupa Michała G o d le w s k ie g o ............................................ 282
https://openalex.org/W3132188769	https://flore.unifi.it/bitstream/2158/1238966/1/geosciences-11-00104.pdf	English	null	A New Magma Type in the Continental Collision Zone. The Case of Capraia Island (Tuscany, Italy)	Geosciences	2,021	cc-by	12,738	A New Magma Type in the Continental Collision Zone. The Case of Capraia Island (Tuscany, Italy) Alba Patrizia Santo Department of Earth Sciences, University of Florence, Via A. La Pira 4, 50121 Florence, Italy; alba.santo@unifi.it Abstract: The Tuscany Magmatic Province consists of a Miocene to Pleistocene association of a wide variety of rock types, including peraluminous crustal anatectic granites and rhyolites, calcal- kaline and shoshonitic suites and ultrapotassic lamproites. In addition to the magma types already recognised, the occurrence of a new, distinct magma type at Capraia and Elba islands and in mafic enclaves in the San Vincenzo rhyolites has been suggested by recent studies. This particular type of magma, represented by intermediate to acidic calcalkaline rocks showing high Sr, Ba, and LREE, is restricted to the northwestern sector of the province and to a time interval of about 8 to 4.5 Ma. New data obtained on rocks from Capraia Island have allowed for the verification of the occurrence of this new magma type, the exploration of its origin and a discussion of its possible geodynamic sig- nificance. The high-Sr-Ba andesite-dacite rocks occurring in the Laghetto area at Capraia display a composition that is intermediate between adakitic and calcalkaline rocks. It is suggested that they represent a distinct type of magma that originated at mantle pressure by melting of the lower con- tinental crust, followed by mixing with other Capraia magmas. The geodynamic model that best explains the composition of the studied rocks is the thickening of the continental crust during con- tinental collision, followed by extension that favoured melting of the lower crust. Keywords: Capraia Island; adakitic rocks; lower continental crust; melting; continental collision Citation: Santo, A.P. A New Magma Type in the Continental Collision Zone. The Case of Capraia Island (Tuscany, Italy). Geosciences 2021, 11, 104. https://doi.org/10.3390/ geosciences11020104 Citation: Santo, A.P. A New Magma Type in the Continental Collision Zone. The Case of Capraia Island (Tuscany, Italy). Geosciences 2021, 11, 104. https://doi.org/10.3390/ geosciences11020104 Academic Editors: Jesus Martinez-Frias and Javier Dóniz- Páez Received: 20 January 2021 Accepted: 17 February 2021 Published: 20 February 2021 1. Introduction The Tuscany Magmatic Province (TMP) is one of the most extensively studied and best known magmatic worldwide areas. It consists of Miocene to Pleistocene (about 8.5 Ma to 300 ka) intrusive and effusive rocks, cropping out in the Tuscan Archipelago, as seamount on the northern Tyrrhenian Sea floor, and at several places in southern Tuscany and northern Latium. In addition, a small peralkaline mafic sill, occurring at Sisco (Cor- sica) and showing an older age of 14.5 Ma [1], is considered as an early part of the Tuscany Province, essentially because of its petrogenic lamproitic affinity close to some occur- rences from southern Tuscany (Orciatico, Montecatini Val di Cecina and Torre Alfina; e.g., [2] and references therein). Received: 20 January 2021 Accepted: 17 February 2021 Published: 20 February 2021 Publisher’s Note: MDPI stays neu- tral with regard to jurisdictional claims in published maps and institu- tional affiliations. Petrological and geochemical studies recognised the occurrence of several types of primary magmas, both of mantle and crustal origin. Mantle-derived magmas show a range of mafic compositions, from calcalkaline and shoshonitic to ultrapotassic lampro- itic; crustal magmas consist of peraluminous granites and rhyolites. A wide variety of hybrid compositions results from the mixing of different types of mantle-derived melts as well as between these and crust-derived end-members [3]. Copyright: © 2021 by the author. Li- censee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and con- ditions of the Creative Commons At- tribution (CC BY) license (http://crea- tivecommons.org/licenses/by/4.0/). Despite the high number of existing studies on the Tuscan Magmatic Province, there are still several petrological, volcanological and geodynamic aspects that have been not adequately considered in the past and that deserve further attention. In particular, the number of end-member magma compositions occurring in the area and their significance for the structural evolution of the northern Apennines and back-arc area deserves clarifi- cation. Such a subject is also of interest to other complex orogenic settings. www.mdpi.com/journal/geosciences Geosciences 2021, 11, 104. https://doi.org/10.3390/geosciences11020104 Geosciences 2021, 11, 104 2 of 26 Recent reconsideration of the literature data has revealed a new magma type in Tus- cany, in addition to those recognised by previous studies. This supposedly new end-mem- ber is characterised by andesitic s.l. composition and anomalously high Sr and Ba contents [3]. 1. Introduction It crops out as lavas at Capraia Island, as dykes at Elba Island and as mafic micro- enclaves in the San Vincenzo (southern Tuscany) rhyolites [4]. However, its occurrence, nature, origin and geodynamic significance are still ambiguous. g g y g g This paper reports on new data on rocks from Capraia Island, with the aims of veri- fying the occurrence of this new magma type, exploring its origin and discussing its pos- sible geodynamic significance. 2. The Tuscany Magmatic Province The Tuscany Magmatic Province (Figure 1) is a magmatic setting characterised by the occurrence of a wide variety of rock types, including peraluminous granites and rhyolites, calcalkaline and shoshonitic suites, and ultrapotassic lamproites (e.g., [5] and references therein). Acid compositions, such as the monzodiorites to leucogranites occurring at Mon- tecristo, Giglio and Elba Islands, and the rhyolitic lava flows and domes found at San Vincenzo, Roccastrada, and Tolfa-Manziana-Cerite areas, are the most abundant in the Tuscany Province. Mafic-intermediate calcalkaline-shoshonitic to ultrapotassic rocks oc- cur as small lava and hypabyssal bodies at Montecatini Val di Cecina, Orciatico, Torre Alfina, Radicofani, Monti Cimini, Amiata, Capraia Island, Campiglia, central-eastern Elba Island, and Sisco. Mafic-intermediate compositions are also found as enclaves within some granitoid and extrusive rocks. g According to Poli and Peccerillo [3] and Peccerillo [6], three different extreme end- members can be recognised within the wide range of compositions. One has a silicic per- aluminous composition and is derived from the melting of crustal metapelitic rocks, as indicated by incompatible element contents and a Sr-Nd-Pb isotope signature, all similar to pelites ([3] and references therein); the other two end-members are of mantle origin and have mafic calcalkaline-shoshonitic (CA-SHO), and lamproitic (LMP) compositions, re- spectively. Mixing between the three end-members and fractional crystallisation or assim- ilation-fractional crystallisation (AFC) of mafic magmas would be responsible for the origin of the other rocks in Tuscany. These processes generated a continuum for the main petrological and geochemical characteristics of magmatism as a whole, which makes it hard discriminating between different magma types and understanding their genetic re- lationships. Silicic rocks are characterised by variable geochemical-petrological compositions, which suggests the occurrence of two distinct groups. One group represents primary crus- tal anatectic magmas related to the melting of metasediments (micaschists, gneisses) of the Tuscany basement. In contrast, another group consists of evolved melts derived from intermediate parents via fractional crystallisation or AFC (see [3] and references therein). The two groups overlap each other for several petrological and geochemical characteris- tics but have distinct Sr isotopic signatures. Geosciences 2021, 11, 104 3 of 26 Figure 1. (a) Geological map of Capraia Island (modified from [4]). Legend: A = Monte Rosso Synthem, B = Monte Cam- panile Synthem, C = Monte Castello Synthem, D = Monte Rucitello Synthem, E = Punta dello Zenobito Synthem. (b) Loca- tion of the magmatic centres of Tuscany Magmatic Province (TMP). 2.1. The Capraia Island The island of Capraia is located at the northwestern sector of the Tuscan Archipelago. It is a large asymmetric cone (Figure 1) consisting of a partially collapsed stratovolcano, on which the small late-stage lateral centre of Punta dello Zenobito formed at the south- ernmost end of the island (e.g., [4,21–25]). The island was built up during two distinct periods of magmatic activity, at 7.7–7.2 Ma and 4.6 Ma [4,26,27]. Therefore, Capraia Island is the oldest volcanic centre in the Tuscan Magmatic Province, and its activity is slightly younger to contemporaneous to the magmatism of Elba. y g p g Aldighieri et al. [4] reconstructed the volcanic evolution of the island (Figure 1). They grouped the deposits of the first phase of activity (Capraia Supersynthem) in five different synthems. The deposits of the second activity phase were included in the Punta dello Ze- nobito Synthem. Accordingly, it is possible to distinguish the following: (a) the oldest vol- canic products (lava flows, breccia and scoria) represented by the Punta del Trattoio Syn- them (southwestern cliff, not visible on the map in Figure 1); (b) the andesitic domes and deposits (block, ash and pyroclastic flow) belonging to the Monte Rosso Synthem, visible along the western sector of the island; (c) the thick lava domes associated with lava flows and pyroclastics of Monte Campanile Synthem on the eastern side of the island; (d) lava flows and breccias of Monte Castello Synthem, which cover most of the island; (e) the lava flows forming the Monte Rucitello Synthem, which partially fill a large depression due to a flank collapse of the eastern part of the Monte Castello Synthem. In the same period, a new flank collapse, possibly due to the intrusion of a cryptodome, occurred toward the east; lava flows, breccias, scoriae and small lava domes were emplaced in the northern sector of the island as well as two lava domes in the central area. After a long quiescence period, the eruptive activity resumed in the southern area of the island (Punta dello Ze- nobito Synthem). The Capraia stratovolcano is predominantly composed of massive high-K calcalka- line andesite and dacite lavas, with minor ash and block flows and subordinate pyroclastic flows, emplaced during the first phase of activity. 2. The Tuscany Magmatic Province The orange areas indicate the occurrence of adakite- like rocks. Figure 1. (a) Geological map of Capraia Island (modified from [4]). Legend: A = Monte Rosso Synthem, B = Monte Cam- panile Synthem, C = Monte Castello Synthem, D = Monte Rucitello Synthem, E = Punta dello Zenobito Synthem. (b) Loca- tion of the magmatic centres of Tuscany Magmatic Province (TMP). The orange areas indicate the occurrence of adakite- like rocks. Based on the available data, CA-SHO and LMP mafic end-members were likely formed by melting of anomalous mantle rocks contaminated by variable amounts of sub- ducted sediments (see [7–9] and others). The compositions falling in between CA and LMP mafic magmas might result from mixing between the two end-members. An alternative hypothesis is that the entire range of mafic magmas is primary and was generated in a mantle source containing variable proportions of veins and geochemically barren mantle rocks entering into the melt (e.g., [10]). According to Poli [11,12] and Poli and Peccerillo [3], the compositions falling between mafic and crustal anatectic rocks (i.e., the bulk of outcropping rocks) represent mixtures between crustal and mafic melts. In contrast, Farina et al. [13] suggested that the primary magmas were generated by different degrees of melting of sedimentary to igneous crustal protoliths, with variable amounts of residual phases being incorporated in the melts. A distinct group of intermediate to acidic calcalkaline rocks showing high Sr, Ba and LREE has been distinguished by Poli and Peccerillo [3] among the Orano dykes at Elba Island, for mafic enclaves in the San Vincenzo rhyolites, and at Capraia Island. It was sug- gested that this represents a distinct type of magma, which had not been recognised by Geosciences 2021, 11, 104 4 of 26 previous studies. Actually, the high-Sr-LREE of these rocks had been already pointed out (e.g., [14–16]), but petrogenetic significance and regional distribution were not explored. previous studies. Actually, the high-Sr-LREE of these rocks had been already pointed out (e.g., [14–16]), but petrogenetic significance and regional distribution were not explored. These rocks show very similar abundances of most major and trace elements (SiO2 = 60–65 wt%; MgO ~ 3–4 wt%; TiO2 = 0.6–0.8 wt%; Th = 40–60 ppm; Zr ~ 200–250 ppm; etc.) as other intermediate rocks from the same region. However, Sr (>700 ppm), Ba (>1000 ppm), Sr/Y (>30) and LREE are higher. 2. The Tuscany Magmatic Province Peccerillo [6] noticed that these characteristics are typical of adakites, as defined by several authors [17–20], although typical low Y contents (<15 ppm) are only found in a few samples from Orano dykes. 87Sr/86Sr ratios (~0.708– 0.712) plot at the low end of the Tuscany mafic-intermediate compositional range, whereas Nd-isotope ratios have an opposite behaviour. Such a particular type of magma seems to be restricted to the northwestern sector of the Tuscany Magmatic Province; it was emplaced over a time interval of about 8 to 4.5 Ma, decreasing in age from Elba to Capraia and San Vincenzo. 2.1. The Capraia Island The second eruptive phase mostly con- sists of strombolian scoriae and a few lava flows and dykes of Punta dello Zenobito centre which show a shoshonitic basaltic composition [25,26,28]. p Prosperini [28] and Poli et al. [24] distinguished six different groups of rocks: (1) Older Series, belonging to the main and older emission stage, whose lava flows cover the largest part of the island; (2) Monte Campanile and Porto volcanites, cropping out in the central part of the eastern coast; (3) Zurletto pyroclastics, probably originating from an external submerged centre, which crop out in the eastern coast; (4) San Rocco and Piano volcanites, erupted by small eruptive centres, disposed in a NE–SW direction; (5) Laghetto volcanites, cropping out in a narrow depressed area on the top of the volcano; (6) Zenobito volcanites, in the southern part of the island, forming a neck of porphyritic lava sur- rounded by stratified scoria deposits. A petrographic and geochemical overview of the Capraia lavas is reported in Prosperini [28] and Poli and Perugini [25]. 5 of 26 Geosciences 2021, 11, 104 The bulk of the rocks making up the stratovolcano have mostly a high-K andesite- dacite composition with a slight enrichment in potassium (K2O = 2.5–4.0 wt%). The basal- tic-andesites from Punta dello Zenobito display moderate concentrations of CaO (around tic-andesites from Punta dello Zenobito display moderate concentrations of CaO (around 7–8 wt%) and Na2O (around 3 wt%) and variable MgO, Ni, and Cr content. Sr isotope ratios of Capraia rocks have partially overlapping values. Punta dello Ze- nobito shows 87Sr/86Sr ~ 0.7071–0.7074; high-K andesites and dacites show a large range of values with 87Sr/86Sr = 0.7073–0.71102. Nd isotope ratios are poorly variable in the range 143Nd/144Nd = 0.51224–0.51235 [9,28]. 3. Materials and Methods Among the Capraia products, a group of rocks shows Sr, Ba and LREE contents dis- tinctively higher than other rocks with similar silica contents [3,6]. They come from the “Laghetto volcanic group” [24,28]. This group has been sampled with the aim of provid- ing further data and investigating the origin of high Sr-Ba rocks. In the Laghetto area (Figure 2), a series of lava flows crops out in a small depression. Several samples have been collected from various lava levels and have been analysed for major and trace elements. Mineral chemistry and Sr-Nd radiogenic isotope analyses have been carried out for selected samples. Figure 2. Panoramic view of the Laghetto area. Figure 2. Panoramic view of the Laghetto area. 3.1. Analytical Methods The major element composition of selected mineral phases has been obtained on thin polished sections (30 µm) through the Electron Microprobe JEOL JXA-8600 (IGG-CNR of Firenze). The instrument is equipped with four WDS spectrometers and an EDS detector; it is controlled by the software XMas. The system operates at 10 nA beam current and 15 kV accelerating voltage, with counting times of 15 s on the peak (10 s for Na) and of 5 s on each background. The data acquired have been processed using the PAP correction sys- tem [29]. Major and trace element analyses of the collected rock samples were carried out at the Activation Laboratories, Ancaster, Ontario, Canada. For major oxides and selected trace elements (Ba,Sr,Y,Zr,Sc,V), the samples were prepared and analysed in a batch sys- tem. Each batch contained a method reagent blank, certified reference material and 17% replicates. Samples were mixed with a flux of lithium metaborate and lithium tetraborate and fused in an induction furnace. The molten melt was immediately poured into a solu- tion of 5% nitric acid containing an internal standard and mixed continuously until com- pletely dissolved (~30 min). The samples were run on a combination simultaneous/se- quential Thermo Jarrell-Ash ENVIRO II ICP (inductively coupled plasma). Calibration was performed using 7 prepared USGS and CANMET certified reference materials. One Geosciences 2021, 11, 104 6 of 26 of the 7 standards was used during the analysis for every group of ten samples. Detection limit was in the range between 0.01 and 0.001 (%) for major elements and between 1 and 5 (ppm) for trace elements. The same sample solution was spiked with internal standards to cover the entire mass range and was further diluted and introduced into a Perkin Elmer SCIEX ELAN 6000, 6100 or 9000 ICP/MS (inductively coupled plasma/mass spectrometry) using a proprietary sample introduction methodology. The detection limits in ppm were: Pb = 5 ppm; Ni, Co, Cr, Rb = 1 ppm; Nb= 0.2; Cs, Hf, Ta = 0.1; La, Ce, Nd, Eu, Th = 0.05; Lu = 0.02; Pr, Sm, Gd, Tb, Dy, Ho, Eb, Yb, U = 0.01; Eu, Tm = 0.005. Radiogenic isotope ratio measurements were performed at IGG–CNR of Pisa (Italy). Sr and REE fractions were separated from the matrix using conventional cation columns with AG 50W-X8 200–400 mesh resin. 3.1. Analytical Methods Strontium separates were then loaded onto 99.98% Re filaments, and isotope ratios were measured using a Finnigan MAT 262 multiple col- lector thermal ionisation mass spectrometer (TIMS) for running in dynamic mode. Instru- mental mass bias correction was done by internal normalisation to 86Sr/88Sr = 0.1194. Rep- licate measurements of NIST SRM 987 (SrCO3) standard gave an average value of 0.710229 ± 11 (2SD, n = 16). Throughout the full chemical process, the Sr blanks were approximately 0.3 ng, which were negligible for the analysed samples (0.1–0.2 g of sample). Nd vas ex- tracted from REE eluates using Eichrom Ln resin. Nd eluates were dried, then redissolved in 2% HNO3 solution and measured in static mode with a Thermo Neptune Plus, MC-ICP- MS equipped with 1011 and 1012 Ohm resistors. Blank was negligible; thus, no blank cor- rection was performed. 144Sm interference over 144Nd was calculated using 147Sm; however, this correction also had also a negligible effect on the final ratio. Instrumental mass bias correction was done by internal normalis ation to 146Nd/144Nd = 0.7219. Seventeen repli- cates of the standard J Nd Replicate analyses of JNdi-1 standard [30] gave an average value of 0.512098 ± 6 (2SD). 4.1. Petrography and Mineral Chemistry The collected samples show hypocrystalline, moderately porphyritic (Porphyritic In- dex = 20–30), textures, with phenocrysts of plagioclase, clinopyroxene, and biotite; amphi- bole, K-feldspar, opaque minerals and apatite are present in accessory amounts. The mi- cro- to crypto-crystalline groundmass consists of the same mineral phases as the pheno- crysts, with abundant microlites of plagioclase and biotite. The composition of the main mineral phases is reported in Table 1. Plagioclase is the most abundant mineral (40–65% of total phenocryst abundance) and consists of euhedral or subhedral twinned crystals; in a few cases, phenocrysts display a sieved mantle. Both phenocryst core (An37-49) and rim (An44-47) have andesinic composition. The micropheno- crysts (An36-57) and groundmass (An40-64) plagioclase span a larger range of compositions, from poorer to richer in Anorthite. Clinopyroxene is ubiquitous; it appears as colourless or lightly brown-coloured euhedral to rounded crystals, showing at times reaction rims. Phenocryst core composition is in the range Wo43-45, En35-50, Fs5-21; zoning is generally slightly normal (more Mg-rich cores), even though, in a few cases, crystals with reverse zoning are observed. Some microphenocrysts and groundmass clinopyroxene show dise- quilibrium evidence, having higher Enstatite content than phenocrysts, in agreement with the higher Anorthite content in plagioclase microliths. When plotted in the quadrilateral classification diagram [31], the analysed pyroxenes fall in the augite field or on the bound- ary between the augite and diopside fields; they form two slightly distinct groups (Figure 3), each including phenocrysts, microphenocrysts, and groundmass clinopyroxene. Biotite phenocryst may display a ragged habit and variable extent of resorption; both biotite and amphibole often display opacitic rims or are almost entirely transformed to opaque min- erals. Geosciences 2021, 11, 104 7 of 26 Table 1. Representative chemical analyses of the main mineral phases from the Laghetto rocks. 4.1. Petrography and Mineral Chemistry Plagioclase CA3-1 CA3-1 CA4-8 CA4-11 CA5-1 CA5-1 CA5-6 CA3-9 CA3-15 CA3-19 CA4-5 CA4-13 CA5-3 CA3-14 CA4-6 CA5-16 Ph core Ph rim Ph core Ph core Ph core Ph rim Ph core Micro core Micro core Micro core Micro core Micro core Micro core Gdm Gdm Gdm SiO2 57.2 57.5 59.8 59 55.9 56.2 56.6 59.8 53 59.4 58.7 59.3 57 59.4 54.7 51.8 Al2O3 26.1 26.6 25.2 25.9 27.7 26.9 28 25.9 28.6 24.7 26.3 25.3 26.9 25.9 29 30.3 FeO 0.11 0.11 0.17 0.07 0.65 0.16 0.15 0.13 0.20 0.19 0.19 0.12 0.07 0.77 0.66 0.14 CaO 9.2 9.3 7.5 8 10.2 9.7 10 7.9 11.3 7.5 8.3 7.2 9.1 8.2 11.5 13.5 Na2O 6.1 6.1 7.1 7 5.5 5.3 6.2 6.9 4.5 6.6 6.6 6.5 6 6.4 5.1 4 K2O 0.54 0.68 0.76 0.66 0.43 1.10 0.54 0.78 0.42 1 0.89 1.02 0.58 0.71 0.38 0.27 Total 99.2 100.3 100.5 100.7 100.4 99.2 101.5 101.5 98 99.4 100.9 99.4 99.7 101.5 101.4 100 An 43.9 43.8 35.4 37.4 49.3 47 45.8 37.2 56.8 36.6 39.1 35.8 44.1 39.8 54.4 63.9 Ab 53.1 52.3 60.3 58.9 48.3 46.7 51.2 58.5 40.6 57.7 55.9 58.2 52.6 56.2 43.5 34.6 Or 3.1 3.8 4.3 3.7 2.4 6.3 2.9 4.3 2.5 5.8 5 6 3.3 4.1 2.1 1.5 Clinopyroxene CA3-3 CA3-3 CA4-1 CA4-1 CA4-12 CA4-17 CA4-16 CA5-9 CA5-9 CA4-4 CA5-7 CA5-11 CA5-11 CA3-5 CA4-3 CA5-15 Ph core Ph rim Ph core Ph rim Ph core Ph core Ph core Ph core Ph rim Micro core Micro core Micro core Micro rim Gdm Gdm Gdm SiO2 52.3 52.6 51.7 52.4 53.3 53.9 53.1 53.2 54.8 54.4 54.4 51.6 53.3 53.2 53.2 54.3 TiO2 0.18 0.14 0.17 - 0.09 0.36 0.29 0.24 0.18 0.16 0.21 0.02 0.39 0.25 0.21 0.24 Al2O3 0.80 0.52 0.43 0.54 0.56 1.34 1.24 1.30 1.34 0.70 0.60 0.41 1.84 1.55 1.37 0.78 FeO 11.6 11.9 12.7 12 11.6 4.3 4.8 3.4 4 4.7 3.8 11.8 5.4 5.9 7.7 5.6 MnO 0.46 0.61 0.68 0.61 0.57 0.11 0.13 0.06 0.11 0.14 0.17 0.67 0.16 0.14 0.26 0.16 MgO 13 12.4 12.4 12 12.9 17.3 16.8 17.7 18.2 18.7 19.1 11.9 17.2 17.1 17.6 18.7 CaO 21.7 21.5 21.4 22.2 22.1 22.1 22.1 21.7 22.2 20.8 20.6 21.6 21.5 21.7 19.8 20 Na2O 0.26 0.17 0.15 0.20 0.30 0.25 0.27 0.27 0.14 0.18 0.16 0.26 0.29 0.26 0.29 0.20 K2O - 0.02 - 0.02 0.01 0.01 - - 0.07 - 0.02 0.04 0.01 - - 0.01 Cr2O3 0.02 0.01 - - 0.02 0.53 0.33 0.67 0.31 0.15 0.48 0.01 0.02 0.11 0.02 - Total 100.2 99.9 99.7 100 101.5 100.1 99.1 98.5 101.4 100 99.5 98.3 100.1 100.3 100.4 99.9 Wo 44.1 44.3 43.5 45.6 44.5 44.5 44.7 44.3 43.7 41.1 41.1 45.1 43.1 43.2 39.2 39.6 En 36.7 35.7 35.2 34.2 36.2 48.5 47.4 50.2 49.7 51.4 52.8 34.5 48.1 47.4 48.4 51.5 Fs 19.2 20.1 21.3 20.2 19.2 7 7.8 5.5 6.4 7.5 6.1 20.4 8.7 9.5 12.4 8.9 Biotite Amphibole Opaque minerals CA3-6 CA3-11 CA3-20 CA4-9 CA5-8 CA3-16 CA5-5 CA5-14 Micro core Ph core Ph core Ph core Ph core Ph core Gdm Gdm SiO2 37.8 40.3 37 43.7 38.7 54.0 - 0.01 TiO2 3 3.9 2.8 3.6 4.6 0.18 2.1 2 Al2O3 14.7 17.8 14.5 14.7 14 1.3 0.83 2 FeO 8.5 5.2 10.7 7.2 8.2 3.4 85.5 89 MnO 0.12 0.04 0.10 0.02 0.05 0.09 0.27 0.14 MgO 21.6 18.4 20.5 17 19 18.5 0.85 0.30 CaO 0.12 0.06 0.14 0.14 0.09 20.64 0.35 0.07 Na2O 0.68 0.66 0.66 0.78 0.29 0.21 - - K2O 9.8 6.8 8.9 9.3 10.1 0.04 0.04 0.03 Cr2O3 - 0.03 0.13 0.02 0.06 1.05 0.06 0.09 Total 96.3 93.1 95.4 96.4 95.1 99.4 90 93.6 Ph = phenocryst; Micro = microphenocryst; Gdm = groundmass. 4.1. Petrography and Mineral Chemistry Table 1. Representative chemical analyses of the main mineral phases from the Laghetto rocks. 12 of 26 Geosciences 2021, 11, 104 Figure 3. Classification diagram of pyroxene [31] from the Laghetto rocks. Figure 3. Classification diagram of pyroxene [31] from the Laghetto rocks. 4.2. Geochemistry Major and trace element compositions of the analysed samples are reported in Table 2, along with a few representative analyses of other Capraia rocks [9,28]. The Total-Alkali versus Silica (TAS) [32] and K2O vs. SiO2 [33] classification diagrams are shown in Figure 4. Major and trace element diagrams are shown in Figures 5–7. Other rocks occurring at Capraia, as dykes at Elba Island and as mafic enclaves in the San Vincenzo rhyolites, are also reported [15,16,34,35]. p The Capraia rocks are mostly high-K calcalkaline andesites and dacites. The samples from Punta dello Zenobito are more mafic and fall at the boundary between shoshonitic and high-K basaltic andesites. The Zurletto pyroclastics display more evolved composi- tion plotting between dacite and rhyolite fields (Figure 4b). The investigated samples from the Laghetto Series plot at the boundary between the trackyandesite and trachydacite fields in the TAS diagram (Figure 4a) and mostly in the high-K calcalkaline andesite and dacite fields in the K2O vs. SiO2 diagram (Figure 4b). g g g The variation diagrams for major elements (Figures 4 and 5) show a similarity of the analysed rocks with other andesite-dacites from Capraia for most elements, although they exhibit slightly higher K2O and lower TiO2 and FeOt. g y g Trace elements against SiO2 (Figures 6 and 7) show that the Laghetto samples have similar abundances as other Capraia intermediate rocks. However, they display distinct spikes in Sr and Ba, LREE, Th, U and Pb. In contrast, HREE have slightly lower values, and consequently, La/Yb and Tb/Yb ratios are higher. High Sr-Ba compositions in the Laghetto Series were also found by Prosperini ([28] and Table 2). High Sr-Ba rock samples from Orano dykes and San Vincenzo mafic enclaves display compositions in the range of intermediate Capraia rocks for many major and trace elements (Figures 4–7). 13 of 26 Geosciences 2021, 11, 104 Figure 4. (a) TAS and (b) K2O vs. SiO2 classification diagrams for Capraia rocks. Open circles (first activity phase) and triangles (second activity phase): data from the literature [9,28]; full green circles: this work. Some Elba dykes [15,35] and mafic enclaves from San Vincenzo [16,34] are also plotted for comparison. Data are plotted on water-free basis. Figure 4. (a) TAS and (b) K2O vs. SiO2 classification diagrams for Capraia rocks. Open circles (first activity phase) and triangles (second activity phase): data from the literature [9,28]; full green circles: this work. 4.2. Geochemistry Some Elba dykes [15,35] and mafic enclaves from San Vincenzo [16,34] are also plotted for comparison. Data are plotted on water-free basis. Figure 5. Major element variation diagrams for Capraia rocks. 1 2 3 4 5 6 7 8 9 10 MgO wt% 2 3 4 5 6 7 8 9 CaO wt% 0.0 0.5 1.0 1.5 2.0 TiO2 wt% 2 3 4 5 6 7 8 9 10 11 FeOtot wt% 2.2 2.5 2.8 3.1 3.4 3.7 4.0 4.3 50 52 54 56 58 60 62 64 66 68 70 72 SiO2 wt% Na2O wt% 50 52 54 56 58 60 62 64 66 68 70 72 0.1 0.2 0.3 0.4 0.5 0.6 0.7 P2O5 wt% SiO2 wt% Capraia Isl. first activity phase San Vincenzo mafic enclave Capraia Isl. second activity phase Elba Isl. Orano porphyry Capraia Isl. Laghetto (this work) Elba Isl. Chiessi dyke Figure 5. Major element variation diagrams for Capraia rocks. 14 of 26 Geosciences 2021, 11, 104 Figure 6. Trace element variation diagrams for Capraia rocks. 200 500 800 1100 1400 1700 Sr ppm 0 100 200 300 400 500 Cr ppm 75 125 175 225 275 Rb ppm 0 30 60 90 120 150 180 210 La ppm 15 20 25 30 35 40 45 50 55 60 Th ppm 400 600 800 1000 1200 1400 1600 1800 Ba ppm 3 5 7 9 11 13 50 52 54 56 58 60 62 64 66 68 70 72 SiO2 wt% U ppm 50 52 54 56 58 60 62 64 66 68 70 72 0 10 20 30 40 50 60 70 80 90 100 110 Pb ppm SiO2 wt% Capraia Isl. first activity phase San Vincenzo mafic enclave Capraia Isl. second activity phase Elba Isl. Orano porphyry Capraia Isl. Laghetto (this work) Elba Isl. Chiessi dyke Figure 6. Trace element variation diagrams for Capraia rocks. Figure 7. Trace element and element ratio variation diagrams for Capraia rocks. 4.2. Geochemistry 200 500 800 1100 1400 Sr ppm 0 100 200 300 400 500 Cr ppm 75 125 175 225 Rb ppm 0 30 60 90 120 150 180 210 La ppm 15 20 25 30 35 40 45 50 55 60 Th ppm 400 600 800 1000 1200 1400 1600 1800 Ba ppm 3 5 7 9 11 13 50 52 54 56 58 60 62 64 66 68 70 72 SiO2 wt% U ppm 50 52 54 56 58 60 62 64 66 68 70 72 0 10 20 30 40 50 60 70 80 90 100 110 Pb ppm SiO2 wt% 0.5 1.0 1.5 2.0 2.5 3.0 3.5 Yb ppm 0 20 40 60 80 100 120 140 50 52 54 56 58 60 62 64 66 68 70 72 SiO2 wt% La/Yb 50 52 54 56 58 60 62 64 66 68 70 72 0.3 0.4 0.5 0.6 0.7 0.8 0.9 Tb/Yb SiO2 wt% 0 10 20 30 40 Y ppm Capraia Isl. first activity phase San Vincenzo mafic enclave Capraia Isl. second activity phase Elba Isl. Orano porphyry Capraia Isl. Laghetto (this work) Elba Isl. Chiessi dyke Figure 6. Trace element variation diagrams for Capraia rocks. Figure 7. Trace element and element ratio variation diagrams for Capraia rocks. 0.5 1.0 1.5 2.0 2.5 3.0 3.5 Yb ppm 0 20 40 60 80 100 120 140 50 52 54 56 58 60 62 64 66 68 70 72 SiO2 wt% La/Yb 50 52 54 56 58 60 62 64 66 68 70 72 0.3 0.4 0.5 0.6 0.7 0.8 0.9 Tb/Yb SiO2 wt% 0 10 20 30 40 Y ppm Capraia Isl. first activity phase San Vincenzo mafic enclave Capraia Isl. second activity phase Elba Isl. Orano porphyry Capraia Isl. Laghetto (this work) Elba Isl. Chiessi dyke Figure 7. Trace element and element ratio variation diagrams for Capraia rocks. 15 of 26 Geosciences 2021, 11, 104 Table 2. Major (%), trace (ppm) and isotopic compositions of representative rocks from Capraia Island. 4.2. Geochemistry Rock Sample CA4 CA2 CA5 CA1 CA6 CA7 CA3 CP25 CP22 CP50 CP9 CP28 CP7 CP6 CP30 CP14 CP 101 CP110 CP54 RZ05E PP-180C PP-180R DG-27 DG-28 Locality Capraia Island South- ern Tus- cany Elba Island Laghetto Laghetto Laghetto Laghetto Laghetto Laghetto Laghetto Laghetto Laghetto Monte Campa- nile Monte Campa- nile Porto Zurletto Zurletto San Rocco- Piano San Rocco- Piano Capo Fer- raione Peraiola Zeno- bito San Vin- cenzo enclave Orano porphyry Orano porphyry Orano dykes (Chiessi) Orano dykes (Chiessi) Data Source This Work This Work This Work This Work This Work This Work This Work [28] [28] [28] [28] [28] [28] [28] [28] [28] [9] [9] [9] [16] [15] [15] [35] [35] SiO2 60.2 61.1 61.2 61.4 61.5 62.3 63.3 62.2 63 62 63.9 59.1 67.6 68.4 62.2 62.5 61.9 62.3 50.7 55.2 64.5 63.3 61.2 59.8 TiO2 0.66 0.65 0.67 0.64 0.62 0.61 0.65 0.67 0.67 0.97 0.94 0.81 0.51 0.56 0.71 0.73 0.69 0.68 1.65 0.82 0.62 0.69 0.67 0.67 Al2O3 14.8 16.1 15.4 16.7 15.6 15.7 16.1 16 16 16.5 16 16.5 13.6 14.3 15.8 15.8 15.3 15.2 15.5 15.6 16.1 15.2 14.6 14.5 Fe2O3 4.72 4.68 4.71 4.71 4.54 4.51 4.63 4.66 4.60 5.35 4.99 5.97 3.26 3.38 5.32 5.50 5.39 5.24 10.20 6.55 4.23 4.64 5.01 5.11 MnO 0.09 0.07 0.08 0.06 0.07 0.05 0.07 0.09 0.07 0.06 0.06 0.07 0.06 0.06 0.06 0.06 0.07 0.07 0.13 0.05 0.06 0.06 0.11 0.10 MgO 3.07 2.96 3.02 2.72 3.04 2.08 2.26 3.06 3.38 2.60 2.09 4.16 1.36 1.33 3.53 3.18 3.48 2.88 6.41 3.70 3.06 4.33 5.78 5.76 CaO 5.06 4.29 4.91 4.50 4.89 4.56 4.46 4.92 4.32 4.34 3.74 5.66 2.42 2.72 5.35 5.14 5.48 4.59 7.92 6.26 2.37 3.49 4.11 4.68 Na2O 3.09 3.01 3.27 3.06 3.18 3.32 3.35 3.27 3.17 3.53 3.61 3.20 3.82 3.10 2.88 2.87 3.14 3.14 2.83 2.27 3.27 2.92 3.28 3.20 K2O 4.26 4.14 4.27 4.07 3.99 4.15 4.26 3.95 3.75 3.71 4 2.82 3.86 3.95 3.04 3.12 3.33 3.90 2.42 3.78 3.87 3.95 3.25 3.09 P2O5 0.31 0.27 0.33 0.30 0.28 0.29 0.26 0.29 0.29 0.36 0.34 0.26 0.14 0.18 0.23 0.25 0.18 0.29 0.48 0.61 0.28 0.27 0.25 0.24 LOI 1.97 1.85 1.06 1.83 1.46 1.99 0.66 1.10 0.88 0.70 0.45 1.69 3.41 2.14 1.14 1.10 1.36 2.19 1.13 4.98 1.99 1.41 2.02 3.21 Total 98.3 99.1 98.9 99.9 99.2 99.6 100 100.2 100.1 100.2 100.1 100.3 100.1 100.1 100.3 100.2 100.3 100.5 99.4 99.8 100.3 100.3 100.3 100.3 Sc 14 14 15 14 14 13 13 13.2 - 13.8 - 17.4 6.8 - 16 - - - 23 19 11 12 - - V 106 92 94 93 92 86 86 88 88 99 78 101 24 27 123 127 128 137 166 - 75 88 118 124 Cr 120 120 100 110 120 120 100 100 123 94 56 243 20 34 144 168 177 132 400 310 86 144 248 243 Co 16 14 15 15 14 10 13 8 14 11 11 18 8 7 14 16 11.9 15.2 30 19 12.8 16.3 18 19 Ni 20 20 20 20 30 30 < 20 25 26 22 10 84 8 9 13 13 29 - 69 90 43.1 83.7 101 112 Rb 164 169 162 166 163 172 180 164 153 187 191 125 182 205 122 122 116 161 115 129 166 206 189 189 Sr 1092 908 1164 1004 947 932 914 1110 1042 498 489 489 341 383 852 849 733 933 399 1634 920 1461 1563 1602 Y 20.9 19.4 22.7 18.7 19.2 19.7 21.5 31 20 25 24 22 18 20 19 23 19.3 31 20 30 20 20.1 10 8 Zr 227 247 245 236 222 252 255 206 223 259 232 207 170 176 198 197 185 234 221 226 192 213 219 217 Nb 11.3 9.8 11.3 10.3 12.9 12.7 12.9 8 13 20 17 13 10 12 11 9 11.9 14.3 15 7 12.4 10.9 9 8 Cs 11.7 11.5 11 11.5 12.1 13.4 12.3 12 - - - - - - - - 9.2 15.2 4 - 12 30 14 - Ba 1537 1342 1574 1318 1259 1296 1278 1220 1367 828 820 738 563 650 866 853 711 1410 540 1510 970 1278 1528 1470 La 129 121 145 119 114 119 107 127 128 68 63 68 44 49 57 59 58.5 165 29.3 181 101 120 146 148 Ce 232 228 259 237 213 210 202 224 246 119 129 106 89 86 102 98 109 297 68.4 333 186 205 263 295 Nd 85.2 81.3 96.8 79.5 77.7 76.9 74.6 103 - 49 - 43 34 - 39 - 43.6 105 51.9 160 80 94 107.3 - Sm 12.8 12.2 14.4 11.7 11.8 11.5 11.6 13.9 - 10.7 - - 6.7 - 8.6 - 8.01 16.4 9.6 25 12.6 14.4 15.7 - Eu 2.38 2.32 2.72 2.33 2.33 2.3 2.3 2.62 - 1.63 - 1.67 1.05 - 1.63 - 1.76 3 2.09 4.88 2.05 2.76 3 - Gd 7.25 6.54 7.93 6.48 6.5 6.36 6.65 8.1 - - - - - - - - 5.3 9.5 6.2 14 7.89 9.38 10.3 - Tb 0.86 0.79 0.89 0.71 0.74 0.76 0.83 1.1 - 0.82 - 0.72 0.57 - 0.71 - 0.77 1.24 1 1.5 0.88 0.92 1 - Dy 4.03 3.64 4.23 3.59 3.77 3.63 4.05 4.9 - - - - - - - - 3.6 5.6 4.9 6.9 4.25 4.36 4.9 - Ho 0.67 0.62 0.71 0.61 0.6 0.6 0.7 0.91 - - - - - - - - 0.7 1.01 0.99 1.1 0.74 0.73 0.6 - Er 1.92 1.77 2.12 1.79 1.72 1.79 1.94 2.3 - - - - - - - - 2.05 2.85 2.7 2.6 1.83 1.69 1.7 - Tm 0.24 0.23 0.29 0.26 0.23 0.25 0.27 0.30 - - - - - - - - 0.29 0.40 0.40 - 0.26 0.24 0.20 - Yb 1.68 1.58 1.82 1.56 1.50 1.53 1.84 2.30 - 2.10 - 2 1.80 - 1.80 - 1.84 2.49 2.30 2.10 1.67 1.37 1.20 - Geosciences 2021, 11, 104 16 of 26 16 of 26 Lu 0.24 0.25 0.29 0.22 0.24 0.23 0.28 0.35 - 0.35 - 0.36 0.28 - 0.23 - 0.28 0.36 0.35 0.30 0.22 0.18 0.20 - Hf 5.6 5.5 6.2 5.7 5.6 5.9 5.9 5.3 - 7.3 - 4.6 5.9 - 5 - 5.4 6.3 5.6 6.5 - - - - Ta 1.07 1 0.97 1 1.03 1.11 1.17 1 - 1.8 - 1.3 1.5 - 1.08 - 0.9 1.11 1.2 1.1 1.54 1.3 - - Pb 72 74 78 77 78 72 70 53 59 41 42 38 51 52 48 46 61 82 12 - 36 101 77 61 Th 51.5 53.7 56.8 52.5 50 52.5 48.3 44 46 44 43 26 34 34 25 23 23.1 49.4 24 51.5 37 35 43 51 U 10.5 10.4 10.6 10.9 10.5 11.8 9.9 - - - - - - - - - 6.4 11.7 3.8 - 11 9.9 - - 87Sr/86Sr 0.709642 - 0.710009 - - - 0.709553 0.710260 0.707314 0.709628 0.709811 0.709175 - - 0.708782 0.707933 0.708719 0.710213 0.708135 - - 0.711500 0.711348 - 143Nd/144Nd 0.512343 - 0.512331 - - - 0.512339 0.512243 - - - - - - - - 0.512346 0.512239 0.512254 - - 0.512270 0.512330 - Geosciences 2021, 11, 104 17 of 26 Chondrite-normalised REE patterns of the analysed samples are fractionated for both LREE and HREE (Figure 8a). 4.2. Geochemistry Incompatible element abundances normalised to primordial mantle compositions show strong negative spikes of HFSE, high positive anomalies of Pb and other LILE, and a small negative anomaly of Ba (Figure 8b). Similar patterns are shown by the Elba and San Vincenzo rocks. Figure 8. (a) Chondrite normalised REE patterns and (b) Primitive mantle normalised trace ele- ment patterns for the Laghetto rocks. Patterns of other Capraia rocks [9,28], Elba dykes [15,35] and a San Vincenzo mafic enclave [16] are shown for comparison. Normalising values from Sun and McDonough [36]. La Ce Pr Nd Pm Sm Eu Gd Tb Dy Ho Er Tm Yb Lu 1 10 100 1000 Rock/Chondrite CsRbBa Th U NbTa K La CePb Pr Sr P Nd Zr SmEuGd Ti Dy Y Tb Lu 1 10 100 1000 Rock/Primitive mantle (a) (b) Capraia Isl. first activity phase San Vincenzo mafic enclave Capraia Isl. second activity phase Elba Isl. Orano porphyry Capraia Isl. Laghetto (this work) Elba Isl. Chiessi dyke 1 10 100 1000 Rock/Chondrite (a) Rock/Chondrite CsRbBa Th U NbTa K La CePb Pr Sr P Nd Zr SmEuGd Ti Dy Y Tb Lu 1 10 100 1000 Rock/Primitive mantle (b) (b) CsRbBa Th U NbTa K La CePb Pr Sr P Nd Zr SmEuGd Ti Dy Y Tb Lu CsRbBa Th U NbTa K La CePb Pr Sr P Nd Zr SmEuGd Ti Dy Y Tb Lu 1 Figure 8. (a) Chondrite normalised REE patterns and (b) Primitive mantle normalised trace ele- ment patterns for the Laghetto rocks. Patterns of other Capraia rocks [9,28], Elba dykes [15,35] and a San Vincenzo mafic enclave [16] are shown for comparison. Normalising values from Sun and McDonough [36]. Figure 8. (a) Chondrite normalised REE patterns and (b) Primitive mantle normalised trace ele- ment patterns for the Laghetto rocks. Patterns of other Capraia rocks [9,28], Elba dykes [15,35] and a San Vincenzo mafic enclave [16] are shown for comparison. Normalising values from Sun and McDonough [36]. Sr-Nd isotope ratios of the analysed samples fall in the range of 87Sr/86Sr = 0.709553– 0.710009 and 143Nd/144Nd = 0.512331–0.512343. Sr-isotope ratios overlapped or were some- what higher than other Capraia rocks reported in the literature [9,28]. 4.2. Geochemistry Overall, the Capraia rocks, as well as the Elba dykes and San Vincenzo enclaves, plot near to the lowest Sr and highest Nd isotope distribution range of the Tuscany Province, whose main isotopic char- acteristic is the intermediate composition between typically mantle-derived and crustal anatectic magmas (Figure 9). The few published Pb isotope ratios show poorly radiogenic 18 of 26 Geosciences 2021, 11, 104 18 of 26 values, as typically found in other rocks from the Tuscany Province (206Pb/204Pb = 18.65– 18.73; 207Pb/204Pb = 15.64–15.70; 208Pb/204Pb = 38.96–39.09; [9] and references therein). values, as typically found in other rocks from the Tuscany Province (206Pb/204Pb = 18.65– 18.73; 207Pb/204Pb = 15.64–15.70; 208Pb/204Pb = 38.96–39.09; [9] and references therein). 18.73; Pb/ Pb 15.64 15.70; Pb/ Pb 38.96 39.09; [9] and references therein). Figure 9. Nd vs. Sr isotope ratios for Laghetto rocks. In grey is shown the Sr and Nd isotope area of the Tuscan province. Data from other adakite localities (orange areas) are reported for compari- son [19]. 0.705 0.710 0.715 0.720 0.725 0.5120 0.5121 0.5122 0.5123 0.5124 0.5125 143Nd/144Nd 87Sr/86Sr Adakitic rocks northern Tibet Eastern Yank Tse Block adakites Tuscany Province Capraia Isl. first activity phase San Vincenzo mafic enclave Capraia Isl. second activity phase Elba Isl. Orano porphyry Capraia Isl. Laghetto (this work) Elba Isl. Chiessi dyke 0.705 0.710 0.715 0.720 0.725 0.5120 0.5121 0.5122 0.5123 0.5124 0.5125 143Nd/144Nd 87Sr/86Sr Adakitic rocks northern Tibet Eastern Yank Tse Block adakites Tuscany Province Capraia Isl. first activity phase San Vincenzo mafic enclave Capraia Isl. second activity phase Elba Isl. Orano porphyry Capraia Isl. Laghetto (this work) Elba Isl. Chiessi dyke Figure 9. Nd vs. Sr isotope ratios for Laghetto rocks. In grey is shown the Sr and Nd isotope area of the Tuscan province. Data from other adakite localities (orange areas) are reported for compari- son [19]. 5. Discussion 5.1. Classification, Occurrence and Genetic Processes of Sr-Ba-LREE–Rich Rocks The analysed rocks of the Laghetto Series differ from other intermediate calcalkaline rocks from Capraia and most of the other centres of the Tuscany Magmatic Province for showing anomalous enrichments in Sr and Ba [3]. Moreover, they also show enrichment in LREE, Th, U, and Pb, and strong REE fractionation (high La/Yb and La/Y). A few dykes from Elba Island (Table 2) also show similar characteristics to the Laghetto samples [15,35]. [ ] Intermediate to felsic high-Sr rocks are found in many recent and old subduction zones and have been named adakites. They were interpreted early on as representing magmas formed directly by melting of MOR basaltic crust at high pressures along sub- duction zones ([17,18] and references therein). Successively, several occurrences of rocks with high Sr-Ba, with variable isotopic compositions, have been reported both in some circum-Pacific arc systems and in conti- nental collision environments ([36,37] and references therein). Summarising previous studies on various occurrences, Richard and Kerrich [38] and Castillo [19] defined the adakites and adakitic rocks as characterised by intermediate to felsic compositions, high Sr (≥ 400–500 ppm), Sr/Y (≥ 20), and La/Yb (≥ 2) ratios, and low Y (≤ 18 ppm) and HREE (Yb ≤ 1.9 ppm). pp The analysed Laghetto rocks share several compositional features of adakites, as de- fined by Richard and Kerrich [38] and Castillo [19]. These include high Sr, Sr/Y, Ba and La/Yb (Figures 7 and 10). On the other side, Y contents are also somewhat higher in the Capraia rocks with respect to typical adakites, although low Y and HREE are found at Elba (Figure 10). Sr (and Nd) isotopic ratios differ from typical island-arc adakite values (87Sr/86Sr ~ 0.704) [38]; however, they are close to some Yang Tse Block and Northern Tibet adakite values (Figure 9). Based on this evidence, the Laghetto rocks can be considered as members of the adakitic rock clan or as transitional rocks between adakitic and arc calcal- kaline rocks. They represent a new magma type for Central Italy, which had not been Geosciences 2021, 11, 104 19 of 26 highlighted by previous studies. Similar rocks occur in at least one other place in Tuscany: at Elba, a few dykes from Chiessi (Orano dykes) have similar characteristics as Laghetto, in addition to lower abundances of Y and HREE [35]. 5. Discussion Some mafic enclaves from San Vin- cenzo rhyolites, southern Tuscany, likely also belong to the same magma type, as will be discussed later. There is a general agreement that the high Sr-Ba contents of adakitic rocks point to genetic processes that occurred at high pressure, outside the stability field of feldspars. In addition to the early hypothesis of a direct derivation by partial melting of the MORB-type crust along subduction zones [17], other processes include high-pressure am- phibole-dominated crystal fractionation of arc hydrous basalts, a derivation from anoma- lous mantle rocks contaminated by upper crustal material coming from the slab, or melt- ing of thickened lower continental crust in zones of continental collision (e.g., [18–20,37– 44]). In all cases, Sr and Ba behave as incompatible elements and, therefore, are enriched in the melts. Figure 10. Y vs Sr/Y diagram to discriminate adakitic from typical arc rocks. Fields from Ribeiro et al. [20]. Dashed lines are two mixing curves between Elba dykes (Chiessi) and different Capraia samples. Note that the samples with high Sr/Y plot on these curves, and the Laghetto rocks plot at the transition between adakite and calcalkaline rocks. 0 10 20 30 40 0 50 100 150 200 Sr/Y Y ppm Calcalkaline rocks Adakites Capraia Isl. first activity phase San Vincenzo mafic enclave Capraia Isl. second activity phase Elba Isl. Orano porphyry Capraia Isl. Laghetto (this work) Elba Isl. Chiessi dyke 0 10 20 30 40 0 50 100 150 200 Sr/Y Y ppm Calcalkaline rocks Adakites Capraia Isl. first activity phase San Vincenzo mafic enclave Capraia Isl. second activity phase Elba Isl. Orano porphyry Capraia Isl. Laghetto (this work) Elba Isl. Chiessi dyke Capraia Isl. second activity phase p y p Capraia Isl. Laghetto (this work) Calcalkaline rocks Figure 10. Y vs Sr/Y diagram to discriminate adakitic from typical arc rocks. Fields from Ribeiro et al. [20]. Dashed lines are two mixing curves between Elba dykes (Chiessi) and different Capraia samples. Note that the samples with high Sr/Y plot on these curves, and the Laghetto rocks plot at the transition between adakite and calcalkaline rocks. 5.2. Petrogenesis of Laghetto Rocks Sharp spikes of some elements in the Laghetto rocks might suggest a shallow-level magma chamber process, for instance, the accumulation of Sr-Ba-LREE–rich phases such as feldspars, biotite, and accessory apatite. All feldspars contain high concentrations of Sr; K-feldspars and biotite are enriched in Ba (e.g., [45,46]); apatite contains high amounts of all REE (e.g., [47]). The analysed samples contain apatite and biotite. There is a positive correlation between P2O5 and LREE (not shown), which would support accumulation of apatite. However, apatite is rich in both Light and Heavy REE (e.g., [48]) and should pro- duce an increase of all REE, which is not observed. Also, accumulation of phlogopite should lead to exceedingly high Rb contents, which is not observed in our rocks. Moreo- ver, the porphyritic index of Laghetto rocks is moderate (about 20%) and much lower than other intermediate rocks from the same island [28]. Therefore, mineral accumulation is unlikely to be responsible for anomalous enrichment in the investigated rocks, based on petrographic and geochemical evidence. Their anomalous composition as compared to Geosciences 2021, 11, 104 20 of 26 20 of 26 other andesite-dacite rocks in Tuscany must reflect processes occurring during melting or/and during the ascent of magma to the surface. Clear evidence emerging from isotopic data is that the Laghetto rocks differ from typical MORB rocks. Moreover, the Sr-Nd iso- tope signature clearly requires the participation of both mantle and crustal components in its origin and/or evolution. This excludes any direct genetic relationship with mid-ocean ridge basalts (MORB), e.g., by melting of the subducted oceanic slab. These conclusions are valid for most, if not all, Tuscany magmatic rocks (e.g., [3]). Therefore, an origin from melting of the subducting oceanic basaltic slab is unlikely. Other possible processes in- clude high-pressure fractional crystallisation of basaltic magmas, melting of mixtures of crustal and mantle rocks above subduction zones (subduction mélange bodies), and melt- ing of the lower continental crust, thickened during collision. These processes will be tested by trace element modelling. High-pressure fractional crystallisation. Such a process has been tested by assuming a calcalkaline-shoshonitic mafic magma with a composition as the rocks from Punta dello Zenobito. Models have been carried out for both compatible and incompatible trace ele- ments. Fractionation of feldspar-free mineral assemblages dominated by mafic minerals amphibole and/or clinopyroxene has been assumed. 5.2. Petrogenesis of Laghetto Rocks This mineral assemblage has a low bulk partition coefficient for Sr and Ba and other incompatible elements during fractiona- tion and can explain the enrichments in the Laghetto rocks. However, amphiboles and clinopyroxenes also incorporate significant amounts of ferromagnesian elements, such as Cr, Co, and Sc, which have partition coefficients much higher than unity (see GERM web- site [49]). For modelling, Sr and Rb have been used as incompatible elements, whereas Cr has been selected as representative of compatible elements. Partition coefficients have been set at DSr = 0.1, DRb = 0.01, DCr = 2 and 20; a very low partition coefficient for Rb has been assumed because this element is one of the most incompatible in both mafic and felsic systems. Partition coefficients for Cr are variable, and two extreme values are used for modelling (see GERM website [49]). The model of Cr vs. Sr (Figure 11a) shows that the separation of a mafic mineral assemblage from basaltic Capraia magmas generates high- Sr residual liquid as the Laghetto rocks, after about 50 to 70% crystal fractionation. How- ever, the Cr contents are matched only if the minimum value of DCr = 2 is assumed. For higher and more realistic values of DCr (= 5–10), the decrease in Cr is extreme and produces residual melts that are away from Laghetto rocks. This argues against fractional crystallisation of mafic phases. The same conclusion is suggested by Rb-Sr modelling (Fig- ure 11b), which is unable to generate high-Sr and moderately Rb-rich residual liquids starting from Zenobito basalts unless the unrealistic assumption is made of a nearly com- patible behaviour for Rb (i.e., DRb = 0.9–1). A compatible behaviour of Rb would imply fractionation of either amphibole and/or phlogopite, which would not explain the increase in Ba. 21 of 26 Geosciences 2021, 11, 104 Figure 11. Model of fractional crystallisation and equilibrium melting for the Laghetto magmas. (a,b) Fractional crystalli- sation models from Capraia Zenobito basalts; (c,d) Batch melting model of lower crust. Numbers along lines indicate amount of liquid. Some Elba dykes [15,35] and mafic enclaves from San Vincenzo [16,34] are also plotted for comparison. For further explanation, see text. Figure 11. Model of fractional crystallisation and equilibrium melting for the Laghetto magmas. (a,b) Fractional crystalli- sation models from Capraia Zenobito basalts; (c,d) Batch melting model of lower crust. Numbers along lines indicate amount of liquid. 5.2. Petrogenesis of Laghetto Rocks Some Elba dykes [15,35] and mafic enclaves from San Vincenzo [16,34] are also plotted for comparison. For further explanation, see text. Lower crust melting. Alternative processes could be melting of the lower continental crust or a derivation from mixtures between mantle peridotite and upper crustal rocks, i.e., subduction mélanges of peridotite and metapelites [50]. Both occur at high pressure outside the stability field of feldspars (i.e., thickened crust), making Sr behave as an in- compatible element. Lower crust batch melting processes of Sr vs. the compatible element Cr are reported in Figure 11c. The lower crust composition is from Wedepohl [51]. Mod- elling shows that, assuming an incompatible behaviour (i.e., few or no feldspars in the residue, compatible with high-P melting in the deep crust), Sr increases with a decreasing degree of melting, whereas compatible elements are not strongly depleted. Overall, a com- positional trend that resembles the Laghetto rocks can be obtained. However, the adakite- like rocks from Elba and the San Vincenzo mafic enclave have exceedingly high Cr con- tents to be derived from the lower crust. Such a problem does not necessarily rule out lower crust melting. However, a contribution of mantle material has to be assumed in the origin of these rocks. g Crustal melting has been also tested using couples of incompatible elements (Sr vs. Rb). If partition coefficients of 0.1 and 0.01 are respectively assumed for Sr and Rb, crustal melting can generate liquids that resemble some of the most Sr-rich among the analysed Geosciences 2021, 11, 104 22 of 26 22 of 26 samples but does not explain the entire range of the Laghetto rocks. However, the com- positional range of the latter rocks can be matched by assuming that Sr-rich magmas mixed with other Capraia liquids to form a suite of hybrid magmas matching the trend of the analysed samples. In conclusion, the most Sr-rich rocks from Capraia and Elba can be formed by melting of the lower crust. However, a contribution from upper mantle peri- dotite is required to obtain the observed values of compatible elements such as Cr. q p Melting of mantle–upper crust mixtures (subduction mélange). The occurrence of up- per mantle rocks strongly contaminated by upper crustal material in the Roman Province has been suggested by many papers ([8] and others). A hybridism of mantle and crustal rocks is probably common along the entire Alpine-Himalayan orogenic belt [52]. 5.2. Petrogenesis of Laghetto Rocks Mantle- crust mixing can occur by sediment transport along subduction zones, melting and vein- ing by these melts inside the overlying mantle wedge ([4] and references therein). Other models assume that basalts, sediments, and slices of continental crust are transported along the subduction channel and are added to mantle wedge to form hydrous mélange bodies at the slab–mantle interface. The low density of these bodies favours ascent as plumes toward the hotter corner of the mantle wedge, where they dehydrate and melt (e.g., [50,53–55]). The melting of different portions of diapirs generates various types of magmas, with variable enrichments in incompatible elements; melting of dehydrated sec- tors of the diapirs can generate magmas enriched in poorly mobile elements such as Th and REE [56]. These magmas can also be rich in Ba-Sr if melting occurs at high pressure, outside the stability field of feldspars. Quantitative modelling of hybrid mantle-crust rocks melting is very difficult to per- form because of the large number of assumptions that have to be made on the composition of the crustal rocks, the amounts of crustal material entering into the mantle, and the role of fluid in modifying different portions of the mantle wedge. Because of these difficulties, only some aspects will be explored. As stated earlier in this paper, Sr-Nd isotopic signatures on the Laghetto rocks re- quire contributions by both mantle and upper continental crust, as amply recognised for other mafic to intermediate magmas in the Tuscany province and along the Alpine-Him- alayan belt. The amount of crustal material involved in the mixing with mantle is uncer- tain. If a composition as that reported for the average gneiss from Southern Tuscany is assumed (Sr = 180 ppm, 87Sr/86Sr = 0.723 [57]), the contribution of the continental crust end- members to the Laghetto source is around 5%, starting from an upper mantle composition of Sr = 20 ppm and 87Sr/86Sr = 0.703 (see GERM website [49]). A mixture of 95% mantle material and 5% crustal material is still ultramafic in composition. Its melting generates a liquid containing high contents of compatible elements Ni and Cr (Ni around 300–400 ppm and Cr around 1000–1100 ppm, assuming batch melting), which differ from the stud- ied rocks. Role of magma mixing. 5.2. Petrogenesis of Laghetto Rocks The Laghetto volcanics display evidence of textural and min- eralogical disequilibrium, such as the sieve-texture in plagioclase, the reaction rim and variable composition of clinopyroxene and plagioclase (Table 1), and the resorption rims in biotite and amphibole, which suggest mixing between compositionally different magma batches. To highlight this process, plots of elements vs. element ratios (i.e., Rb vs. Rb/Sr) are reported in Figure 12. The analysed samples and other Capraia rocks define different trends that are typical of mixing processes. These trends are consistent with the hypothesis that a range of high-Sr and low Rb/Sr magmas was formed during lower crus- tal melting (dash-dot line). These subsequently mixed with low-Sr and high-Rb/Sr melts, which represent the bulk of Capraia rocks and might be derived from mafic parents fol- lowing shallow-level fractional crystallisation processes. The circled symbols in Figure 12 represent the different end members. 23 of 26 Geosciences 2021, 11, 104 Figure 12. Element vs. element ratio diagrams for the Laghetto and other Capraia rocks. Some Elba dykes [15,35] and mafic enclaves from San Vincenzo [16,34] are also plotted for comparison. The dash-dot line is the melting curve of the lower continental crust. The full lines are possible mixing trends between different end members (circled symbols). 0 100 200 300 0.0 0.1 0.2 0.3 0.4 0.5 0.6 Rb/Sr Rb ppm Lower Crust Capraia Isl. first activity phase San Vincenzo mafic enclave Capraia Isl. second activity phase Elba Isl. Orano porphyry Capraia Isl. Laghetto (this work) Elba Si. Chiessi dyke 0 100 200 300 0.0 0.1 0.2 0.3 0.4 0.5 0.6 Rb/Sr Rb ppm Lower Crust Capraia Isl. first activity phase San Vincenzo mafic enclave Capraia Isl. second activity phase Elba Isl. Orano porphyry Capraia Isl. Laghetto (this work) Elba Si. Chiessi dyke San Vincenzo mafic enclave Elba Isl. Orano porphyry Elba Si. Chiessi dyke Figure 12. Element vs. element ratio diagrams for the Laghetto and other Capraia rocks. Some Elba dykes [15,35] and mafic enclaves from San Vincenzo [16,34] are also plotted for comparison. The dash-dot line is the melting curve of the lower continental crust. The full lines are possible mixing trends between different end members (circled symbols). In conclusion, the melting of the lower continental crust seems the most plausible genetic mechanism for the investigated rocks. These processes occurred at high pressure, probably at the base of a continental crust that was thickened during continental collision. 5.2. Petrogenesis of Laghetto Rocks Mixing between these magmas and other mantle-derived calcalkaline liquids, showing lower Sr contents, modified primary lower crustal melts, thus giving the range of compo- sitions observed at Laghetto. 5.3. Regional Distribution and Geodynamic Significance 5.3. Regional Distribution and Geodynamic Significance High-Sr-Ba andesites, similar to the analysed samples from Capraia, occur elsewhere in the Tuscany Magmatic Province. In particular, a group of dykes from Elba share many characteristics with the Laghetto rocks, but have lower Y and HREE contents [35] and can be also classified as adakitic rocks. The Elba and Capraia adakitic rocks have been erupted around 7–8 Ma and represent some of the oldest rocks of the Tuscany Magmatic Province. Other adakitic compositions might be represented by mafic enclaves in the San Vincenzo rhyolites. These also have high Sr contents, but the available data are insufficient to com- pare these enclaves with the Capraia and Elba high-Sr rocks [14,16,34]. The San Vincenzo rhyolites are sited on mainland Tuscany, east of Capraia and Elba. Their age is 4.5 Ma, younger to coeval to Elba and Capraia activity. Overall, the episode of adakitic-type mag- matism seems to be restricted to the westernmost and oldest sector of the Tuscany Prov- ince. There is a general agreement that the mafic-intermediate magmas in Tuscany are generated in a heterogeneous and anomalous upper mantle, modified by the addition of subduction-related upper crustal material such as pelitic sediments or their metamorphic equivalents. According to some authors [2,3,6,50], modification of the upper mantle oc- curred during southeast directed Alpine subduction of the European plate beneath the Adriatic-African margin. Geosciences 2021, 11, 104 24 of 26 24 of 26 Starting from the Oligocene, there was an inversion of the subduction, with the Adri- atic-African plate immerging westward beneath the older collision zone. New subduction and back-arc extension induced melting of the previously contaminated mantle, which gave several compositionally different magma types, ranging from calcalkaline to potassic lamproitic, according to the depth and degrees of partial melting. Continental collision and inversion of subduction immersion were followed by east- ward migration of the magmatic arc, along with back-arc extension. Collision probably generated a thickening of the continental crust, which was the site of melting and for- mation of adakitic-like magmas. Melting was favoured by the post-collisional extension, which migrated eastward, behind the subduction zone shifting in the same direction. Therefore, these episodes of magmatism occurred in the westernmost sectors where the crustal thickness was produced by the collision. By contrast, they are not present in other younger manifestations of the Tuscany magmatism, probably because back-arc extension reduced the crustal thickness of the continental crust. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Informed Consent Statement: Not applicable. Acknowledgments: The author would like to thank Enrico Pandeli (Department of Earth Sciences, University of Florence) for the fieldwork support. Acknowledgments: The author would like to thank Enrico Pandeli (Department of Earth Sciences, University of Florence) for the fieldwork support. Conflicts of Interest: The author declares no conflict of interest. 6. Conclusions  A series of high-Sr-Ba andesite-dacite rocks occur at Capraia in the Laghetto area, representing a particular stage of calcalkaline activity at ~ 7 Ma. They have a compo- sition that is intermediate between adakitic and calcalkaline rocks.  Geochemical data suggest that these rocks cannot be related to other calcalkaline rocks occurring on the island by fractional crystallisation but represent a distinct type of magma. g  Geochemical modelling suggests that the most likely hypothesis is the generation at mantle pressure by melting of the lower continental crust, followed by mixing with other mantle-derived Capraia magmas.  A similar rock type also occurs at Elba Island and is almost coeval with those from Capraia and possibly at San Vincenzo, on the mainland Tuscany. In contrast, it seems to be absent in other sectors of the Tuscany magmatic province.  The geodynamic model that best explains the composition of the studied rocks is the thickening of the continental crust during a continental collision, followed by exten- sion that favoured melting of the lower crust. 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https://openalex.org/W1860055066	http://www.scielo.br/pdf/reben/v57n1/a06v57n1.pdf	Portuguese	null	The performance of mastectomized women in group activities	null	2,004	cc-by	3,468	* Enfermeira. Professora Doutora do Departamento de Enfermagem da Universidade Federal do Ceará. *Acadêmica de Enfermagem da Universidade Federal do Ceará – Bolsista PIBIC/CNPq. E-mail do autor: afcana@ufc.br Resumo Title: The performance of mastectomized women in group activities Title: The performance of mastectomized women in group activities Descritores: comportamento; câncer de mama; mulheres mastectomizadas; atividades grupais Título: Comportamiento de la mujer mastectomizada frente a las actividades de grupo Título: Comportamiento de la mujer mastectomizada frente a las actividades de grupo 1 Introdução O valor dos grupos de auto-ajuda é destacado quando “o compartilhar experiências comuns, proporciona aos seus integrantes uma enorme energia, que pode ser carreada para as exigências da vida, para a ressocialização e para a recuperação”(3:77). A retirada da mama é um processo cirúrgico, considerado agressivo, visto ser acompanhado de conseqüências, muitas vezes, traumatizantes, nas experiências de vida, e por afetar a saúde da mulher acometida de câncer. Os grupos de auto-ajuda são considerados componentes importantes no processo de reabilitação da mulher mastectomizada, assim como, na sua aceitação do câncer e da condição de mulher que foi submetida a uma mastectomia. Proporcionam o compartilhar de experiências de vida, relacionadas à enfermidade e à busca coletiva de meios de soluções para os problemas. A doença expõe as mulheres a uma série de dificuldades, tais como o desajuste psicológico devido essa doença crônica e mutilante. Ocasiona e limita os movimentos, no caso do membro superior correspondente à mama afetada, impossibilitando a mulher de desenvolver trabalhos domésticos e atividades profissionais exercidas anteriormente. O câncer é uma das doenças que mais induz sentimentos negativos em qualquer um de seus estágios: o choque emocional causado pelo impacto ao tomar conhecimento do diagnóstico; o medo da cirurgia; a incerteza do prognóstico e de uma recorrência deste câncer; os efeitos do tratamento da radioterapia e da quimioterapia; o medo da dor e o pavor de encarar a morte nas circunstâncias com essa que em geral se apresenta(1) . Portanto, diante da importância e necessidade de assistência à mulher mastectomizada, propusemos-nos a estudar a problemática dessa mulher através dos seguintes objetivos: descrever as reações da mulher frente à mastectomia e os sentimentos emergidos a partir da interação grupal. COMPORTAMENTO DA MULHER MASTECTOMIZADA FRENTE ÀS ATIVIDADES GRUPAIS Ana Fátima Carvalho Fernandes Maria Socorro Pereira Rodrigues* Pacífica Pinheiro Cavalcanti** Resumo We aim to describe the reactions of women confronting mastectomy, and the feelings that emerged from group interaction. 10 women from the GEPAM group (Teaching, Research, Self-help and Assistance to Mastectomized Women Group) were interviewed in November and December 2001 and January 2002. Reactions in the face of mastectomy can be singled out as: depression, discrimination, adoption of new behavior and the need for social interaction. Group interaction revealed feelings of friendship and curiosity. These aspects lead us to understand that group activities are important in minimizing the conflicts associated with mastectomy and also in valuing of the feelings of these women, to provide more effective rehabilitation and better quality of life. Es nuestro objetivo describir las reacciones de la mujer frente a la mastectomía y los sentimientos resultantes de la interacción grupal. Se entrevistaron a 10 mujeres integrantes del grupo GEPAM los meses de noviembre y diciembre de 2001 hasta enero de 2002. De las reacciones se puede señalar: la depresión, la discriminación, la adopción de nuevas conductas y necesidad de interacción social. La amistad y la curiosidad son sentimientos que se revelan en la interacción grupal. Dichos aspectos nos hacen comprender que las actividades de grupo ejercen fuerte importancia para minimizar los conflictos que decurren de la mastectomía, así como, para valorizar los sentimientos de esas mujeres, de tal forma que les proporcione una rehabilitación más eficaz y con mejor calidad de vida. Objetivamos descrever as reações da mulher frente à mastectomia e sentimentos emergidos da interação grupal. Foram entrevistadas 10 mulheres integrantes do grupo GEPAM (Grupo de Ensino, Pesquisa, Auto-ajuda e Assistência à Mulher Mastectomizada) nos meses de novembro e dezembro de 2001 a janeiro de 2002. Das reações frente a mastectomia, destacamos: depressão, discriminação, adoção de novos comportamentos e necessidade de interação social. A amizade e a curiosidade foram sentimentos revelados na interação grupal. Estes aspectos levam a compreender que as atividades grupais exercem forte importância na minimização dos conflitos decorrentes da mastectomia, assim como, na valorização dos sentimentos dessas mulheres, de forma a proporcionar uma reabilitação mais eficaz e melhor qualidade de vida. Descriptors: behavior; breast cancer; group activities; mastectomized women Descriptores: comportamiento; cáncer de mama; mujeres mastectomizadas; actividades grupales. 3.1 Reações frente à mastectomia Essa categoria foi subdividida em subcategorias pertinentes às reações que a mulher apresenta em decorrência à retirada de sua mama. 3.1.2 Discriminação Após as exigências regimentares de trâmites no Comitê de Ética, e a autorização concedida pelas participantes, passou- se à coleta de dados, a qual foi realizada semanalmente, nos meses de novembro de 2001 a janeiro de 2002. Apesar de todo avanço na área científica, as pessoas portadoras de alguma neoplasia maligna, ainda, se ressentem de algum tipo de discriminação, como por exemplo, o isolamento social, o que pode ser evidenciado no seguinte discurso: Utilizamos um roteiro de entrevista semi-estruturado, contendo questões abertas, oferecendo ao entrevistador uma certa liberdade para fazer adaptações conforme ocorresse necessidade, sem ter que seguir rigidamente uma ordem pré- estabelecida. Oferece também ao entrevistado a possibilidade de discorrer sobre o tema proposto, sem respostas prefixadas pelo entrevistador(5). [...] É muito ruim a gente ter um caso desse e viver isolada. Às vezes a gente está cheia de problemas em casa [...] (Medusa). O próprio nome câncer é tido como possuidor de um danoso poder mágico, como se a simples pronúncia dessa palavra acarretasse o agravamento ou até mesmo a transmissão daquela doença(8). As respostas aos questionamentos foram anotadas pelo pesquisador, conforme a permissão, concedida pelas mulheres, tendo em vista a importância de se obter uma descrição detalhada das informações fornecidas pelas mulheres. Nas reuniões do GEPAM as mulheres interagem mutuamente, compartilhando experiências de forma que possibilitam a compreensão e a solução de seus problemas e facilitam enfrentar a condição de ser mastectomizada. Após a leitura dos depoimentos das mulheres, estes foram organizados em categorias de acordo com a similaridade de idéias contidas nos mesmos, conforme método de análise de conteúdo, que define categorização como uma operação que classifica elementos constitutivos de um conjunto, por diferenciação e por reagrupamento, considerando suas características particulares e conforme critérios previamente definidos(6). Comportamento da mulher mastectomizada... - Mulheres que estivessem participando do grupo há pelo menos três meses, e que mantivessem uma freqüência regular no grupo. g g p As dez mulheres, incluídas no estudo, receberam nomes fictícios de personagens femininas da mitologia grega, visando preservar o anonimato. 3 Análise e discussão dos resultados q Essas mudanças são enfatizadas nos depoimentos que seguem: Conforme o plano de organização e análise dos dados, esses vêm assim categorizados: [...] Eu não me frustro, eu falo, eu me abro (Pandora). [...] Eu era muito fechada, eu mudei muito (Artêmis). [...] Eu não me frustro, eu falo, eu me abro (Pandora). [ ] E i f h d d i i (A ê i ) [...] Eu não me frustro, eu falo, eu me abro (Pandora). [...] Eu era muito fechada, eu mudei muito (Artêmis). Vê-se pelas falas a disposição renovada de Pandora e Artêmis em exteriorizar seus sentimentos, estando conscientes dos benefícios que podem surgir em decorrência desse ato. As reuniões grupais facilitam a exteriorização de sentimentos e aumentam a relação de ajuda com os membros do grupo. 3.1.3 Adoção de novos comportamentos O câncer é uma patologia em que não se pode, ainda, determinar com precisão a causa, mas vários estudos comprovam a estreita ligação existente entre o câncer e a repressão de sentimentos. É difícil para muitos acreditar que o câncer possa oferecer algum tipo de ganho secundário, mas a experiência de se defrontar com uma doença grave, possibilita ao paciente se beneficiar de mudanças positivas em sua vida, tais como: ir ao encontro de alguns desejos não realizados, priorizar a busca de uma melhor qualidade de vida, exprimir sentimentos que antes eram reprimidos, satisfazer necessidades que antes eram renegadas, desenvolver a capacidade de dizer não e perceber mais claramente que é um ser mortal(7). As categorias foram criadas, a partir do significado emergido dos discursos e categorizadas nas seguintes temáticas: Reações frente a Mastectomia e Sentimentos emergidos da Interação Grupal. Foram posteriormente analisados, tendo-se em vista conceitos e circunstâncias, conforme o referencial teórico da psicologia social. 2 Metodologia Esta pesquisa é de caráter descritivo e exploratório, que tem como enfoque essencial conhecer traços, características e problemas de um indivíduo, grupo ou comunidade, e visa aumentar a experiência do pesquisador em torno do assunto(4). A presente proposta se reporta à importância de grupos de auto-ajuda na reabilitação da mastectomizada, tendo em vista a problemática decorrente desse evento, o qual repercute nos mais diversos aspectos da vida de uma pessoa, desde os psicológicos, passando pelo sociofamiliar e, em particular, pelo espiritual. Com a finalidade de auxiliar essas mulheres mastectomizadas na resolução dos problemas decorrentes do câncer e da mastectomia, têm sido criados, atualmente, muitos grupos de auto-ajuda, os quais são grupos terapêuticos e homogêneos, que visam congregar pessoas que passam pela experiência de ser acometida por um câncer. Esses trabalhos em grupo são conhecidos como self-help, fundamentam-se em ajudar as pessoas a resolver ou minimizar problemas relacionados a traumas decorrentes do acometimento de doenças de natureza aguda, e, em especial, crônicas(2). Rev Bras Enferm, Brasília (DF) 2004 jan/fev;57(1):31-4 31 Comportamento da mulher mastectomizada... Comportamento da mulher mastectomizada... Este estudo foi desenvolvido com mulheres que freqüentam um Grupo de Ensino, Pesquisa e Assistência à Mulher Mastectomizada (GEPAM), o qual funciona na área física do Departamento de Enfermagem da Universidade Federal do Ceará. maioria dos casos existe uma grande tendência para reprimir emoções como a raiva, a tristeza e o medo, podendo gerar um estado de depressão crônica. p Para Simonton(7) “a própria natureza do câncer, com seus altos e baixos, sua incerteza quanto ao futuro, a possibilidade de uma recidiva e da morte cria períodos de depressão”(7:49). Sabendo-se ainda que a depressão acarreta estados de distonias, assim como, estados de amnésias passageiras e de outras intercorrências. Constituindo, por esse motivo, um momento em que a paciente necessita de redobrada atenção, a fim de prevenir-se atitudes de auto ou hetero-destruição. As mulheres participantes do estudo expressaram claramente esse momento de depressão, nos seguintes termos: [...] Porque eu estava muito sensível, qualquer coisa eu estava chorando, me aborrecendo facilmente. Pensei até em me suicidar (Medusa). Participam do GEPAM mulheres mastectomizadas do tipo unilateral e bilateral, algumas passando pela experiência de reconstrução mamária, cuja faixa etária varia de 36 a 70 anos. O estado civil das mulheres varia em casadas, solteiras, viúvas e amasiadas. Suas ocupações variam entre dedicação ao lar, confecção e venda de bijuterias e aposentadas sem uma ocupação mais definida. Foram incluídas na pesquisa mulheres de qualquer idade, crença, procedência, nível socioeconômico ou educacional, tendo sido estabelecidos os seguintes critérios: - Mulheres que apresentassem disponibilidade e aceitação para participar voluntariamente do estudo, concedendo autorização prévia verbal e por escrito para a efetivação da entrevista; - Mulheres que apresentassem disponibilidade e aceitação para participar voluntariamente do estudo, concedendo autorização prévia verbal e por escrito para a efetivação da entrevista; Sabe-se que a mastectomia, cirurgia em que se retira a mama, é em geral indicada em decorrência de uma neoplasia maligna. O fato de constituir um processo cirúrgico agressivo pode desencadear desajuste psicológico na mulher, tendo em vista o caráter mutilador dessa cirurgia. Nos primeiros anos que se seguem a mastectomia, a depressão é, via de regra, uma reação comum, podendo atingir até a metade das mulheres que se submetem a essa cirurgia(1). - Mulheres que estivessem participando do grupo há pelo menos três meses, e que mantivessem uma freqüência regular no grupo. 3.2.2 Curiosidade Troca de experiências, sinto-me muito bem (no grupo) É onde eu me identifico (Pandora). As mulheres, quando submetidas à retirada da mama, sentem necessidade de se manterem informadas sobre tudo que diz respeito à sua saúde. As mulheres que tratam com sucesso o câncer de mama buscam informações, de modo particular sobre seu problema, comportamento esse que parece estar relacionado com a resistência à doença física(1). As palestras, o relaxamento e a troca de experiências (Helena). É As palestras, o relaxamento e a troca de experiências (Helena). É muito bom (o grupo), fico torcendo que chegue sexta- ( g p ), q g feira. Esse ambiente de conversar, de dialogar, é muito bom (Pandora). feira. Esse ambiente de conversar, de dialogar, é muito bom (Pandora). ç As informações. Sou muito curiosa, quanto mais eu sei, mais quero saber sobre essa doença (Afrodite). [...] Aqui (no grupo) a pessoa se distrai, não fica pensando besteira (Hera). q ç ( ) [...] Sinto-me muito bem informada e feliz (no grupo). (Hera) A convivência entre as mulheres que vivenciaram situação com o câncer de mama parece ser um ponto chave na reabilitação das mastectomizadas. Além de diminuir o estigma e o isolamento social associados à doença(3). Uma coisa maravilhosa [...] As informações sobre saúde que aqui recebo (Céris). Nas reuniões, as mulheres manifestam necessidade de interação social, de se relacionarem com outras pessoas que passaram pelo mesmo problema e de buscarem esclarecimentos para as eventuais dúvidas que surgem em decorrência do câncer e o que fazer para continuar vivendo de forma mais saudável e mais feliz. Os profissionais do GEPAM mostram-se disponíveis e capacitados para ajudar as mulheres a se sentirem capazes e responsáveis para contribuir com sucesso no tratamento e conseqüentemente, viver de forma mais saudável. Temos percebido através das situações práticas desenvolvidas com as mulheres que participam do GEPAM, que essas valorizam muito os momentos que são dedicados aos esclarecimentos das suas dúvidas. Os profissionais, sempre que possível, realizam cursos, seminários e palestras que objetivam deixar essas mulheres bem informadas e instruídas a respeito de sua condição no que tange a serem mastectomizadas. As entrevistadas referiram que a integração grupal funciona, também, como um espaço de equilibro. Muitas enfatizam, que o tempo em que estão no grupo é um dos poucos momentos de suas vidas em que se sentem bem e se divertem. 3.1.4 Necessidade de interação social A depressão está intimamente relacionada ao câncer, pois estudos demonstram que mesmo antes do diagnóstico, a pessoa portadora de câncer já se encontra deprimida, pois na As mulheres mastectomizadas entrevistadas revelam que a interação e a partilha de experiências com pessoas que sofrem dos mesmos problemas é uma forma de se incluir no 32 Rev Bras Enferm, Brasília (DF) 2004 jan/fev;57(1):31-4 Fernandes AFC, Rodrigues MSP, Cavalcanti PP. Fernandes AFC, Rodrigues MSP, Cavalcanti PP. Fernandes AFC, Rodrigues MSP, Cavalcanti PP. grupo, de serem apoiadas e a partir daí conseguirem externar seus sentimentos. Neste sentido, foram destacados os seguintes discursos: que uma mais experiente pode esclarecer dúvidas da outra, aconselhar e até mesmo apoiar, dependendo da ocasião. A partir desses momentos, surge então a descoberta de sentimentos, tais como é o caso da amizade. Muito importante [...] a gente partilha [...] tem necessidade de ouvir a outra.É uma forma de apoio (Atena). 3.2 Sentimentos emergidos da interação grupal Nesta categoria destacamos, em particular, os sentimentos das mulheres eclodidos no grupo, elementos de fundamental importância em seu processo de adaptação social. 3.2.2 Curiosidade Vale ressaltar que a grande maioria não dispõe de condições socioeconômicas que favoreçam qualquer tipo de lazer. Pode-se perceber, inclusive, que essas mulheres utilizam termos bastante eloqüentes, do tipo: coisa maravilhosa, é muito importante, para traduzir seu contentamento relacionado ao fato de pertencerem ao grupo. São, portanto, esses aspectos que nos levam a compreender que elas consideram o grupo como um espaço de real significado para suas vidas. A psicoterapia tem por objetivo ensinar essas mulheres a se cuidarem da maneira que elas acreditem que irão se curar, facilitando assim as intervenções médicas tradicionais(1). 4 Considerações Finais As mulheres que tiveram a experiência de serem acometidas por um câncer de mama apresentam muitas reações atribuídas ao fato de terem sido mastectomizadas, tais como: a depressão e a discriminação devido ao caráter estigmatizante que o câncer, ainda, possui. Para minimizar os efeitos negativos que essas reações representam, essas mulheres procuram um grupo de auto-ajuda, que trabalha tendo em vista criar situações para ajudar seus participantes a exteriorizar mutuamente suas dificuldades, o que as ajudará a transpor com melhor desempenho os vários obstáculos surgidos em decorrência da mastectomia.As mulheres são forçadas a adotarem mudanças de comportamento, em decorrência do câncer, o que tem um aspecto positivo, que é favorecer a essas mulheres uma melhor qualidade de vida. Elas admitem que, a partir da doença, passaram a ter vidas melhores e mais plenas.Pode-se afirmar que a interação grupal favorece um espaço em que se percebe claramente o bem- estar das integrantes, expandindo horizontes e estabelecendo laços de amizade fortalecida com a vivência grupal. O grupo GEPAM utiliza um espaço amplo, com condições adequadas para a realização das atividades. São realizados exercícios corporais, com intuito de promover relaxamento físico e mental, e promover a reabilitação do membro superior afetado pela mastectomia. Além dos exercícios físicos, a equipe multiprofissional integrante do GEPAM oferece atividades que favorecem uma melhoria da saúde mental, com a finalidade de diminuir o máximo possível, de forma significante, as crises de depressão e de angústia. 3.2.1 Amizade O compartilhamento de sentimentos e reações, a partir de um relacionamento que favoreça a discussão e exploração das idéias dos participantes constitui formas poderosas de terapia para o câncer de mama. Conseqüentemente o fortalecimento do relacionamento com outras pessoas implica em uma resposta favorável do sistema imunológico, tanto mais forte e eficaz quanto possa ser reforçado pelos demais sistemas do organismo(1). A enfermagem exerce fundamental importância nos trabalhos realizados nesses grupos, em especial no sentido de minimizar os conflitos identificados, através do ensino do autocuidado e da valorização do indivíduo como um ser único, com seus medos e suas dúvidas, visando promover um crescimento individual, a partir da aceitação espontânea de cada indivíduo em sua singularidade, dando-lhe estímulo e apoio. Cabe também à enfermagem buscar a elucidação de dúvidas que surgem em decorrência do tratamento oncológico, podendo deixar a paciente menos apreensiva e mais crente no seu processo de cura. É muito bom conversar com as outras, a amizade com as meninas (Helena). ( ) É uma hora de lazer, de amizade (Afrodite). É uma hora de lazer, de amizade (Afrodite). É É muito importante, venho para cá, sinto-me bem, a amizade (Medusa). Rotineiramente, nas reuniões do GEPAM, são reservados momentos em que as mulheres interagem, particularmente, entre elas mesmas. É nessas ocasiões que elas percebem, que não são as únicas a ter câncer. A partir dessa percepção, elas descobrem que podem se ajudar mutuamente, uma vez Reforçamos a importância da atuação do enfermeiro no preparo do paciente com câncer, para enfrentar dificuldades emocionais, visto que o cuidado deve abranger o apoio emocional, através de um profissional presente e atencioso, Rev Bras Enferm, Brasília (DF) 2004 jan/fev;57(1):31-4 33 Rev Bras Enferm, Brasília (DF) 2004 jan/fev;57(1):31-4 Referências requisito característico da arte de cuidar em enfermagem, e que caracteriza assistência como humanizada. O papel expressivo da enfermeira consiste em cuidar das pessoas de forma integral, total, reconhecendo os principais fatores psicológicos, sociais, espirituais e ambientais, que afetam o bem- estar da paciente, ajudando-a viver de forma mais saudável. 1. Deitos TFH, Gaspary JFP. Efeitos biopsicossociais e psiconeurológicos do câncer sobre pacientes e familiares. Revista Brasileira de Cancerologia; Rio de Janeiro 1997;43(2):117-26. 2. Zimerman DE, Osório LC. Como trabalhamos com grupos. Porto Alegre (RS): Artes Médicas; 1997. A assistência à mulher mastectomizada não deve focalizar apenas a doença e a reabilitação física; deve abranger um contexto amplo, que envolva os aspectos culturais, educacionais, econômicos e sociais de cada uma das mulheres envolvidas no trabalho do grupo(6). É 3. Zukerfield R. Act bulímico, cuerpo y tercera tópica. Buenos Aires: Ricardo Vergara; 1992. 4. Triviños ANS. Introdução à pesquisa em ciências sociais. São Paulo: Atlas;1993.175p. 5. Ludke M, André MED. A Pesquisa em educação: abordagens qualitativas. São Paulo: EPU;1986. É importante que o enfermeiro saiba identificar todas as necessidades que uma paciente mastectomizada apresenta, para então estabelecer cuidados mais efetivos no programa de assistência, respeitando a integralidade e a individualidade do ser humano em questão, facilitando, dessa forma, uma completa reabilitação, tanto física quanto psicológica promovendo uma melhor qualidade de vida e de saúde. 6. Rodrigues MSP, Leopardi MT. O Método de análise de conteúdo: uma versão para enfermeiros. Fortaleza (CE): Fundação Cearense de Pesquisa e Cultura;1999.118 p. 7. Simonton SM. A família e a cura. São Paulo: Summus; 1990. 8. Sontag S. A Doença como metáfora. Rio de Janeiro: Edições Graal; 1984.108 p. Data de Recebimento: 20/05/2003 Data de Aprovação: 30/04/2004 Data de Recebimento: 20/05/2003 Data de Aprovação: 30/04/2004 34 Rev Bras Enferm, Brasília (DF) 2004 jan/fev;57(1):31-4
https://openalex.org/W2079870775	http://cds.cern.ch/record/1747248/files/jpconf15_587_012012.pdf	English	null	Performance of the LHCb calorimeters during the period 2010-2012	Journal of physics. Conference series	2,015	cc-by	4,480	Home Search Collections Journals About Contact us My IOPscience Performance of the LHCb calorimeters during the period 2010-2012 This content has been downloaded from IOPscience. Please scroll down to see the full text. 2015 J. Phys.: Conf. Ser. 587 012012 (http://iopscience.iop.org/1742-6596/587/1/012012) View the table of contents for this issue, or go to the journal homepage for more Download details: IP Address: 128.141.192.149 This content was downloaded on 16/11/2016 at 13:26 Please note that terms and conditions apply. 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Any further distribution of this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI. Published under licence by IOP Publishing Ltd 1 IP Address: 128.141.192.149 This content was downloaded on 16/11/2016 at 13:26 SIMD studies in the LHCb reconstruction software Daniel Hugo Cámpora Pérez and Ben Couturier 16th International Conference on Calorimetry in High Energy Physics (CALOR 2014) IOP Publishing Journal of Physics: Conference Series 587 (2015) 012012 doi:10.1088/1742-6596/587/1/012012 Pascal Perret1 and Xavier Vilas´ıs-Cardona2, on behalf of the LHCb Collaboration. 1 Clermont Universit´e, Universit´e Blaise Pascal, CNRS/IN2P3, LPC, Clermont-Ferrand, France 2 GR-SETAD, La Salle, Universitat Ramon Llull, Barcelona, Spain E-mail: perret@in2p3.fr, xvilasis@salleurl.edu Abstract. The calorimeter system of LHCb is subdivided into four sub-detectors which provide its longitudinal segmentation: a Scintillator Pad Detector, followed by a Preshower and, then, an electromagnetic, an hadronic calorimeter. After a description of these detectors, procedures developed to calibrate their response are discussed together with the degradation of response observed due to the harsh conditions (high radiation and current levels). First results on the calorimeter performance over the two most productive years of physics data taking, 2011 and 2012 are given. Performance of the LHCb calorimeters during the period 2010-2012 Pascal Perret1 and Xavier Vilas´ıs-Cardona2, on behalf of the LHCb Collaboration. 1 Clermont Universit´e, Universit´e Blaise Pascal, CNRS/IN2P3, LPC, Clermont-Ferrand, France 2 GR-SETAD, La Salle, Universitat Ramon Llull, Barcelona, Spain E-mail: perret@in2p3.fr, xvilasis@salleurl.edu 2. Description of the LHCb Calorimeters p The LHCb calorimeter [5] has two main goals: the ﬁrst one is to provide information to the hardware trigger level and the second is to measure the neutral particle energy and to take part to particle identiﬁcation for oﬄine analysis. To reach them it is subdivided into four systems. From the interaction point particles encounter at 12,3 m the SPD, which is designed to identify charged particles, followed by a wall of lead and the PS, then the ECAL and the HCAL. This ensures the characterisation of the longitudinal proﬁle of the showers, along the LHC beam axis. All calorimeters use the same energy detection principle: scintillation light is transmitted to a photomultiplier tube (PMT) by wavelength-shifting (WLS) ﬁbres. The single ﬁbres for the SPD/PS cells are read out using multianode 64 channel photomultiplier tubes (MAPMT), while the ﬁbre bunches in the ECAL and HCAL modules require individual phototubes. Optimal energy resolution requires the full containment of the showers from high energy photons. For this reason, the thickness of ECAL was chosen to be 25 radiation lengths (X0). The PS is 10% as thick as the ECAL. The SPD/PS, ECAL and HCAL adopt a variable lateral segmentation into square cells since the hit density varies by two orders of magnitude over the calorimeter surface. A segmentation into three diﬀerent sections has been chosen for the ECAL and projectively for the SPD/PS. Given the dimensions of the hadronic showers, the HCAL is segmented into two zones with larger cell sizes. The outer dimensions of the ECAL match projectively those of the tracking system, θx < 300 mrad and θy < 250 mrad while the square hole around the beampipe limits the inner acceptance approximately to projective polar angles θx,y > 25 mrad. The main parameters of the sub-detectors are summarised in Table 1. Table 1. 16th International Conference on Calorimetry in High Energy Physics (CALOR 2014) IOP Publishing Journal of Physics: Conference Series 587 (2015) 012012 doi:10.1088/1742-6596/587/1/012012 16th International Conference on Calorimetry in High Energy Physics (CALOR 2014) IOP Publishing Journal of Physics: Conference Series 587 (2015) 012012 doi:10.1088/1742-6596/587/1/012012 introduced. By adjusting the transverse overlap of the beams at LHCb the instantaneous luminosity could be kept stable to within about 5% during a ﬁll. The two most productive years were 2011 and 2012, when 1.107 fb−1 and 2.082 fb−1 of integrated luminosity have been recorded at beam energies 3.5 TeV and 4 TeV correspondingly. introduced. By adjusting the transverse overlap of the beams at LHCb the instantaneous luminosity could be kept stable to within about 5% during a ﬁll. The two most productive years were 2011 and 2012, when 1.107 fb−1 and 2.082 fb−1 of integrated luminosity have been recorded at beam energies 3.5 TeV and 4 TeV correspondingly. The bb cross-section measured by LHCb at 7 TeV constitutes 79 µb [2] in LHCb acceptance, implying ∼100 000 bb pairs produced every second. In each p-p collision about 1500 charged particles are produced resulting in sizeable radiation levels on detectors and readout electronics. In general, the apparatus has shown robust performance even in such a challenging environment. The performance of the calorimeters relies both on their time alignment [3] and calibration [4] which require diﬀerent approaches for each sub-detector and are described in section 3. Section 4 describes the detector ageing. Some performances in terms of particle identiﬁcation are illustrated in section 5. Additional results are available in Ref. [4] and references therein. 1. Introduction The LHCb experiment [1] is one of the four major experiments at the Large Hadron Collider (LHC) of CERN. The detector is a single-arm forward spectrometer covering the pseudorapidity range 2 < η < 5, designed for the study of particles containing b or c quarks. It includes a high-precision tracking system consisting of a silicon-strip vertex detector surrounding the pp interaction, a large-area silicon-strip detector located upstream of a dipole magnet with a bending power of about 4 Tm, and three stations of silicon-strip detectors and straw drift tubes placed downstream. The combined tracking system has a momentum resolution ∆p/p that varies from 0.4% at 5 GeV/c to 0.6% at 100 GeV/c, and an impact parameter (IP) resolution of 20 µm for tracks with high transverse momentum (pT). Charged hadrons are identiﬁed using two ring- imaging Cherenkov detectors (RICH). Photon, electron and hadron candidates are identiﬁed by a calorimeter system consisting of scintillating-pad (SPD) and preshower (PS) detectors, an electromagnetic calorimeter (ECAL) and a hadronic calorimeter (HCAL) which is described in details in the next section. Muons are identiﬁed by a system composed of alternating layers of iron and multiwire proportional chambers. The trigger consists of a hardware stage, based on information from the calorimeter and muon systems, followed by a software stage which applies a full event reconstruction. 32 The LHC delivered instantaneous luminosity at the LHCb interaction point of up to 4×1032 cm−2s−1 for proton energies up to 8 TeV, which was equivalent to the average number of interactions per beam crossing 1.8, while the design value for the LHCb luminosity was 2×1032 cm−2s−1 resulting in an average number of interactions per beam crossing of 0.4, much smaller than what the LHC is capable to provide. In 2011 a luminosity leveling procedure was 1 16th International Conference on Calorimetry in High Energy Physics (CALOR 2014) IOP Publishing Journal of Physics: Conference Series 587 (2015) 012012 doi:10.1088/1742-6596/587/1/012012 3. Monitoring and calibration The SPD calibration is performed by adjusting the discriminator threshold for each channel to yield a pre-deﬁned eﬃciency to identify a charged track. The eﬃciency is computed from the ratio between the number of hits with a track pointing to a given cell over the total number of tracks pointing to it. The result of the eﬃciency as a function of the threshold can be compared with the theoretical expectation given by the integral of the convolution of a Landau and a Poisson distribution. The Landau distribution describes energy lost by charged particle traversing a thin layer of material while the Poisson distribution describes the ﬂuctuations in the number of photoelectrons generated at the photocathode. The resolution in the electronics to set the threshold is 5% of EMIP which directly sets the objective resolution at the 5% level. MIP y j The charge response of the 6016 PS channels is equalised according to the most probable energy deposition of minimum ionising particles (MIPs). The goal is to set the MIPs peak value to 10 ADC counts in the Front-End electronics (FE). It results in a 100 MIPs full dynamic range. Discrimination of e and γ’s at the hardware trigger is then achieved with a typical threshold of 5 - 10 MIP. By tuning the high voltage (HV) of the MAPMT’s one can adjust the gain of a group of 64 PS channels simultaneously. The FE numeric gain with tunable values in the range [1; 2] further allows a ﬁne adjustment of the 64 channels response within a given board. This procedure results in a calibration of the PS better than 5%. Both ECAL and HCAL are equipped with monitoring systems on the basis of light emitting diodes (LEDs). Each photomultiplier is illuminated by light ﬂashes of ﬁxed intensity, so the value of average PMT response can be used to follow up the behaviour of each readout channel (mostly deﬁned by the stability of the PMT gain). In order to eliminate the eﬀects, coming from possible variations of the intensity of LED ﬂash, LEDs themselves are monitored by means of very stable silicon PIN photodiodes. The monitoring of ECAL and HCAL responses is performed online in parallel with data taking. 16th International Conference on Calorimetry in High Energy Physics (CALOR 2014) IOP Publishing Journal of Physics: Conference Series 587 (2015) 012012 doi:10.1088/1742-6596/587/1/012012 16th International Conference on Calorimetry in High Energy Physics (CALOR 2014) IOP Publishing Journal of Physics: Conference Series 587 (2015) 012012 doi:10.1088/1742-6596/587/1/012012 The SPD/PS detector consists of a 15 mm lead converter 2.5 X0 thick, that is sandwiched between two almost identical planes of rectangular scintillator pads of high granularity with a total of 12032 detection channels. The ECAL uses the shashlik calorimeter technology, i.e. a sampling scintillator/lead structure readout by plastic WLS ﬁbres. The energy resolution of the ECAL modules was determined at the test beam, σE/E =a/ √ E⊕b⊕c/E (E in GeV), where a, b and c stand for the stochastic, constant and noise terms respectively. Depending on the type of module and test beam conditions the stochastic and constant terms were measured to be 8.5% < a < 9.5% and b ∼0.8%. The noise term corresponds to 1.2 ADC count. The pedestal subtraction scheme accounts for an underestimation of the pedestal over a 12bit -10 GeV range and corresponds to c = 0.003 GeV. The hadron calorimeter (HCAL) is a sampling device made from iron and scintillating tiles, as absorber and active material respectively. Its thickness is limited to 5.6 nuclear interaction lengths (λint) due to space limitations and the limited performance required. The special feature of this sampling structure is the orientation of the scintillating tiles that run parallel to the beam axis. A resolution of σE/E = (69 ± 5)%/ √ E ⊕(9 ± 2)% (E in GeV) was determined. 2. Description of the LHCb Calorimeters Main parameters of the LHCb calorimeter sub-detectors sub-detector SPD/PS ECAL HCAL number of channels 2×6016 6016 1488 overall lateral 6.2 m × 7.6 m 6.3 m × 7.8 m 6.8 m × 8.4 m dimension in x, y cell size (mm) Inner 39.66 (SPD), 39,84 (PS) 40.4 131.3 Middle 59.5 (SPD), 59.76 (PS) 60.6 Outer 119 (SPD), 119.5 (PS) 121.2 262.6 depth in z 180 mm, 835 mm, 1655 mm, 2 X0, 0.1 λint 25 X0, 1.1 λint 5.6 λint light yield ∼20 p.e./MIP ∼3000 p.e./GeV ∼105 p.e./GeV dynamic range 0 - 100 MIP 0 - 10 GeV ET 0 - 20 GeV ET 10 bits (PS), 1 bit (SPD) 12 bits 12 bits 2 3. Monitoring and calibration LEDs ﬂash with the frequency of about 11 kHz synchronously with one of accelerator service time slot not ﬁlled with protons and a subset at about 50 Hz is sent to a monitoring farm for analysis. The LED system allow to follow fastly the ageing of the detector. It is also used to detect malfunctioning cells and bad cable connections. 0 The ﬁne calibration of ECAL uses ﬁts to the invariant mass distribution of π0 →γγ decays. Photons are deﬁned from 3 × 3 neutral clusters consistent with single photon signals. The cell with the highest energy deposit is called the seed. Each π0 candidate mass is assigned to two cells, one for each photon seed. When enough statistics is collected (for the most peripheral cells it amounts up to few hundred millions of interactions), the π0 mass distribution is ﬁtted for each 3 16th International Conference on Calorimetry in High Energy Physics (CALOR 2014) IOP Publishing Journal of Physics: Conference Series 587 (2015) 012012 doi:10.1088/1742-6596/587/1/012012 cell, and the energy-scale of the cell is corrected to center the mass peak onto the nominal π0 mass (135 MeV/c2). In addition, samples of pure electrons selected from conversions are used to follow fast ageing of ECAL (Section 4) by studying the energy to momentum ratio (E/p) distributions. This last method requires precise information from the tracking system. The ﬁnal precision of the calibration is estimated to be better than 2%. cell, and the energy-scale of the cell is corrected to center the mass peak onto the nominal π0 mass (135 MeV/c2). In addition, samples of pure electrons selected from conversions are used to follow fast ageing of ECAL (Section 4) by studying the energy to momentum ratio (E/p) distributions. This last method requires precise information from the tracking system. The ﬁnal precision of the calibration is estimated to be better than 2%. In addition to use of the LED system, more precise HCAL calibration is performed on the regular basis with a cesium radioactive source calibration system. Two 10 mCi 137Cs sources (one per each detector half) are moved by a hydraulic system through each scintillator tile. The PMT response is measured with a dedicated system of current integrators. Relation between anode current and the measured particle energy is known from test beam studies, which showed also that this method facilitates a cell-to-cell intercalibration level 2-3%. 3. Monitoring and calibration Such precision is more than enough to fulﬁll all HCAL objectives in LHCb. 4. Detector ageing Over the ﬁrst years of almost continuous LHCb operation, signs of the degradation of detector response was observed in all four calorimeter sub-detectors. So far, two major reasons for it have been identiﬁed. The ﬁrst is the radiation damage of scintillator tiles and WLS ﬁbers, which manifests itself in the areas with largest hit densities, especially in the innermost regions around the beam pipe. This eﬀect is proportional to the particle ﬂux (charged and neutral particles). In the ECAL inner most region, up to ∼0.25 Mrad/year are observed. For instance it was found that the precision of the ECAL monitoring based on LED suﬀers from the radiation degradation of transparency of polystyrene clear ﬁbers used to transport the LED light to the ECAL photomultipliers. In order to improve the performance of the monitoring system, and especially in view of signiﬁcant increase of LHCb working luminosity foreseen after 2018, the present plastic ﬁbers were replaced by radiation hard quartz ﬁbers [6]. [ ] For now, the most extensive studies of its eﬀects was done for HCAL, where light yields can be controlled directly with the 137Cs sources of the calibration system. The average relative decrease in the light yield for 44 most central HCAL cells is shown in Fig. 1 (left) as a function of the luminosity, delivered by LHC over 2011 and 2012, separately for each layer of scintillator tiles. As expected, the most aﬀected by radiation layer is the frontal one, which corresponds to the shower maximum position, thanks to the additional 1.1 λint of ECAL. The second factor is Figure 1. Left: relative decrease of HCAL light yield for diﬀerent layers of scintillator tiles, averaged over the 44 innermost cells, as a function of the delivered luminosity. Right: PMT gain degradation as a function of the integrated anode current measured at lab. Figure 1. Left: relative decrease of HCAL light yield for diﬀerent layers of scintillator tiles, averaged over the 44 innermost cells, as a function of the delivered luminosity. Right: PMT gain degradation as a function of the integrated anode current measured at lab. PMT sensitivity losses. The eﬀect was studied in laboratory, the tested photomultiplier being PMT sensitivity losses. 4. Detector ageing The eﬀect was studied in laboratory, the tested photomultiplier being 4 16th International Conference on Calorimetry in High Energy Physics (CALOR 2014) IOP Publishing Journal of Physics: Conference Series 587 (2015) 012012 doi:10.1088/1742-6596/587/1/012012 kept under continuous illumination by short light pulses to emulate the LHCb environment. Figure 1 (right) shows the relative change of PMT response with the integrated charge, which has passed through the device since the beginning of the test. The initial sharp decrease of the gain noticeably slows down after 150 coulombs, though it should be kept in mind, that this test did not reproduce exactly the real operational conditions with periods of inactivity between ﬁlls, allowing some recuperation of PMT sensitivities. This eﬀect depends upon cell size and location. The actual amounts of integrated charge, obtained over 2011 and 2012, is estimated with current integrators, which readings are continuously logged. In case of HCAL, the total charge, which passed through the innermost photomultipliers during 2011, was on the level of 100 C. Prior to the beginning of 2012 data taking, the gains of all HCAL PMTs were decreased by a factor of two in order to reduce the sensitivity losses and also to keep the PMT anode currents within the recommended limits in the face of the increase of luminosity-setting. For ECAL, the maximal integrated charge was on the level of 20 C for 2011 and 37 C for 2012. These are well known unavoidable eﬀects which aﬀect the trigger rate based on calorimeter information as well as detector performances. They required regular calibrations using the methods described in Sect. 3 to restore uniform detector responses. In addition the cumulative change in the response of the calorimeter sub-detectors could be estimated with cell occupancies in minimum bias events, thanks to the luminosity leveling system providing relatively stable conditions over long periods of data taking. This method is very promising and preliminary studies have shown that a precision of ∼1% could be achieved after few hours of data taking. It will be implemented for an automatic and online calibration for the next data taking period (Run 2) to adjust PMT high voltage after each LHC ﬁll of data taking to obtain a stable trigger. 5. Performance of the LHCb calorimeters 5.1. Electron identiﬁcation 2 Candidates / 50 MeV/c 0 20 40 60 80 100 120 140 160 (b) LHCb Data Full fit γ φ → 0 s B Combinatorial 0 π - K + K → s 0 B X 0 π V → B γ * Λ → b 0 Λ ) 2 ) (MeV/c γ - K + M(K 4500 5000 5500 6000 -5 0 5 2 Candidates / 25 MeV/c 0 100 200 300 400 500 600 (a) LHCb Data Full fit γ * K → 0 d B Combinatorial γ 0,+ π 0 K → 0,+ B 0 π -π + K → 0 B γ Λ → 0 b Λ X 0 π V → B 0 π -π + K → 0 s B γ 0 K → 0 s B ) 2 ) (MeV/c γ π M(K 4500 5000 5500 6000 -5 0 5 Figure 2. Mass distribution of the reconstructed B0 →K∗0(K+π−)γ candidates (a) and B0 s → φ(K+K−)γ candidates (b) obtained with an integrated luminosity of L=1.0 fb−1 [7]. The black points represent the data and the ﬁt result is represented as a solid blue line. The signal (green dotted line) and the various background contaminations are shown. Neutral pions with low transverse energy are mostly reconstructed as a resolved pair of well separated photons. A mass resolution of 8 MeV/c2 is obtained for such neutral pions. However, due to the ﬁnite ECAL granularity, photon pairs from the decay of suﬃciently high momentum π0 cannot be resolved as individual clusters. This is essentially the case for all π0 decays with transverse momentum above 2 GeV/c. To reconstruct such π0 candidates a procedure has been design to identify merged clusters. The algorithm consists of splitting each single ECAL cluster into two 3 × 3 subclusters built around the two highest deposits of the original cluster. The energy of the common cells is then distributed over the two assumed subclusters according to an iterative procedure based on the expected transversal shape of photon showers. The division of the energy depends on the barycenter position of each subcluster. Since the latter is a function of the energy distribution, this procedure requires a few iterations, but it is found to converge quickly. 5. Performance of the LHCb calorimeters 5.1. Electron identiﬁcation The electron identiﬁcation (ID) in the calorimeter system uses the background and signal likelihood distributions constructed for the ECAL, the PS and the HCAL sub-detector [4]. The performance is evaluated using the data recorded in 2011. The available statistics allows the electron ID eﬃciency to be measured using a tag-and-probe method. The method is applied to B± →J/ψK± candidates having J/ψ →e+e−, where one of the electrons is required to be identiﬁed by electron ID (etag) while the second electron is selected without using any information from the calorimeter system (eprobe). This second electron is used to estimate the eﬃciency of the electron ID and the performance of the electron hypothesis test. The eﬃciency and misidentiﬁcation rate are obtained as a function of the eprobe momentum. By way of illustration of the typical performance, for electrons from J/ψ →e+e−decay in B± →J/ψK± events the average identiﬁcation eﬃciency in the complete calorimeter system is (91.9±1.3)% for a misidentiﬁcation rate of (4.54±0.02)%. In addition information from the RICH detectors can be used to improved these performances allowing to reach an average identiﬁcation eﬃciency up to 97% for a misidentiﬁcation rate lower than 2%. 5.2. Isolated photon and neutral pion reconstruction Energy deposits in ECAL cells are clusterised applying a 3x3 cell pattern around local maxima of energy deposition. Consequently the centers of the reconstructed clusters are always separated at least by one cell. If one cell is shared between several reconstructed clusters, the energy of the cell is redistributed between the clusters proportionally to the total cluster energy. The process is iterative and it converges rapidly due to relatively small ratio of the Moliere radius (3.5 cm) to the cell size. After the redistribution of energy of shared cells, the energy-weighted cluster moments up to the order 2 are evaluated to provide the (hypotheses-independent) cluster parameters, namely, the total energy, the energy barycenter position and the two-dimensional 5 16th International Conference on Calorimetry in High Energy Physics (CALOR 2014) IOP Publishing Journal of Physics: Conference Series 587 (2015) 012012 doi:10.1088/1742-6596/587/1/012012 16th International Conference on Calorimetry in High Energy Physics (CALOR 2014) IOP Publishing Journal of Physics: Conference Series 587 (2015) 012012 doi:10.1088/1742-6596/587/1/012012 energy spread matrix. Neutral clusters are identiﬁed as those clusters that do not match to charged tracks. energy spread matrix. Neutral clusters are identiﬁed as those clusters that do not match to charged tracks. 6. References [1] LHCb Collaboration, Jr. A. A. Alves et al., The LHCb detector at the LHC, JINST, 3:S08005, 2008. [2] LHCb Collaboration, R. Aaij et al., Phys. Lett., B694 (2010) 209. [3] C.Abellan Beteta et al., JINST, 7:P08020, 2012. [4] C.Abellan Beteta et al., Performance of the LHCb Calorimeters, CERN-LHCb-DP-2013-004, 2013. [5] S. Amato et al., LHCb calorimeters: Technical Design Report, CERN, Geneva, LHCb-TDR-2, 2000. [6] Y. Guz and LHCb collaboration, CALOR 2014 conference proceedings, LHCb-PROC-2014-015. [7] LHCb Collaboration, R. Aaij et al., Nucl. Phys. B867 (2013) 1. 5. Performance of the LHCb calorimeters 5.1. Electron identiﬁcation The photon energy, Ec, is evaluated from the total cluster energy and the reconstructed energy deposit in the PS. The performance of high energy photon reconstruction is illustrated by the reconstructed B0 →K∗0γ and B0 →φγ mass distribution [7] shown in Fig. 2. The mass resolution obtained for this radiative decay, ∼92 MeV/c2, is dominated by the ECAL energy resolution. A comparison of the data to simulated samples shows that this corresponds to an intrinsic resolution in the photon energy of around 2% [4]. 2 Candidates / 25 MeV/c 0 100 200 300 400 500 600 (a) LHCb Data Full fit γ K → 0 d B Combinatorial γ 0,+ π 0 K → 0,+ B 0 π -π + K → 0 B γ Λ → 0 b Λ X 0 π V → B 0 π -π + K → 0 s B γ 0 K → 0 s B ) 2 ) (MeV/c γ π M(K 4500 5000 5500 6000 -5 0 5 2 Candidates / 50 MeV/c 0 20 40 60 80 100 120 140 160 (b) LHCb Data Full fit γ φ → 0 s B Combinatorial 0 π - K + K → s 0 B X 0 π V → B γ Λ → b 0 Λ ) 2 ) (MeV/c γ - K + M(K 4500 5000 5500 6000 -5 0 5 Figure 2. Mass distribution of the reconstructed B0 →K∗0(K+π−)γ candidates (a) and B0 s → φ(K+K−)γ candidates (b) obtained with an integrated luminosity of L=1.0 fb−1 [7]. The black points represent the data and the ﬁt result is represented as a solid blue line. The signal (green dotted line) and the various background contaminations are shown. 5. Performance of the LHCb calorimeters 5.1. Electron identiﬁcation A π0 →γγ invariant mass resolution of ∼30 MeV/c2 is obtained for these ‘merged’ π0 candidates while it is close to ∼20 MeV/c2 for ‘resolved’ π0. Acknowledgements Work supported by CNRS/IN2P3, Region d’Auvergne (France); MICINN and GENCAT (Spain 6. References 6
https://openalex.org/W2775056057	https://revistas.javeriana.edu.co/index.php/MAGIS/article/download/20612/15799, https://dialnet.unirioja.es/descarga/articulo/6230814.pdf	es		El camino de la resiliencia: del sujeto individual al sujeto político	Magis	2,017	cc-by	11,971	El camino de la resiliencia: magis del sujeto individual al sujeto político The Path of Resilience: from the Individual Subject to the Political Subject Le chemin de la résilience: du sujet individuel au sujet politique O caminho da resiliência: do sujeito individual ao sujeito político Fecha de recepción: 27 DE ENERO DE 2016/Fecha de aceptación: 25 DE JULIO DE 2017/Fecha de disponibilidad en línea: 1 DE DICIEMBRE DE 2017 Encuentre este artículo en http://magisinvestigacioneducacion.javeriana.edu.co/ doi: 10.11144/Javeriana.m10-20.crsi Escrito por lUiS FernanDo granaDoS-oSpina, S.j. ponTiFicia UniverSiDaD javeriana c ali, c olomBia lfgranados@javerianacali.edu.co Sara vicToria alvaraDo-SalgaDo cinDe-UniverSiDaD De m aniZaleS m aniZaleS, c olomBia doctoradoumanizales@cinde.org.co jaime c armona-parra Resumen Este artículo de revisión parte de algunas definiciones de la categoría de resiliencia e identifica en ellas diversas tendencias y enfoques. Desde una valoración de la experiencia humana y de la significación que tienen las interacciones sociales y las acciones comunitarias para la construcción de la resiliencia, el artículo permite explorar las fronteras donde la resiliencia se encuentra con la política, entendida en el sentido amplio como acción colectiva. La respuesta resiliente buscaría configurar sentidos y prácticas personales y comunitarias para afrontar la adversidad, desde una acción política comprometida con la dignidad humana. Los métodos utilizados son una revisión documental y un análisis categorial, que buscan ofrecer elementos teóricos para poner en diálogo la resiliencia con la política. cinDe-UniverSiDaD De m aniZaleS m aniZaleS, c olomBia coor.investigaciones@cinde.org.co Palabras clave Resiliencia; jóvenes; política; acción comunitaria Para citar este artículo / To cite this article / Pour citer cet article / Para citar este artigo Granados-Ospina, S.J., Luis Fernando; Alvarado-Salgado, Sara Victoria & Carmona-Parra, Jaime (2017). El camino de la resiliencia: del sujeto individual al sujeto político. magis, Revista Internacional de Investigación en Educación, 10 (20), 49-68. https://doi.org/10.11144/Javeriana.m10-20.crsi VOLUMEN 10 / NÚMERO 20 / JULIO-DICIEMBRE DE 2017 / ISSN 2027-1174 / BOGOTÁ-COLOMBIA / Páginas 49-68 3 Abstract Resilience; young people; politics; community action This is a literature review that starts from some definitions of the category of resilience, identifying in them diverse tendencies and approaches. From an assessment of human experience and the significance of social interactions and community actions for the construction of resilience, the article allows us to explore the frontiers where resilience meets politics, understood in the broad sense as collective action. The resilient response would seek to configure personal and community senses and practices to face adversity, from a political action committed to human dignity. The method used is a literature review and a categorical analysis, which seeks to offer theoretical elements to put the resilience in dialogue with politics. Mots clés Résumé Résilience; jeunes; politique; action communautaire Article de synthèse qui part des définitions de la catégorie de résilience, pour identifier dans celle-ci les différentes tendances et perspectives. A partir d'une appréciation de l'expérience humaine et de la signification qui ont les interactions sociales et les actions communautaires pour la construction de la résilience, l'article permet explorer les lisières où la résilience se trouve avec la politique, en tant qu'action collective; c'està-dire dans sa signification la plus ample. La réponse résiliente cherche façonner les sens et les pratiques personnelles et communautaires pour faire face à l'adversité, à partir d'une action politique engage avec la dignité humaine. La méthode utilisée est une révision documentaire et une analyse de catégories, qui vise à fournir les éléments théoriques pour mettre en place un dialogue entre la résilience et la politique. Palavras-chave Resumo Resiliência; jovens; política; ação comunitária Artigo de revisão que parte de algumas definições da categoria resiliência, identificando nelas diversas tendências e enfoques. Desde uma avaliação da experiência humana e da significação que têm as interações sociais e as ações comunitárias para a construção da resiliência, o artigo permite explorar as fronteiras onde a resiliência se encontra com a política, entendida no sentido amplo como ação coletiva. A resposta da resiliência procuraria configurar sentidos e práticas pessoais e comunitárias para enfrentar a adversidade, desde uma ação política comprometida com a dignidade humana. O método utilizado é uma revisão documental e uma análise categorial, que procura oferecer elementos teóricos para pôr em diálogo resiliência y política. magis Keywords VOLUMEN 10 / NÚMERO 20 / JULIO-DICIEMBRE DE 2017 / ISSN 2027-1174 / BOGOTÁ-COLOMBIA / Páginas 49-68 PÁGINA 50 Introducción magis Este artículo parte de una mirada crítica sobre algunas definiciones de resiliencia, para reconocer el valor que estos acercamientos conceptuales tienen en la comprensión de los tránsitos de la categoría desde una visión más individualista centrada en el sujeto heroico, hacia una visión más social y política centrada en el sujeto histórico. Aquí se valoran los procesos de socialización e interacción, como lugares experienciales donde los sujetos afrontan la adversidad con otros y desde otros. Estos sujetos son actores sociales situados históricamente, que habitan territorios complejos donde se cruzan intereses y búsquedas, realidades de inequidad e injusticia, como la misma construcción social de la resiliencia que propugna por respuestas creativas a los retos que hoy enfrenta la sociedad en un contexto globalizado. El camino de la resiliencia: del sujeto individual al sujeto político PÁGINA Origen de la categoría de resiliencia: primera y segunda generación de investigadores Es notable que la Real Academia de la Lengua en la 23ª edición (2014) de su diccionario haya incorporado la palabra resiliencia y aunque la definición presentada no dé cuenta del constructo en su complejidad y dinamismo, tiene el valor de ofrecer un reconocimiento y visibilidad a una palabra que, en su uso, se ha ido incorporando en la cotidianidad del lenguaje, para nombrar los tránsitos que experimentan los sujetos y las comunidades a partir del afrontamiento de situaciones de adversidad. Resiliencia. Del ingl. resilience, y este der. del lat. resiliens, -entis, part. pres. act. de resilīre ‘saltar hacia atrás, rebotar’, ‘replegarse’. 1. f. Capacidad de adaptación de un ser vivo frente a un agente perturbador o un estado o situación adversos. 2. f. Capacidad de un material, mecanismo o sistema para recuperar su estado inicial cuando ha cesado la perturbación a la que había estado sometido (Real Academia Española, RAE, 2014). En el campo de las Ciencias Sociales, los estudios en resiliencia han ido cobrando una gran relevancia, como demuestra la producción de trabajos de grado en las universidades colombianas en la última década (Campo-Vásquez, Granados-Ospina, S.J., Muñoz-Ortega, Rodríguez-Arenas & Trujillo-García, 2012). Tal interés está ocupando a psicólogos, médicos, sociólogos, antropólogos, educadores, ecologistas y trabajadores sociales, entre otros, que han venido encontrando en la resiliencia una categoría que en su reflexión va dando luces para comprender cómo los sujetos y las comunidades se transforman y trabajan por una vida digna, en medio de situaciones de adversidad. La resiliencia se presenta, efectivamente, como un verdadero disfraz de Arlequín. Las numerosas disciplinas que la consideran como objeto de estudio y los múltiples paradigmas en los que se inscribe han contribuido a dar de ella una visión polisémica, incluso caótica. Es cierto que su complejidad es tal que ha requerido un análisis en segmentos separados mediante abordajes diferentes. Hoy en día, la tendencia es la pluridisciplinariedad y el planteamiento integrativo: genético, psíquico, medioambiental, biológico, cultural, emocional, educativo, cognitivo (Pourtois, 2014, pp. 71-72). El término que procede del vocablo latino resilio (Kotliarenco, Cáceres & Fontecilla, 1997) está indicando un proceso humano que tiene qué Descripción del artículo \| Article description \| Description de l'article \| Artigo descrição Este artículo de revisión surgió en el trabajo de investigación doctoral La resiliencia y lo político: en estudiantes vinculados al Programa “Ser Pilo Paga” de la Universidad Javeriana de Cali, que tuvo como finalidad ampliar la frontera del saber epistémico sobre la resiliencia, desde el horizonte de lo político y la política, para establecer un diálogo entre resiliencia y política. 51 magis ver con volver atrás, resaltar y rebotar hacia adelante para salir fortalecido. La resiliencia aparece como “resistencia” a los golpes de la vida en la que acontecen conflictos y tensiones. El concepto hizo tránsito de la física a las ciencias sociales, precisamente por su flexibilidad para nombrar experiencias de vida que en sus relatos podrían integrar lo que Boris Cyrulnik llamó oxímoron: “figura retórica que consiste en reunir dos términos antinómicos” (Cyrulnik, 2001, p. 21). Según Boris Cyrulnik, el oxímoron se vuelve característico de una personalidad herida, pero resistente; sufriente, pero fuerte a pesar de todo, una persona que frente a una experiencia dura sigue tejiendo la vida con esperanza, sin dejarse destruir por la desgracia. VOLUMEN 10 / NÚMERO 20 / JULIO-DICIEMBRE DE 2017 / ISSN 2027-1174 / BOGOTÁ-COLOMBIA / Páginas 49-68 PÁGINA Por analogía, en las ciencias humanas y particularmente en la psicología se utilizó este término para dar cuenta de la capacidad humana que permite 52 a las personas, que a pesar de atravesar situaciones adversas, dolorosas o difíciles puedan salir de ellas no solamente a salvo, sino aún enriquecidas por la experiencia (Cyrulnik, Tomkiewicz, Guénard, Vanistendael, Manciaux & Balegno, 2004, p. 12). Cyrulink de manera coloquial comenta cómo fue llegando casi fortuitamente al concepto, al contar la historia de unos marineros que hablaban de la resiliencia. Estos trabajadores explicaban cómo los submarinos eran sometidos a tensión, compresión, golpes, pero que, al pasar las pruebas, mostraban cierta elasticidad para volver a recuperarse. El hecho de que no se fueran al fondo y salieran de nuevo, expresaba su “resiliencia”: una capacidad reconocida para recuperar su forma natural después de haber sido expuestos a fuertes presiones. Una desgracia nunca es maravillosa. Es un fango helado, un barro negro, una escara dolorosa que nos obliga a escoger: someterse o sobreponerse. La resiliencia define el resorte de los que, habiendo recibido un golpe, han podido sobrepasarlo. El oxímoron describe el mundo íntimo de esos vencedores heridos (Cyrulnik, 2001, p. 23). Esta cualidad resiliente de soportar golpes y salir fortalecido, adquiere mucha potencia en el ámbito de la construcción humana y social, e invita a un cambio de actitud frente al propio desarrollo humano y a la manera como los sujetos afrontan la adversidad en medio del dolor de sus heridas. Sin desconocer la complejidad de la adversidad, el concepto en sus orígenes emerge para dar cuenta de los tránsitos del sujeto para hacerse más humano en medio de sus adversidades. En consecuencia, no se trata de decir: usted está herido, por lo tanto está perdido. Sino: usted está herido: ¿qué va a hacer con esta herida? ¿Va usted a gemir, a llorar? ¿Va usted a permanecer prisionero del pasado? ¿Va usted a buscar manera de vengarse? ¿Va usted a buscar el modo de hacer una carrera de víctima, tal como lo proponen todas las sociedades? O, por el contrario, con nuestra ayuda y su trabajo, ¿vamos los dos a comenzar a poner en obra un proceso de reparación y de reanudación del desarrollo? (Cyrulnik, Manciaux, Sánchez-Botero, Colmenares, Olaya & Balegno, 2002, pp. 31-32). Boris Cyrulnik, Michel Manciaux, Stefan Vanistendael y Jacques Lecomte (2003, p. 20) advierten que uno de los primeros que usó el concepto de resiliencia fue el psicólogo John Bowlby (1995). Al respecto, sostienen que Bowlby entendía la resiliencia como un: “resorte moral, cualidad de una El punto clave de mi tesis es que existe una intensa relación causal entre las experiencias de un individuo con sus padres y su posterior capacidad para establecer vínculos afectivos y que ciertas variaciones corrientes de dicha capacidad, que se manifiestan en problemas conyugales y conflictos con los hijos, así como en síntomas neuróticos y trastornos de la personalidad, pueden atribuirse a determinadas variaciones corrientes de los modos de desempeñar los padres sus correspondientes roles (Bowlby 1995, p. 163). Boris Cyrulnik reconoce que John Bowlby “lo único que deseaba al final de su vida era que comenzaran los trabajos sobre resiliencia” (Cyrulnik, 2001, p. 14). Cyrulnik recogió los aportes de la teoría del apego y trabajó este tema en su obra Bajo el signo del vínculo: una historia natural del apego (Cyrulnik, 2005a). En su ejercicio reflexivo sobre los complejos mecanismos de los vínculos atiende la amplia gama de señales que fundan el vínculo con la madre, muestra que solo entre los humanos hace falta algo más que la conducta de la paternidad biológica y alude a la función paternal simbólica, que está en la raíz de la existencia como seres culturales. Cuando las gaviotas y los bebés lloran, nos hacen comprender que toda información está escrita en lo biológico. Y cuando esta estimulación es percibida, adquiere sentido porque es interpretada. La historia de quien percibe da sentido a esa percepción. Lo biológico y lo histórico entremezclados nos permiten asistir al nacimiento del sentido (Cyrulnik, 2005a, p. 66). No obstante, para tener una base sólida acerca del surgimiento de la resiliencia hay que remitirse a los estudios longitudinales realizados por las psicólogas estadounidenses Emmy E. Werner y Ruth S. Smith (1982, 1992, 2001) sobre infancia en situación de riesgo, al acompañar el desarrollo de más de 200 niños que pudieron sobreponerse a pesar de haber tenido una infancia difícil. Francisca Infante-Espínola (2002) reconoce que el trabajo de estas autoras fue el detonante de la preocupación futura sobre la resiliencia. Sin que ese trabajo indagara precisamente sobre esta temática, las observaciones y preguntas que suscitó tuvieron una repercusión definitiva en el surgimiento de la resiliencia como tema investigativo. Un aspecto de la investigación realizada por Emmy E. Werner y Ruth S. Smith (1982) llamó poderosamente la atención: algunos niños supieron “sobreponerse” a partir de una infancia difícil. Las expectativas de fracaso fueron superadas y tuvieron unos desarrollos exitosos. Otros no desarro- magis PÁGINA El camino de la resiliencia: del sujeto individual al sujeto político persona que no se desanima, que no se deja abatir” (Cyrulnik, Manciaux, Vanistendael & Lecomte, 2003, p. 20). Bowlby, que trabajaba en las carencias de cuidados maternales, se inspiró en la etología animal para impulsar sus estudios sobre la vinculación y fundamentar su teoría del apego en la configuración de vínculos seguros con los cuidadores, vínculos que tienden a perpetuarse y a favorecer al sujeto. Bowlby creía que el grado de respuesta frente a los hechos estresantes de la vida se hallaba sólidamente determinado por las estructuras de vinculación que el sujeto desarrollaba en sus primeros años de vida. El apego, según Bowlby (1995), está dirigido hacia uno o algunos individuos, por lo general con un claro orden de preferencia y tiende a persistir durante una gran parte del ciclo vital. Hay una relación causal entre los vínculos establecidos con los padres o cuidadores y la capacidad futura del niño para establecer vínculos afectivos y reaccionar frente a eventos desestabilizadores. Bowlby (1995) pone la génesis de la resiliencia en las relaciones vinculares que construye el sujeto, desde los mecanismos del apego. 53 magis VOLUMEN 10 / NÚMERO 20 / JULIO-DICIEMBRE DE 2017 / ISSN 2027-1174 / BOGOTÁ-COLOMBIA / Páginas 49-68 PÁGINA 54 llaron los trastornos previstos. Antes bien, mostraron una “admirable invulnerabilidad”. La pregunta era obligada: ¿cómo explicar esta realidad? ¿Cómo argumentar el hecho de que algunos individuos resistieron mejor que otros los embates de la vida, la adversidad y la enfermedad? ¿Esto tenía una explicación? Según la mirada de Infante-Espínola (2002), este trabajo inspiró lo que se ha llamado la primera generación de investigadores en resiliencia, venidos especialmente de Estados Unidos y Gran Bretaña. Estos académicos centraron su atención en los atributos individuales, los vínculos afectivos, el ambiente social protector de los sujetos, y en los factores diferenciadores que posibilitan las particulares respuestas de los sujetos frente a condicionamientos de entornos difíciles. A esto se lo llamó el modelo triádico de la resiliencia, que se configuraba desde los atributos personales, el ámbito familiar y los escenarios sociales donde acontece la vida de los sujetos (Infante-Espínola, 2002). Desde un principio, las relaciones sociales amables y seguras, los vínculos afectivos y las expresiones de cariño y acogida fueron factores que protegían al sujeto como un resorte frente al futuro. La capacidad para dar y recibir afecto es la verdadera fuente de la resiliencia. Saber amar, ser solidario, acompañar al otro en su dolor, establecer lazos cálidos y serviciales es el principal factor que promueve la resiliencia: Los primeros trabajos en resiliencia se realizaron en personas con trastornos patológicos como esquizofrenia, personas expuestas al estrés y sujetos en condición de pobreza, en busca de reconocer el funcionamiento de estos frente a situaciones adversas (Cicchetti, 2003). Tanto la escuela anglosajona, como la europea han situado el tema de la resiliencia desde una perspectiva psicosocial. Norman Garmezy (1991), en New York, Estados Unidos, ha sido uno de los pioneros en la conceptualización y el estudio de la capacidad de recuperación de los sujetos sometidos a situaciones de adversidad. Según Garmezy, la resiliencia es entendida como: “la capacidad para recuperarse, y mantener una conducta adaptativa, después del abandono o la incapacidad inicial al iniciarse un evento estresante” (Garmezy, 1991, p. 459). No obstante, uno de los asuntos difíciles de la resiliencia es su propia definición, como lo señala Michel Rutter (2012) al insistir en el carácter dinámico del concepto. El regreso a la vida se realiza en secreto, con el extraño placer que proporciona el sentimiento de vivir una prórroga. El trauma ha hecho añicos la personalidad anterior, y cuando nadie reúne los pedazos para frenar su dispersión, el sujeto queda muerto o no vuelve bien a la vida. Sin embargo, cuando se ve sostenido con la afectividad cotidiana de las personas que están cerca de él, y cuando el discurso cultural da sentido a su herida, consigue retomar un tipo de desarrollo distinto (Cyrulnik, 2005b, p. 21). Elisardo Becoña, psicólogo clínico y profesor de la Universidad de Santiago de Compostela, recoge en su artículo Resiliencia: definición, características y utilidades del concepto (Becoña, 2006), una buena síntesis de la primera generación de investigadores. En su trabajo se advierte que los primeros estudios en resiliencia se concentraron en individuos con esquizofrenia, en personas que habían estado sometidas a situaciones de pobreza extrema y traumas en su vida temprana y que no demostraban en su vida futura problemas psicológicos o de adaptación social. Este énfasis en los vínculos amorosos se hace recurrente en las primeras investigaciones. Stefan Vanistendael y Jacques Lecomte (2002) relatan en sus trabajos cómo muchos niños y jóvenes pudieron surgir y desarrollarse en contextos culturales y sociales diferentes, gracias a la presencia amorosa de alguien que les brindó en un momento de la vida una aceptación incondicional. La resiliencia nos recuerda que todos nosotros, niños y adultos, necesitamos ser amados. El amor significa la aceptación profunda de la personalidad, su reconocimiento estable en la vida del otro, aun cuando se manifiesten comportamientos o rasgos de carácter que no se puedan aceptar (Vanistendael & Lecomte, 2002, p. 29). La resiliencia es un fenómeno que manifiestan sujetos jóvenes que evolucionan favorablemente, aunque hayan experimentado una forma de estrés que en la población generalmente se estima que implica un grave riesgo de consecuencias desfavorables (Rutter, 1993, p. 626). El origen del estudio de la resiliencia en psicología y psiquiatría procede, por tanto, de los esfuerzos por conocer la etiología y el desarrollo de la psicopatología, especialmente de los niños en riesgo de desarrollar psicopatología debido a enfermedades mentales de los padres, problemas perinatales, conflictos interpersonales, pobreza o una combinación de estos factores (Becoña, 2006, p. 126). Aquí se ve el interés de los primeros investigadores por la psicopatología, al pensar la resiliencia desde la experiencia de niños, niñas y jóvenes en situación de riesgo y vulnerabilidad, que han padecido o sufrido enormes problemas, traumas o situaciones estresantes, desgarradoras o desafiantes en su infancia y que han tenido la capacidad de recuperarse y de mantener una conducta normal. Por eso, no es de extrañar que las primeras definiciones de resiliencia de esta generación de investigadores preferentemente anglosajones, hablen de la resiliencia como una capacidad adaptativa que posibilita buenos resultados en los sujetos, a pesar de tener muchas y serias amenazas para su desarrollo (Garmezy, 1991). El concepto no pretende presentar una imagen heroica de unos niños en 1991, p. 459). Desde la mirada adaptativa se ve claro que la resiliencia requiere para su verificación una evaluación cualitativa del funcionamiento del sujeto dentro de los parámetros de lo que se acepta como normal (Masten, 1994). Tal evaluación se hace desde los criterios de normalidad establecidos e implica una visión del desarrollo normalizado en diferentes contextos culturales. La idea es poder determinar lo que se considera “apropiado” y “normal” tanto en el área cognitiva, como en conductas emocionales y sociales. La segunda generación de investigadores fue más allá de las cualidades personales, los factores protectores, los vínculos amorosos y los procesos de “adaptabilidad” y se preguntó por las condiciones psicosociales y aquellos procesos sociales y culturales que favorecen o dificultan una respuesta del sujeto frente a la adversidad. Estos investigadores se distanciaron de una concepción de la resiliencia centrada en la herencia o en los genes del sujeto como algo constitutivo, y la consideraron como algo que está presente en las respuestas que los sujetos dan a determinadas situaciones adversas (Kalawski & Haz, 2003). Las preguntas de los investigadores de esta generación están centradas sobre todo en los procesos sociales y en los factores que inciden en los sujetos al tener que dar una respuesta en situaciones de dificultad, para conceptuar que en la resiliencia se suman factores individuales, familiares y sociales, unidos a factores protectores compuestos por recursos personales y sociales (Gómez & Kotliarenco, 2010). Entre los trabajos investigativos de esta segunda generación son muy bien valorados los de la psicóloga argentina Edith Henderson-Grotberg (2006). Esta autora postula una mirada optimista de la resiliencia y la entiende como una capacidad humana que poseen los sujetos para el afrontamiento de la adversidad. Henderson-Grotberg, desde una perspectiva psicosocial, entiende la resiliencia asociada a factores ambientales contextuales que la posibilitan: Poseemos esa capacidad humana que nos hace resilientes, lo que nos permite poder afrontar el bombardeo de situaciones y acontecimientos estresantes. Al afrontar esos acontecimientos nos volvemos más fuertes, más seguros de nuestras capacidades, más permeables al estrés que otras personas estén experimentando y también más hábiles para producir cambios y minimizar o eliminar aquello que nos hace daño (Henderson-Grotberg, 2006, p. 10). Como bien lo refieren Anna Forés y Jordi Grané (2008), esta segunda generación, liderada por Edith Henderson-Grotberg (2006), se concentra PÁGINA El camino de la resiliencia: del sujeto individual al sujeto político pacidad de recuperación y mantener una conducta adaptativa que puede seguir al retroceso o incapacidad al iniciar un evento estresante (Garmezy, magis comparación con otros que se enfrentan a situaciones similares de riesgo o desesperación. De hecho, hablar de resiliencia no refleja necesariamente una impermeabilidad al estrés. Más bien, está diseñado para reflejar la ca- 55 no tanto en las cualidades individuales y atributos personales, como en los procesos y los vínculos que permiten la configuración de una respuesta que articula lo propio del sujeto con los apoyos del ambiente. Todo individuo tiene una capacidad para la resiliencia que debe ser reconocida. Pueden descubrirse rasgos de resiliencia en casi cualquier persona, si se le examina en búsqueda de signos de resiliencia con la magis misma minuciosidad con la que se procura detectar VOLUMEN 10 / NÚMERO 20 / JULIO-DICIEMBRE DE 2017 / ISSN 2027-1174 / BOGOTÁ-COLOMBIA / Páginas 49-68 PÁGINA 56 La resiliencia no es una sustancia, algo hecho; más problemas y déficit. El proceso de adquirir resiliencia bien es una obra de tejedora que enhebra la seda es, de hecho, el proceso de la vida, dado que toda de las capacidades personales con la seda afectiva y la gente debe superar episodios de estrés, trauma y social en un proceso inacabado. Todas las personas rupturas en el proceso de vivir. Una actitud construc- hemos de tejer nuestra personalidad con el ganchi- tora de resiliencia en la escuela debe buscar “todo llo de un punto de encuentro afectivo y social. Di- indicio” de resiliencia, rastreando ocasiones en las cho con otras palabras, la resiliencia es un proceso que tanto docentes como alumnos sortearon, supe- diacrónico y sincrónico donde las fuerzas biológicas raron, sobrellevaron o vencieron la adversidad que se articulan con el contexto social para posibilitar la enfrentaban (Henderson & Milstein, 2003, p. 22). transformación de la persona traumatizada (Forés & Grané, 2008, p. 32). Los mismos autores entienden la resiliencia como “una metáfora de las posibilidades” (Forés & Grané, 2012, p. 12) y la sitúan en diálogo constructivo con escenarios socioeducativos de socialización, para transitar hacia una perspectiva holística de la resiliencia y destacan tres aspectos básicos observables según ellos en los procesos de resiliencia: la energía de relación, la energía de aprendizaje y la energía creativa. La experiencia de los vínculos y las sinergias nutrientes, los aprendizajes vitales y las formas siempre nuevas de superar las barreras y los obstáculos de la vida. El modelo holístico de resiliencia no está orientado a desarrollar fórmulas o recetas simplificadas aplicables a distintos contextos, sino a ofrecer herramientas que puedan sacar a la luz la riqueza y profundidad de los recorridos resilientes. Precisamente a En los trabajos de Michel Manciaux (2003), Stefan Vanistendael y Jacques Lecomte (2002) y Boris Cyrulnik (2001), investigadores de segunda generación, hace eco la pregunta que ya se habían planteado los primeros investigadores, en relación con las respuestas diferentes de sujetos que viven situaciones adversas similares. Pregunta que, en el caso de los primeros investigadores, se había respondido por la vía de la invulnerabilidad y la resistencia al estrés por parte de niños considerados por ellos como excepcionales (Garmezy, 1991). En estos autores, la fuerza la tienen tanto el sujeto, como los vínculos y las interacciones en los diferentes escenarios donde acontecen los procesos de socialización y convivencia. La resiliencia exige una construcción social, un compartir la vida con otros. No podemos desarrollar resiliencia en el aislamiento radical. Si bien el sujeto puede salir hacia adelante con su esfuerzo personal, la resiliencia se hace más profunda con los otros. través de esta complejidad es posible desvelar dinámicas y sinergias que pueden aplicarse en la promo- No se puede desencadenar un proceso de resiliencia ción de los procesos de resiliencia específicos (Forés si se está solo. La “falta de sentido”, la necesidad de & Grané, 2012, p. 30). construir relatos… El relato que hago yo mismo de lo que me ha ocurrido, el relato que haces tú de lo Otros investigadores reconocidos de esta nueva generación son Nan Henderson y Mike M. Milstein (2003), que llevaron el asunto de la resiliencia a la escuela como escenario formativo, en espera de que allí se estimularan factores protectores, tanto internos como ambientales, que mitigaran el impacto de las situaciones adversas en niños, jóvenes y demás miembros de la comunidad educativa. Desde la perspectiva de estos autores, los trabajos en resiliencia desplazaron el modelo de desarrollo humano centrado en la patología y se aproximaron a un modelo constructivo basado en el trabajo compartido para un mejor vivir. El modelo propuesto desafía los diagnósticos que rotulan y clasifican enfermedades, para fijarse en las fuerzas que mantienen sana a la gente. que me ha ocurrido y el relato que hace la cultura de lo que me ha ocurrido… Mediante la convergencia de todos estos relatos culturales es como voy a tener una representación de mí mismo. Si estás herido, quizás te van a acompañar y te ayudarán a reanudar tu desarrollo. Ahí hay posibilidad de resiliencia (Cyrulnik, 2014, pp. 33-34). Stefan Vanistendael (2014) reconoce que no hay una única definición de resiliencia. Le apuesta a una comprensión más pragmática, al entender que la resiliencia inspira formas creativas para afrontar la vida desde una esperanza realista. La articulación de estas dos realidades estaría en el corazón, en la esencia de la resiliencia. A falta de definición universalmente reconocida de la resiliencia humana, proponemos una simple definición pragmática: la capacidad de una persona o de un grupo para crecer en presencia de muy grandes dificultades… la resiliencia no es fija, sino que varía a lo largo de toda la vida; nunca es absoluta; se construye en interacciones con el entorno… siempre se encuentra en proceso; y, más allá de la simple resistencia, construye o reconstruye la Desde la perspectiva de Boris Cyrulnik, Michel Manciaux, Stefan Vanistendael y Jacques Lecomte (2003), la resiliencia es un proceso de agenciamiento de la vida, un quehacer dinámico, evolutivo en los sujetos, en los que las adversidades pueden ser afrontadas desde recursos propios y apoyos del entorno. magis vida (Vanistendael, 2014, p. 53). La resiliencia es la capacidad de una persona o de un grupo para desarrollarse bien, para seguir proyectándose en el futuro a pesar de acontecimientos desestabilizadores, de condiciones de vida difíciles y de traumas a veces graves… La resiliencia nunca es absoluta, total, lograda para siempre. Es una capacidad que resulta de un proceso dinámico, evolutivo, en la que la importancia de un trauma puede superar los recursos del sujeto; varía según las circunstancias, la naturaleza del trauma, el contexto y la etapa de la vida; puede expresarse de modos muy diversos según la cultura (Cyrulnik, Manciaux, Vanistendael & Lecomte, 2003, pp. 22-23). Boris Cyrulnik, Michel Manciaux, Stefan Vanistendael y Jacques Lecomte (2003) mantienen una mirada crítica y realista sobre los propios límites y posibilidades de la resiliencia. Esto es notable, pues en la literatura estudiada, existe poca capacidad crítica para examinar la resiliencia, al reconocer sus límites tanto en sus fundamentos epistemológicos, teóricos y metodológicos, como en sus intencionalidades éticas, estéticas, políticas y sus usos en el ámbito de la intervención social. Desde una concepción sistémica y después de haber trabajado con familias funcionales y en situaciones de riesgo, Froma Walsh (2004) propone una comprensión sistémica de la resiliencia en estos términos: La resiliencia puede definirse como la capacidad de una persona para recobrarse de la adversidad fortalecida y dueña de mayores recursos. Se trata de un proceso activo de resistencia, autocorrección y crecimiento como respuesta a la crisis y desafíos de la vida (Walsh, 2004, p. 26). Froma Walsh (2004) reconoce que es necesario poner en tensión una concepción tradicional que afirma que no es posible eliminar los efectos de los traumas graves que se han sufrido en la infancia. Esas realidades que pasan y que dejan heridas y huellas profundas difícilmente se reparan. Frente a esta visión dramática, la postura de la autora es optimista y esperanzadora: La resiliencia implica algo más que la mera posibilidad de sobrevivir a un suplicio horrible, atravesarlo o eludirlo… Las virtudes propias de la resiliencia permiten a las personas curar sus dolorosas heridas, hacerse cargo de su vida, amar y desarrollar una existencia plena… La resiliencia se forja cuando el individuo se abre a nuevas experiencias y actúa en forma interdependiente con los demás (Walsh, 2004, p. 27). La resiliencia se entiende aquí como una interacción entre lo innato y lo adquirido entrelazado por adecuadas relaciones de apoyo. No obstante, El camino de la resiliencia: del sujeto individual al sujeto político PÁGINA 57 desde su comprensión sistémica, adquieren relevancia los ámbitos familiares, sociales, económicos, políticos y culturales donde acontece la vida de los sujetos y de las comunidades. Al pensar la resiliencia familiar y con una mirada latinoamericana, se destaca el trabajo de Esteban Gómez y María Angélica Kotliarenco (2010), autores que revisaron los antecedentes de la resiliencia familiar desde sus orígenes y la ponen en diálogo con la intervención clínica y psicosocial, con familias altamente vulnerables. Llama la atención su definición de resiliencia familiar. magis Proponemos que la resiliencia familiar se defina como el conjunto de pro- VOLUMEN 10 / NÚMERO 20 / JULIO-DICIEMBRE DE 2017 / ISSN 2027-1174 / BOGOTÁ-COLOMBIA / Páginas 49-68 PÁGINA cesos de reorganización de significados y comportamientos que activa una familia sometida a estrés, para recuperar y mantener niveles óptimos 58 de funcionamiento y bienestar, equilibrar recursos y necesidades familiares, y aprovechar las oportunidades de su entorno (Gómez & Kotliarenco, 2010, p. 124). Boris Cyrulnik (2001) profundiza en la concepción de proceso y en el poder evocador de las narrativas en resiliencia. Cyrulnik reconoce que la acción del sujeto es vital, pues es capaz de relatar la propia historia, darle un sentido y transformarla. La idea de proceso permite entender la dinámica resiliente en función de un caminar que integra múltiples factores, unos de riesgo y otros protectores. La resiliencia constituye un proceso natural en el que lo que somos en un momento dado necesariamente debe entretejerse con los medios ecológicos, afectivos y verbales. Basta con que uno solo de esos medios falle para que todo se hunda. Basta con que haya un solo punto de apoyo para que la edificación pueda continuar (Cyrulnik, 2001, p. 15). María Cristina García-Vesga y Elsy Domínguez de la Ossa, en su trabajo más cercano a nosotros Desarrollo teórico de la resiliencia y su aplicación en situaciones adversas (García-Vesga & Domínguez de la Ossa, 2013), hacen una revisión analítica de varias definiciones y coinciden en que la mayoría de los teóricos entiende la resiliencia como un proceso social e intrapsíquico que se da en las interacciones entre sujetos. Estas investigadoras reconocen que las definiciones transitan entre las que relacionan el concepto con adaptabilidad, las que introducen el término de capacidad o habilidad, las que enfatizan el nexo entre factores internos y externos y aquellas que definen la resiliencia como adaptación y proceso. También son notables los trabajos de Gema Puig y José Luis Rubio, sobre todo en dos textos: Manual de resiliencia aplicada (2011) y Tutores de resiliencia. Dame un punto de apoyo y moveré MI mundo (2015). En el primer texto, después de un exhaustivo trabajo de documentación en el campo de la resiliencia, los autores advierten que sigue habiendo una dificultad para hablar del concepto, por la proliferación de definiciones. Nos encontramos frente a un constructo complejo, conformado por múltiples componentes, lo cual complica su definición… La resiliencia es un concepto en continua evolución, dado que han sido muchos los autores que han propuesto una definición (Puig & Rubio, 2011, pp. 38, 40). Estos autores reconocen la necesidad de pensar interdisciplinarmente la resiliencia, valoran la vida cotidiana como lugar donde acontece la lucha por la supervivencia propia de la esencia humana, se sitúan en una visión optimista sobre la vida y valoran la búsqueda de la felicidad como un motivador para la acción en medio de la adversidad. Algo interesante es su acercamiento a la construcción social de la resiliencia, al reconocer el papel de la comunidad y las redes vinculares relacionadas con el cuidado, el apoyo y el afecto. El nicho ecológico es la red de relaciones necesarias para la supervivencia. Y es que, gracias a la reciprocidad afectiva y comunicativa, los niños tejen su Cyrulnik define el encuentro con el tutor como algo fugaz. Un encuentro significativo puede ser suficiente. Y, aunque podría pensarse que, al tratarse de un momento tan efímero, algo tan aparentemente insignificante, el efecto fuese mínimo, un encuentro casual de estas características, en determinados momentos se constituye como el detonante de la recuperación, como la chispa que reaviva los rescoldos. Por tanto, con una capacidad reconstructora y regeneradora como para impulsar un proceso de resiliencia (Puig & Rubio, 2015, p. 139). Aunque el encuentro sea casual o anecdótico, no lo es necesariamente la fuerza de la representación que de él se tiene. El recuerdo del encuentro pervive y sigue aconteciendo como dinamismo transformador que impulsa al sujeto a seguir tejiendo su vida en medio de la adversidad. Desde este horizonte relacional se entiende la fuerza que los autores les dan a la dimensión comunitaria y social de la resiliencia y a aquellas redes vinculares que se vuelven significativas en un momento particular de la vida. Por eso no se cansan de insistir en la construcción dialógica de los procesos de vida, que le da a la resiliencia un claro sentido comunitario y social. El concepto de resiliencia no es un atributo personal, nos remite a la importancia de la imbricación de la persona en su matriz social… es falso el dilema establecido entre la aportación personal y la aportación social. Sabemos que las dos cosas están presentes en la resiliencia, y la dosis dependerá de cada caso… podemos afirmar que la dimensión comunitaria es una condición sine qua non (Forés & Grané, 2008, pp. 34-35). El concepto además está reclamando un abordaje interdisciplinar que aporte miradas diferentes que lo enriquezcan (Madariaga, Palma-García, Surjo, Villalba & Arribillaga, 2014). Esto es precisamente lo que había constatado Michel Delage (2010) al reconocer que, para comprender la resiliencia, se ha de recurrir a campos de conocimiento heterogéneos que permitan abarcar especialmente los aspectos relacionales y contextuales. Según la perspectiva de Delage (2010), la resiliencia solo puede comprenderse desde una mirada más compleja y sistémica. PÁGINA El camino de la resiliencia: del sujeto individual al sujeto político En el segundo libro Tutores de resiliencia. Dame un punto de apoyo y moveré MI mundo (2015), un texto escrito en un lenguaje vivencial, los autores reconocen la significación que para la resiliencia tienen los nichos sensoriales, los puntos de apoyo, los vínculos, las interacciones, los encuentros, la presencia de los otros en tiempos de crisis y el fortalecimiento de los vínculos, como potentes mediadores para suscitar la resiliencia. magis red de relaciones, en primer lugar, con sus cuidadores, que les transmiten su experiencia y los protegen de los peligros, y posteriormente con la sociedad (Puig & Rubio, 2011, p. 125). 59 La otra corriente, que podemos calificar de holista o global, sostiene, que ciertos fenómenos solo pueden comprenderse en una perspectiva concentrada en la complejidad, es decir, en la manera en la que se intersecan los diferentes niveles, los diferentes sistemas para producir consecuencias que no pueden reducirse al análisis de las partes separadas. Evidentemente, en el caso de la resiliencia, debe prevalecer magis esta segunda actitud, pues se trata de una noción compleja y que, por lo tanto, requiere de diferentes niveles de abordaje (Delage, 2010, p. 90). VOLUMEN 10 / NÚMERO 20 / JULIO-DICIEMBRE DE 2017 / ISSN 2027-1174 / BOGOTÁ-COLOMBIA / Páginas 49-68 PÁGINA 60 Ámbito latinoamericano: la resiliencia y su dimensión comunitaria, social y política En el ámbito latinoamericano, las investigaciones en resiliencia se han concentrado más en la aplicabilidad del concepto, mediante modelos y programas de intervención que manifiestan un interés más práctico, en la búsqueda de un fortalecimiento de las prácticas de niños, mujeres y familias, en contextos socioculturales específicos (Álvarez & Hurley, 2010). En estos trabajos investigativos y de intervención social, se busca poder entender cómo algunas comunidades, o grupos sociales, incluso ciudades y pueblos enteros, se pueden reponer propositivamente, en medio de calamidades desgarradoras, fuertes catástrofes naturales o en circunstancias agobiantes de violencia social, económica y política. Sin duda, desde la perspectiva comunitaria de la resiliencia, hay que reconocer el aporte del Centro de Estudios y Atención al Niño y la Mujer, CEANIM, organización no gubernamental que, por más de 34 años en Chile, se ha orientado al estudio, diseño y ejecución de programas de desarrollo humano, comprometidos con la realidad de personas que viven en situación de pobreza y de desventajas sociales, económicas y culturales desde el horizonte de la resiliencia. Desde la visión de su directora María Angélica Kotliarenco, la resiliencia se puede definir de la siguiente manera: procesos de desarrollo infantil, se revisaron algunos fundamentos psicológicos del concepto, algunas particularidades metodológicas en los estudios sobre resiliencia y se introdujo propiamente la concepción de la resiliencia desde América Latina. De sus interesantes aportes en cuanto a la comprensión del concepto de resiliencia podemos destacar lo siguiente: La resiliencia se produce en función de procesos sociales e intrapsíquicos. No se nace resiliente ni se adquiere “naturalmente” en el desarrollo: depende de ciertas cualidades del proceso interactivo del sujeto con los otros humanos, responsable de la construcción del sistema humano (Melillo, Estamatti & Cuestas, 2000, p. 28). En sintonía con estos ejercicios reflexivos, es notable el trabajo que realizó María Eugenia Colmenares (2002a, 2002b) con otros académicos sensibles al tema de la resiliencia en Colombia. Sus reflexiones pusieron en tensión la comprensión de la resiliencia con los fenómenos sociales y políticos del país pasados por las violencias, las múltiples pobrezas y la inequidad. Plantear la resiliencia como instrumento teórico de interrogación sobre la coyuntura histórica que vive Colombia señala que este planteamiento no desborda el espacio propio de la reflexión desde la psicología, sino que propone otros elementos a la representación del discurso social para favorecer las rupturas de lugares comunes y permitir la construcción de otras representaciones de lo real que puedan contribuir a modificar apreciaciones y comportamientos (Colmenares, 2002a, p. 17). La definición que aporta María Eugenia Colmenares (2002b) sobre la resiliencia, tiene valor en la medida en que pone a dialogar el concepto con el compromiso ético y político de los sujetos que afrontan la adversidad en búsqueda de sentidos de vida y transformaciones históricas. De acuerdo a los procesos reflexivos realizados, podemos definir la resiliencia como un proceso dinámico, de origen interactivo y sociocultural que conduce a la optimización de los recursos humanos y permite sobreponerse a las situaciones adversas, gracias a la defensa que le brindan los mecanismos protectores, sean estos individuales, familiares, comunitarios y/o culturales (Kotliarenco, 2014. p. 163). Esta autora y otros investigadores muy reconocidos, como Aldo Melillo y Elbio Néstor Suárez-Ojeda publicaron Actualizaciones en resiliencia (Kotliarenco, Mardones, Melillo & Suárez, 2000). En este trabajo se puso de relieve la cercanía de la resiliencia con los Llamamos resiliencia a la capacidad que muestran algunos seres humanos, en medio de las peores adversidades, de tomar el contrapié a partir de sí mismos y que les permite desarrollarse y construir sus sueños conservando la estructura humana en términos de valores éticos de supervivencia (Colmenares, 2002b, p. 22). María Stella Rodríguez-Arenas (2004) hace un interesante trabajo al poner en diálogo la resiliencia con el desplazamiento forzado desde las vivencias de niños víctimas del conflicto armado. A partir de una alternativa pedagógica que tiene en la palabra su fuente de inspiración, permite recuperar al sujeto como actor de su proyecto de vida, emergiendo visiones prospectivas, ideales, sueños e ilusiones, que de alguna manera manifiestan cualidades resilientes, expresadas en la capacidad de estos niños, para convertir su experiencia de vida en narración, que les permita visualizar las experiencias difíciles como una parábola, con la posibilidad de crear un final feliz. La resiliencia no resulta solo de lo individual o lo colectivo, sino que deri- que las personas puedan responder con esta fortaleza a las dificultades de la vida (Rodríguez-Arenas, 2004, p. 78). Los trabajos de Aldo Melillo y Elbio Néstor Suárez-Ojeda (2002) y de estos con Daniel Rodríguez (2004) posicionaron una concepción latinoamericana de la resiliencia en situaciones de exclusión y de pobreza, que acentúa su dimensión comunitaria y la significación que para estas situaciones tienen los procesos resilientes. Tales perspectivas se distanciaban de la mirada clásica de los primeros estudios en resiliencia, cuyo interés se concentraba en los atributos individuales y en la relación de estos con los entornos sociales, y en reconocer el poder de la resiliencia para luchar contra las inequidades de la sociedad actual: A partir de este enfoque, la resiliencia comunitaria desplaza la base epistemológica del concepto inicial, modificando no solo el objeto de estudio, sino también la postura del observador y los criterios de observación y validación del fenómeno… Cada desastre o catástrofe que una comunidad sufre representa un daño en términos de pérdidas de recursos y de vidas… Esa desgracia puede significar el desafío para movilizar las capacidades solidarias de la población y emprender procesos de renovación, que modernicen no solo la estructura física sino toda la trama social en esa comunidad (Suárez, 2002, pp. 70-71). La perspectiva que aporta Elbio Néstor Suárez-Ojeda (2002) permite entonces pensar la posibilidad de una “teoría latinoamericana de la resiliencia, con enfoques más adecuados a esta realidad social” (Suárez, 2002, p. 67) que se sabe imbricada entre los problemas de una sociedad inequitativa, excluyente e injusta y los desafíos que estas realidades plantean a la misma resiliencia y su capacidad para potenciar respuestas que permitan fortalecer las dinámicas de agenciamiento y liberación de los sujetos y las comunidades y sus prácticas de resistencia y liberación frente a poderes que estructuralmente violentan la dignidad humana. En su trabajo, después de reconocer el avance y posicionamiento de la resiliencia en América Latina, Elbio Néstor Suárez-Ojeda (2002) presenta algunos pilares de lo que él llama resiliencia comunitaria. Tales pilares son: “la autoestima colectiva, la identidad cultural, el humor social y la honestidad estatal” (Suárez-Ojeda, 2002, p. 72). De la autoestima colectiva se destacan la actitud y el sentimiento de orgullo por el lugar en que se vive, el aprecio por la geografía, el clima y las tradiciones culturales. Se trata de un reconocimiento de lo que se es, de los orígenes y de las herencias recibidas de los antepasados. No es extraño que aquellas ciudades o localidades en las que se observa PÁGINA El camino de la resiliencia: del sujeto individual al sujeto político muchas de las facultades humanas no es totalmente innata, ni totalmente adquirida más bien requiere de unas condiciones particulares que permitan magis va del interjuego de ambos aspectos. El punto de encuentro de estas dos posiciones radica en que es una posibilidad del ser humano de resignificar la adversidad en términos positivos… pensamos que esta capacidad como 61 magis VOLUMEN 10 / NÚMERO 20 / JULIO-DICIEMBRE DE 2017 / ISSN 2027-1174 / BOGOTÁ-COLOMBIA / Páginas 49-68 PÁGINA 62 una elevada autoestima y orgullo de sus tradiciones tengan mayor capacidad de recuperación frente a la adversidad. Por identidad cultural se reconoce esa persistencia del ser social en su unidad y “mismidad” por medio de cambios y circunstancias diversas. Tiene qué ver con la valoración de los rasgos culturales que nos develan sentido de pertenencia a un territorio y una cultura propia y singular. De allí que aquellas poblaciones que han hecho un baluarte del respeto y la exaltación de sus culturas tradicionales hayan mostrado una mayor capacidad para recomponerse y renacer luego de numerosas adversidades. Otra variable importante en la resiliencia es el humor, que ha merecido un amplio estudio. El humor social como una capacidad propia de la idiosincrasia de personas y grupos de rasgos culturales particulares se potencia al encontrar la “comedia” en la propia tragedia, que se apoya en la flexibilidad y la relajación que puede brindar cierto sentido del humor, en medio de la complejidad de las situaciones, lo cual favorece la capacidad para hallar respuestas originales y soluciones innovadoras en medio de la crisis. Un último componente es el designado como honestidad colectiva o estatal. Más allá de la transparencia en el manejo de la cosa pública, esto implica la existencia de una conciencia colectiva que desaprueba la deshonestidad de los funcionarios públicos y que valora un ejercicio decente de la gestión estatal, al reconocer su esfuerzo por respetar los derechos de los ciudadanos. Se trata de una intolerancia activa frente a la corrupción que se ha venido instalando en muchos niveles de la sociedad, tanto en la esfera pública como en la privada. Otros asuntos como la capacidad de generar liderazgos auténticos y participativos, el ejercicio de una democracia efectiva en la toma de decisiones, en las que se reconozca la diversidad y no haya discriminación, fortalecen también el proceso de la resiliencia comunitaria. En contraste con estos pilares, desde la perspectiva de Suárez-Ojeda (2002), están los “antipilares”. Como antípodas de estos pilares, se han podido Este desplazamiento de las investigaciones centradas en el sujeto herido que afronta la adversidad y en las comunidades sometidas a tensiones y conflictos sociales complejos, se fue abriendo camino, no sin la preocupación que manifestaban algunos académicos por el mismo concepto de resiliencia, su significación y sus usos en algunos programas de atención e intervención a la infancia y adolescencia (Llobet & Wegsman, 2004). Valeria Llobet (2005) más adelante afirma que el mismo concepto puede ser mal interpretado y usado de manera tendenciosa, desde su asociación con intervenciones pobres para pobres, pasando por la promoción de un conformismo con connotaciones conservadoras. Y considera que hay una línea de problemas teóricos, políticos y éticos, cuyo debate según la autora no ha sido saldado entre los investigadores en resiliencia. Es sencillo suponer que, o bien es posible predicar de alguien que “es” resiliente por sus peculiares características personales (inteligencia, astucia, etc.) lo que sería una aproximación psicologista y objetivizante, o que en realidad el ascenso social depende de las mismas capacidades por las cuales alguien puede lograr que la adversidad no se transforme en destino (Llobet, 2005, p. 13). Frente a esta crítica que se deja planteada, la autora reconoce que la resiliencia puede tener una utilidad científica y que en medio de sus tensiones teóricas y de intervención, puede ser significativa para explicar problemas de vida, al favorecer la apertura hacia caminos creativos de reconstrucción personal y social. La resiliencia, entonces, no es un rasgo de personalidad, sino que las personas son actores y fuentes de las actuaciones resilientes, y las familias, escuelas, comunidades, servicios sociales son el escenario de promoción de la resiliencia, y propician el despliegue, proveen de los factores protectores… redefinimos el concepto como procesos resilientes, entendiéndolos como la posibilidad de protección y autonomía frente a situaciones de adversidad apoyados en el proceso de subjetivación (Llobet, 2005, pp. 14-15). detectar condiciones o características que reducen la resiliencia comunitaria o, dicho de otra manera, inhiben la capacidad solidaria de reacción frente a la adversidad colectiva. Entre estos “antipilares” cabe mencionar: malinchismo, fatalismo, autoritarismo, corrupción (Suárez-Ojeda, 2002, p. 76). Las comunidades combinan pilares y “antipilares” y de su reconocimiento se podrán elaborar predicciones, que ayuden a orientar y fortalecer la intervención social para poder dinamizar la resiliencia colectiva o comunitaria. Según Valeria Llobet (2005) es la interfase entre los niveles individuales, comunitarios y sus interacciones, lo que hace más interesante el concepto y lo que permite reconocer la potencia de la resiliencia, para abandonar un determinismo fatal, encontrando en ella una posibilidad para recuperar una visión de la micropolítica como espacio para la resistencia a la reproducción y las dinámicas del poder, que tiende a hegemonizar a los sujetos y las comunidades para que escriban una única historia, lo que les impide la creatividad para escribir su propio relato. La resiliencia está zativa, comunitaria, nacional), entendida como la capacidad de un sistema de absorber los choques o presiones externas y de reorganizarse, sin perder su estructura, función e identidad esencial (Azkarraga, 2014, pp. 92-93). Esta manera de entender la resiliencia deja abierta la posibilidad de poner en diálogo las ciencias sociales con las ciencias naturales pues entiende que la crisis global es una crisis socioecológica, que está reclamando soluciones integradoras en cuanto a una auténtica transición hacia la sostenibilidad responsable del planeta. El aporte latinoamericano propone un desplazamiento de la mirada más centrada en lo individual de los sujetos, hacia el ámbito de lo social, donde acontecen los procesos de interacción y de construcción del tejido comunitario, de participación democrática y acción política. La resiliencia comunitaria implica una conciencia de responsabilidad común frente a lo que afecta al sujeto como colectivo. La resiliencia comunitaria permite explorar las fronteras donde la resiliencia se encuentra con la política. Desde este horizonte comprensivo, se deben reconocer los esfuerzos de los investigadores por enfrentar diversas problemáticas sociales desde la mirada resiliente. El trabajo de Patricia Granada-Echeverry y Sara Victoria Alvarado (2010) se centró precisamente en la relación entre resiliencia y política, y buscaba mostrar la contribución de la resiliencia en el fortalecimiento de la subjetivación política de niños y jóvenes que, en situación de adversidad, son capaces de agenciar experiencias vitales, desde la construcción de narrativas que les permiten actuar políticamente en sus vidas y sus entornos. Lo novedoso de este acercamiento es el de haber puesto en la mesa de la discusión la dimensión sociopolítica de la resiliencia, o la significación de la resiliencia en el horizonte de lo político que acontece en la vida de la gente. Propusimos en este estudio la definición sociopolítica de la resiliencia como: la conservación, despliegue y desarrollo de las dimensiones humanas en contextos de adversidad, entre los que se destacan la dimensión crítica y la dimensión política que no se subordinan a la sobrevivencia, sino que, por el contrario, se transforman en discurso político, en posiciones de denuncia y de acción social transformadora ante el mundo. Esta definición sociopolítica de la resiliencia exigió redefinir el concepto de adversidad a partir de significados que incluyeran los significantes culturales, como las situaciones vividas cuya significación social estuviera ligada al sufrimiento humano como experiencia vital que hunde sus raíces en los mitos tanto de lo sacro como de lo profano que se reproducen en las acciones de protección y vulneración que tienen por sujeto/objeto a los niños y niñas más vulnerables (Granada-Echeverry & Alvarado, 2010, p. 314). PÁGINA El camino de la resiliencia: del sujeto individual al sujeto político Por ello hablamos de la necesidad de construir resiliencia (familiar, organi- magis en esas márgenes y en las líneas de fuga que permiten a los sujetos y las comunidades ser actores de su propia construcción histórica. Desde una mirada social, Joseba Azkarraga (2014) se refiere a la resiliencia en su aspecto local y comunitario, la pone en tensión frente a la crisis sistémica del planeta, y reconoce que es necesario hacerle preguntas a un mundo excesivamente consumidor que produce más residuos de los que puede absorber. Estamos —según Joseba Azkarraga (2014)— en una fase de translimitación, donde se han superado los límites impuestos por la naturaleza. Desde su perspectiva, la resiliencia se puede ubicar en la búsqueda de un cambio medioambiental global, como iniciativas regeneradoras de territorios más resilientes. 63 Lo novedoso de su acercamiento fue concebir los casos ejemplarizantes no como sujetos considerados “resilientes” desde la teoría clásica de la resiliencia, sino como ella lo afirma: “sujetos que están resiliando, es decir, “están-siendo” en la adversidad” (Granada-Echeverry & Alvarado, 2010, p. 315). De esta forma, se buscó abrir la discusión al pensar la resiliencia no como una condición o una característica esperada de un sujeto frente a situaciones de adversidad, para entenderla mejor, sino como un proceso fenomenológico existencial mediante el cual el sujeto se constituye. magis El término resiliencia emerge en esta investigación para dar cuenta, específicamente, del conjunto de estrategias vinculantes que llevan a cabo los VOLUMEN 10 / NÚMERO 20 / JULIO-DICIEMBRE DE 2017 / ISSN 2027-1174 / BOGOTÁ-COLOMBIA / Páginas 49-68 PÁGINA niños, niñas y jóvenes en situación de calle para reparar el vínculo enfermo, enfrentar y resistir la adversidad y desarrollarse humanamente, logrando su 64 inclusión progresiva a los bienes y servicios sociales, así como el ejercicio de su ciudadanía dentro de un proyecto de vida que incluye no solo su propia reivindicación sino la de otros en situaciones de vida semejantes a las suyas. Mediante el vínculo, el sujeto encuentra en el otro el reconocimiento y las oportunidades para lograr una parcela de dominio de sí mismo, de posesión de sí mismo, de placer de sí mismo, a partir del cual es posible la interacción con el otro, con el cuidado del otro y del mundo (GranadaEcheverry & Alvarado, 2010, p. 322). El trabajo de Granada y Alvarado (2010) se inscribe en un amplio espectro de ejercicios reflexivos, que tienen una marcada sensibilidad por comprender las transiciones que viven los sujetos y las comunidades al afrontar adversidades que surgen en contextos de violencia y exclusión. Estos trabajos expresan lo que se podría llamar la dimensión política de la resiliencia. Mencionemos algunos más significativos: Resiliencia y violencia política en niños (Silva, 1999a); Resiliencia y violencia política: la esperanza realista en el siglo XXI (Silva, 1999b); Factores psicosociales asociados con la resiliencia en niños colombianos víctimas de violencia intrafamiliar (Amar-Amar, Kotliarenco & Abello-Llanos, 2003); Evaluación de factores de resiliencia en niños argentinos en condición de vulnerabilidad familiar (Morelato, 2014); Factores de resiliencia asociados al rendimiento académico en estudiantes de contextos de alta vulnerabilidad social (Villalta-Páucar, 2010); Factores que influyen en el pronóstico de recuperación de las familias en riesgo psicosocial: el papel de la resiliencia del menor (RodrigoLópez, Camacho-Rosales, Máiquez-Chávez, Byrne & Benito-Cruz, 2009); Estudio de factores resilientes en familiares de personas con discapacidad (Ponce-Espino & Torrecillas-Martín, 2014); Madres y padres en contextos de riesgo psicosocial (Martín, Cabrera, León & Rodrigo-López, 2013); Construcción de escala de factores personales de resiliencia en mujeres víctimas de desplazamiento forzado (Amar-Amar, Utria-Utria, Abello-Llanos, Martínez-González & Crespo-Romero, 2014); Resiliencia cultural comunitaria como quehacer político femenino de las mujeres williche del Chaurakawin, Región de los Lagos, Chile (Duquesnoy, 2014); Un modelo teórico de la resiliencia familiar en contextos de desplazamiento forzado (Domínguez de la Ossa, 2014); Transversalidad y resiliencia en los relatos de desplazados del Oriente de la Zona Metropolitana de la Ciudad de México (Domínguez-Echeverría & Espinosa-García, 2017). Los ejercicios reflexivos tomados como ejemplo muestran la pertinencia de la resiliencia para la promoción de una salud integral y la recuperación del tejido humano y social; abren la pregunta por las posibilidades de una existencia digna del sujeto en contextos de marginalidad, violencias y poderes de dominación. Es necesario que la resiliencia se plantee Las diversas definiciones sobre resiliencia muestran la proliferación de acercamientos a la categoría, como su complejidad conceptual al entenderla como capacidad, proceso, rasgo, adaptación positiva, habilidad, potencialidad, paradigma, cualidad y estrategia de afrontamiento de la adversidad. Llama la atención la primacía de la comprensión psicológica y el acentuado carácter de la respuesta más individual de los sujetos frente a la adversidad. Este enfoque, si bien reconoce los procesos de interacción y los vínculos de apoyo, concentra la eficacia del proceso resiliente, en las posibilidades del sujeto y su adaptabilidad en medio de situaciones de adversidad. La perspectiva de la resiliencia comunitaria permite reconocer el valor de las redes humanas, la cultura y las comunidades locales, en el fortalecimiento y el florecimiento de una vida digna. En su puesta en escena, no solo favorece la mitigación del impacto de las condiciones sociales y ambientales adversas, sino que abre la posibilidad para el posicionamiento del concepto de resiliencia política, al ser puente entre las acciones de los sujetos por el reconocimiento y las acciones colectivas que jalonan prácticas humanas de agenciamiento y liberación. Se hace necesario pasar del sujeto individual al sujeto histórico, un sujeto social, cultural y político, que permita plantearse la pregunta por la relación entre resiliencia y política. Un estudio de la resiliencia, desde la dimensión más comunitaria y social, ayudaría a deslocalizar al sujeto individual, para poder entender la resiliencia como un proceso humano histórico que acontece en sujetos plurales, con autonomía, conciencia histórica, capacidad ética y estética para imaginar, desde otros y con otros, un mundo donde sea posible reinventarnos para la vida. Sobre los autores Luis Fernando Granados-Ospina es filósofo de la Pontificia Universidad Javeriana. Licenciado en teología, Pontificia Universidad Javeriana. Licenciado en pedagogía, Pontificia Universidad de Comillas (España). Magíster en educación, Pontificia Universidad Javeriana. Candidato a doctor en ciencias sociales, niñez y juventud del Centro de Estudios Avanzados en Niñez y Juventud (alianza CINDE-Universidad de Manizales). Jaime Carmona-Parra es posdoctorado, Universidad de Manizales, CINDE, Universidad Católica de São Paulo, CLACSO. Doctor en psicología social, Universidad Complutense de Madrid. Magíster en ciencias sociales, Universidad de Antioquia. Psicólogo, Universidad Autónoma Monterrey de Costa Rica. Profesor del doctorado en ciencias sociales, niñez y juventud, Universidad de Manizales, Centro de Estudios Avanzados en Niñez y Juventud, CINDE. Director de la Escuela de Psicología, Universidad de Manizales. Referencias Álvarez, Liliana H. & Hurley, Dermot J. (2010). Desde la zona de riesgo a la zona de resiliencia. Revista Científica de UCES, Universidad de Ciencias Empresariales y Sociales, 14 (2), 13-36. Disponible en: http://dspace.uces.edu.ar:8180/xmlui/bits tream/handle/123456789/890/Desde_la_zona_ de_riesgo_Alvarez.pdf?sequence=1 Amar-Amar, José Juan; Kotliarenco, María Angélica & Abello-Llanos, Raimundo (2003). Factores psicosociales asociados con la resiliencia en niños colombianos víctimas de violencia intrafamiliar. Investigación & Desarrollo. Universidad del Norte, 11 (1), 162-197. Disponible en: http://rcientificas. uninorte.edu.co/index.php/investigacion/article/ view/1134/710 magis Conclusiones Sara Victoria Alvarado-Salgado es psicóloga, Pontificia Universidad Javeriana. Magíster en ciencias del comportamiento y doctora en educación, Nova University-CINDE. Posdoctora en Ciencias Sociales, Niñez y Juventud, Universidad Católica de São Paulo, Universidad de Manizales, Centro de Estudios Avanzados en Niñez y Juventud, Consejo Latinoamericano de Ciencias Sociales, CINDE-CLACSO. Directora del Centro de Estudios Avanzados en Niñez y Juventud, Universidad de Manizales-CINDE y directora del doctorado en ciencias sociales, niñez y juventud. Coordinadora Red Iberoamericana de Posgrados en Infancia y Juventud, RedINJU. Integrante del comité académico del programa posdoctoral de investigación en ciencias sociales, niñez y juventud, y coordinadora académica de la Escuela Internacional de Posgrados en Infancia y Juventud. Gerente del programa de investigación cofinanciado por Colciencias: Sentidos y prácticas políticas de niños y jóvenes en torno a la democracia, la reconciliación y la paz. Actualmente, investigadora de las investigaciones Procesos de construcción social de la niñez en contextos de conflicto armado: la paz, la reconciliación y la democracia desde la perspectiva de narrativas generativas de niños y niñas y Narrativas colectivas de paz y conflicto armado desde las voces de los niños y niñas de la primera infancia, familias y agentes relacionales en el marco del posconflicto/ posacuerdo, del grupo de investigación Perspectivas políticas, éticas y morales de la niñez y la juventud (categoría A en Colciencias) adscrito al Centro de Estudios Avanzados en Niñez y Juventud, CINDE-Universidad de Manizales. Fundadora del programa Niños, Niñas y Jóvenes Constructores de la Paz, creado en 1998. PÁGINA El camino de la resiliencia: del sujeto individual al sujeto político su responsabilidad ética y política, que implica ir más allá del afrontamiento o la resistencia frente a la adversidad, hacia un compromiso con las transformaciones históricas necesarias para una auténtica humanización de la sociedad. Esta convicción deja instalada la pregunta por la dimensión política de la resiliencia y su significación en el ámbito de la acción política. 65 magis VOLUMEN 10 / NÚMERO 20 / JULIO-DICIEMBRE DE 2017 / ISSN 2027-1174 / BOGOTÁ-COLOMBIA / Páginas 49-68 PÁGINA 66 Amar-Amar, José Juan; Utria-Utria, Leider Miguel; Abello-Llanos, Raimundo; Martínez-González, Marina Begoña & Crespo-Romero, Fernando Alexis (2014). Construcción de la Escala de Factores Personales de Resiliencia (FPR-1) en mujeres víctimas del desplazamiento forzado en Colombia. Universitas Psychologica, 13 (3), 853-864. doi:10.11144/Javeriana. UPSY13-3.cefp. 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Construir la resiliencia. Barcelona: Gedisa. Villalta-Páucar, Marco Antonio (2010). Factores de resiliencia asociados al rendimiento académico en estudiantes de contextos de alta vulnerabilidad social. Revista de Pedagogía, Caracas, Escuela de Educación, Universidad Central de Venezuela, 31 (88), 159-188. Disponible en: http://www.re dalyc.org/pdf/659/65916617007.pdf Walsh, Froma (2004). Resiliencia familiar: estrategias para su fortalecimiento. Buenos Aires: Amorrortu. Werner, Emmy E. & Smith, Ruth S. (1982). Vulnerable but Invincible: A Study of Resilient Children. New York: McGraw-Hill. Werner, Emmy E. & Smith, Ruth S. (1992). Overcoming the Odds: High Risk Children from Birth to Adulthood. Ithaca, New York: McGraw-Hill. Werner, Emmy E. & Smith, Ruth S. (2001). Journeys from Childhood to Midlife. Risk, Resilience, and Recovery. Ithaca, New York: Cornell University Press.
https://openalex.org/W1888758454	https://ruj.uj.edu.pl/xmlui/bitstream/handle/item/18280/artymowski_lalak_lewicki_saddle_point_inflation_from_f-r_theory_2015.pdf?sequence=1&isAllowed=y	English	null	Saddle point inflation from<mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" altimg="si1.gif" overflow="scroll"><mml:mi>f</mml:mi><mml:mo stretchy="false">(</mml:mo><mml:mi>R</mml:mi><mml:mo stretchy="false">)</mml:mo></mml:math>theory	Physics letters. B	2,015	cc-by	5,216	1. Introduction Cosmic inﬂation [1–3] is a theory of the early universe which predicts cosmic acceleration and generation of seeds of the large scale structure of the present universe. It solves problems of classi- cal cosmology and it is consistent with current experimental data [4]. The ﬁrst theory of inﬂation was the Starobinsky model [5,6], which is an f (R) theory [7] with R + R2/6M2 Lagrangian density. In such a model the acceleration of space–time is generated by the gravitational interaction itself, without a need to introduce any new particles or ﬁelds. The embedding of Starobinsky inﬂation in no-scale SUGRA has been discussed in Ref. [8]. Recently the whole class of generalisations of the Starobinsky inﬂation have been dis- cussed in the literature [9–15], also in the context of the higher order terms in Starobinsky Jordan frame potential [16–18]. φ ≃ N 0 r 8dN , φ ≃ N 0 r 8dN , (1) (1) which for nearly scale-invariant power spectrum gives φ < Mp for r < 0.002. Small φ seems to be preferable from the point of view of the naturalness principle, since Mp is the cut-off scale of the theory. The value of r determines the scale of inﬂation, since V /r (where V is the potential of the inﬂaton) at the scale of inﬂa- tion is set by the normalisation of CMB anisotropies. Therefore in order to obtain small r one needs a low-scale inﬂation, which may be provided by a potential with a saddle point. The typical scale of inﬂation is set around the GUT scale, which is of the order of (1016 GeV)4. Such a high scale of inﬂation seems to be a disadvantage of inﬂationary models. First of all inﬂation- ary physics is very far away from scales which can be measured in accelerators and other high-energy experiments. The other issue is, that high scale of inﬂation enables the production of super- heavy particles during the reheating [19]. Those particles could be in principle heavier than the inﬂaton itself, so particles like mag- netic monopoles, which abundant existence is inconsistent with A separate issue related with f (R) inﬂation is related with loop corrections to the f (R) function. In order to obtain quasi de Sitter evolution of space–time one needs a range of energies for which the R2M−2 term dominates the Lagrangian density. a r t i c l e i n f o Article history: Received 2 September 2015 Received in revised form 28 September 2015 Accepted 30 September 2015 Available online 3 October 2015 Editor: M. Cvetiˇc We analyse several saddle point inﬂationary scenarios based on power-law f (R) models. We investigate inﬂation resulting from f (R) = R + αnM2(1−n)Rn + αn+1M−2nRn+1 and f (R) = l n αnM2(1−n)Rn as well as l →∞limit of the latter. In all cases we have found relation between αn coeﬃcients and checked consistency with the PLANCK data as well as constraints coming from the stability of the models in question. Each of the models provides solutions which are both stable and consistent with PLANCK data, however only in parts of the parameter space where inﬂation starts on the plateau of the potential, some distance from the saddle. And thus all the correct solutions bear some resemblance to the Starobinsky model. © 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Funded by SCOAP3. observations, could be produced after inﬂation. Another argument, which supports low-scale inﬂation is the Lyth bound [20], which is the relation between variation of the inﬂaton during inﬂation in Planck units (denoted as φ) and tensor-to-scalar ratio r, namely Michał Artymowski b,∗, Zygmunt Lalak a, Marek Lewicki a,c a Institute of Theoretical Physics, Faculty of Physics, University of Warsaw, ul. Pasteura 5, 02-093 Warsaw, Poland b Institute of Physics, Jagiellonian University, Łojasiewicza 11, 30-348 Kraków, Poland c Michigan Center for Theoretical Physics, University of Michigan, Ann Arbor MI 48109, USA a Institute of Theoretical Physics, Faculty of Physics, University of Warsaw, ul. Pasteura 5, 02-093 Warsaw b Institute of Physics, Jagiellonian University, Łojasiewicza 11, 30-348 Kraków, Poland c Michigan Center for Theoretical Physics, University of Michigan, Ann Arbor MI 48109, USA Physics Letters B 750 (2015) 595–600 Physics Letters B 750 (2015) 595–600 * Corresponding author. E-mail address: michal.artymowski@uj.edu.pl (M. Artymowski). * Corresponding author. E-mail address: michal.artymowski@uj.edu.pl (M. Artymowski). all higher order terms. From this perspective it would be better to generate inﬂation in f (R) theory without the Starobinsky plateau, which in principle could be obtained in the saddle point inﬂation. http://dx.doi.org/10.1016/j.physletb.2015.09.076 0370-2693/© 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Funded by SCOAP3. .076 by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Funded by 2 Some higher order corrections to Starobinsky inﬂation discussed here have al- ready been analysed in Refs. [24–26], however not in the context of the saddle-point inﬂation. In addition, in our analysis we take into account at least 2 higher order terms, which is not the case in papers cited above. 1 Differences between Einstein and Jordan frame in loop quantum cosmology are described in Ref. [23]. 2. Introduction to f (R) theory, inﬂation and primordial inhomogeneities The EF potential around the saddle point (up to the maximal allowed value of φ) for f (R) = R + α2R2/M2 + R3/M4 + α4R4/M6 has been shown in Fig. 1. We have rescaled M to obtain α3 = 1. The R2 term in not necessary to obtain a saddle point, but we include it to combine the inﬂation on the Starobinsky plateau with the saddle point inﬂation. From Eq. (6) one ﬁnds the value of α4, normalisation of inhomogeneities gives M as a function of α2. The EF potential around the saddle point (up to the maximal allowed value of φ) for f (R) = R + α2R2/M2 + R3/M4 + α4R4/M6 has been shown in Fig. 1. We have rescaled M to obtain α3 = 1. The R2 term in not necessary to obtain a saddle point, but we include it to combine the inﬂation on the Starobinsky plateau with the saddle point inﬂation. From Eq. (6) one ﬁnds the value of α4, normalisation of inhomogeneities gives M as a function of α2. 2. Introduction to f (R) theory, inﬂation and primordial inhomogeneities where F = f ′ and prime denotes the derivative with respect to the Ricci scalar. Let us assume the following form of f (R) The f (R) theory is one of the simplest generalisations of general relativity (GR). It is based on Lagrangian density S = 1 2 d4√−g f (R) and it can be expressed using the so-called aux- iliary ﬁeld ϕ deﬁned by ϕ = F(R) := df dR . In such a case the Jor- dan frame (JF) action is equal to S = √−g(ϕR/2 −U(ϕ), where U = (R F −f )/2 is the JF potential. For F = 1 one recovers GR, so the GR vacuum of the JF potential is positioned at ϕ = 1. The same model can be expressed in the Einstein frame (EF), with the metric tensor deﬁned by ˜gμν = ϕgμν. This is purely classical trans- formation of coordinates and results obtained in one frame are perfectly consistent with the ones from another frame.1 The EF action is equal to S = −˜g( ˜R/2 + (∂μφ)2/2 −V (φ)), where ˜R, φ := √ 3/2 log F and V := (R F −f )/(2F 2) are the EF Ricci scalar, ﬁeld and potential respectively. The EF potential should have a minimum at the GR vacuum, which is positioned at φ = 0. f (R) = R + α2 R2 M2 + αn Rn M2(n−1) + αn+1 Rn+1 M2n , (5) (5) where n > 2 is a given number. In such a case the saddle point appears for R = Rs = M2 (n −2)αn n −1 n−1 , αn+1 = − (n −2)αn n n n−1 . (6) (6) Equations above are α2 independent, because any R2 can satisfy Eq. (4). In order to keep Rs and αn+1 real we need to assume that αn > 0 and αn+1 < 0. Then for suﬃciently big R one ﬁnds F < 0 and the gravity becomes repulsive. This instability becomes an issue for R ≃M2 αnn n+1 (αn(n −2)/n) −n n−1 , which is typically of the same order of magnitude as Rs. By redeﬁning M we can always set one of αn to be any given constant. For negative αn one can satisfy Eq. (4) for n < 2. 1. Introduction This would re- quire all higher order corrections (such as R3, R4, etc.) [21] to be suppressed by a mass scale much bigger than M. One naturally expects all higher order correction to GR to appear at the same energy scale if one wants to avoid the ﬁne-tuning of coeﬃcients of all higher order terms. From this perspective it would be better to generate inﬂation in f (R) theory without the Starobinsky plateau, which in principle could be obtained in the saddle point inﬂation. M. Artymowski et al. / Physics Letters B 750 (2015) 595–600 596 The similar case of the inﬂection point inﬂation from f (R) theory has been partially analysed in Ref. [22]. is a mass scale, which suppresses deviations from GR.2 There- fore, in order to obtain suﬃciently long Starobinsky plateau one needs a broad range of energy scales on which R2 dominates over all higher order corrections. This requires a ﬁne-tuning of inﬁnite number of αn coeﬃcients. To avoid that we will consider an in- ﬂationary scenario in which different higher-order corrections can become relevant at the same energy scale, namely the saddle-point inﬂation from a power-law f (R) theory. For general form of f (R) one obtains a saddle point of the EF potential for Vφ = Vφφ = 0, which corresponds to In what follows we use the convention 8πG = M−2 p = 1, where Mp ∼2 × 1018 GeV is the reduced Planck mass. The outline of the paper is as follows. In Section 2 we give short introduction to f (R) and its description as a Brans–Dicke theory. In Section 3 we discuss three saddle point f (R) scenarios, namely: i) two higher order terms Rn and Rn+1, ii) at least 4 higher or- der terms with powers bigger than 2, iii) inﬁnite number of higher order terms with ﬁnite sum at every energy scale. Finally we sum- marise in Section 4 R F = 1 2 f , R F ′ = F , (4) (4) 2. Introduction to f (R) theory, inﬂation and primordial inhomogeneities Nevertheless the saddle point would lie in the repulsive gravity regime, where F < 0. Thus in the follow- ing analysis n < 2 is excluded. Note that for non-zero value of α2 the value of M grows with α2. This comes from the fact that for α2 ≫αn one obtains inﬂationary plateau followed by the saddle point, due to growing value of Rs with respect to α2. The α2 de- pendence of M is shown in Fig. 1. Big α2 term means that the last 60 e-folds of inﬂation happen on the Starobinsky plateau, so one does not obtain signiﬁcant deviations from the R2 model. In the EF the gravity obtains its canonical form and this is why the EF is usually used for the analysis of inﬂation and generation of primordial inhomogeneities. The cosmic inﬂation proceeds when both slow-roll parameters ϵ and η are much smaller than unity. These parameters are, as usual given by ϵ = 1 2 Vφ V 2 , η = Vφφ V , (2) (2) where Vφ and Vφφ are the ﬁrst and the second derivative of the EF potential with respect to φ. During inﬂation ϵ and η can be inter- preted as deviation from the de Sitter solution for FRW universe. During each Hubble time the EF scalar ﬁeld produces inhomoge- neous modes with an amplitude of the order of the Hubble pa- rameter. From them and from the scalar metric perturbations one constructs gauge invariant curvature perturbations, which are di- rectly related to cosmic microwave background anisotropies. Their power spectrum PR, their spectral index ns and their tensor to scalar ratio are as follows The model descried in Eq. (5) can be generalised into f (R) = R + α2R2M−2 + αnRnM2(1−n) + αmRmM2(1−m). Then, for αn = 1 one ﬁnds Rs = M2 (n −2)(m −n) m −1 − 1 n−1 , Rs = M2 (n −2)(m −n) m −1 − 1 n−1 , αm = −(n −1)(n −2) (m −1)(m −2) (n −2)(m −n) m −1 n−m n−1 (7) PR ≃ V 24π 2ϵ , ns ≃1 −6ϵ + 2η , r ≃16ϵ. (3) (3) (7) In the low scale inﬂation one obtains ϵ ≪\|η\|, which for η < 0 gives 1 −ns ≃2\|η\|. In the low scale inﬂation one obtains ϵ ≪\|η\|, which for η < 0 gives 1 −ns ≃2\|η\|. 3.1. Saddle point with vanishing two derivatives As mentioned in the introduction, the loop corrections to the Starobinsky model are of the form ∞ n=2 αnRnM2(1−n), where M M. Artymowski et al. / Physics Letters B 750 (2015) 595–600 597 Fig. 1. Left panel: the Einstein frame potential for the (5) model around the saddle point for n = 3, α3 = 1 and different values of α2. The α4 coeﬃcient is set from Eq. (6) for n = 3. The maximal allowed value of φ is very close to the saddle point. Right panel: the scale of new physics M as a function of α2. Dotted green line represent Starobinsky limit. (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article.) Fig. 1. Left panel: the Einstein frame potential for the (5) model around the saddle point for n = 3, α3 = 1 and different values of α2. The α4 coeﬃcient is set from Eq. (6) for n = 3. The maximal allowed value of φ is very close to the saddle point. Right panel: the scale of new physics M as a function of α2. Dotted green line represent Starobinsky limit. (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article.) Fig. 2. Tensor to scalar ratio r and spectral index ns as a function of α2 for the (5) model with n = 3. One can ﬁt the PLANCK data for α2 ≳100, which means that the saddle point is preceded by the inﬂationary plateau. Fig. 2. Tensor to scalar ratio r and spectral index ns as a function of α2 for the (5) model with n = 3. One can ﬁt the PLANCK data for α2 ≳100, which means that the saddle point is preceded by the inﬂationary plateau. x ns as a function of α2 for the (5) model with n = 3. One can ﬁt the PLANCK data for α2 ≳100, which means that the saddle r ratio r and spectral index ns as a function of α2 for the (5) model with n = 3. One can ﬁt the PLANCK data for α2 ≳100, which m the inﬂationary plateau. Fig. 3. Both panels present ns(n) for the model from Eq. (5) for N⋆= 50 and α2 = 0. 3.1. Saddle point with vanishing two derivatives (For interpretation of the references to colour in this ﬁgure legend, the reader is ref Fig. 6. Numerical results for the model (12) for N⋆= 60 The ns ﬁts the PLANCK data for 0 < α < 1.4 and for α2 ≳34, when the α2 term dominates the inﬂationary evolution. Fig. 6. Numerical results for the model (12) for N⋆= 60 The ns ﬁts the PLANCK data for 0 < α < 1.4 and for α2 ≳34, when the α2 term dominates the inﬂationary evolution. Fig. 6. Numerical results for the model (12) for N⋆= 60 The ns ﬁts the PLANCK data for 0 < α < 1.4 and for α2 ≳34, when the α2 term dominates the inﬂationary evolution. Fig. 6. Numerical results for the model (12) for N⋆= 60 The ns ﬁts the PLANCK data for 0 < α < 1.4 and for α2 ≳34, when the α2 2) for N⋆= 60 The ns ﬁts the PLANCK data for 0 < α < 1.4 and for α2 ≳34, when the α2 term dominates the inﬂationary evolution. R = Rs = √p M2 , where p = (l −1) l 2 −1 . (9) The αn coeﬃcients satisfy + R lM2√pR + 2M4p2 M4p R = Rs = √p M2 , where p = (l −1) l 2 −1 . (9) The αn coeﬃcients satisfy 2(l 3)! 3 + R lM2√pR + 2M4p2 1 − R M2√p l −1 −(l −1)R2 M4p −M2√pR . (11) + R lM2√pR + 2M4p2 1 − R M2√p l −1 −(l −1)R2 M4p −M2√pR . The αn coeﬃcients satisfy The αn coeﬃcients satisfy (11) αn = (−1)n−1 2(l −3)! (l −n)!(n −1)! p 3−n 2 for n = {3,...,l}. (10) αn = (−1)n−1 2(l −3)! (l −n)!(n −1)! p 3−n 2 for n = {3,...,l}. (10) 3.3. The l →∞limit αn = (−1)n−1 2(l −3)! (l −n)!(n −1)! p 3−n 2 for n = {3,...,l}. (10) 3.3. The l →∞limit (10) Note that Eqs. (9) and (10) are completely independent of α2. Since αl < 0 one obtains F < 0 for suﬃciently big R. Alike the model from Eq. (5) the biggest allowed value of R is slightly big- ger than Rs. Using Eq. 3.1. Saddle point with vanishing two derivatives / Physics Letters B 750 (2015) 595–600 598 598 M. Artymowski et al. / Physics Letters B 750 (2015) 595–600 Fig. 4. Left panel: Numerical results for the model (11) for N⋆= 50 and N⋆= 60 (red and blue dots respectively). Right panel: EF potential for the model (11) for l = 6, l = 8, l = 10, l = 12 and l = 14 (orange, green, red, brown and blue lines respectively). The saddle point lies close to the right edge of the potential, beyond which one obtains a second branch of V , which leads to repulsive gravity. (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article.) Fig. 4. Left panel: Numerical results for the model (11) for N⋆= 50 and N⋆= 60 (red and blue dots respectively). Right panel: EF potential for the model (11) for l = 6, l = 8, l = 10, l = 12 and l = 14 (orange, green, red, brown and blue lines respectively). The saddle point lies close to the right edge of the potential, beyond which one obtains a second branch of V , which leads to repulsive gravity. (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article ) Fig. 5. Numerical results for the model (11) for N⋆= 50 and N⋆= 60 (red and blue dots respectively). All values of r obtained in this analysis are consistent with PLANCK, but ns ﬁts the PLANCK data only for N⋆≃60. (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article.) Fig. 5. Numerical results for the model (11) for N⋆= 50 and N⋆= 60 (red and blue dots respectively). All values of r obtained in this analysis are consistent with PLANCK, but ns ﬁts the PLANCK data only for N⋆≃60. (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article.) Fig. 5. Numerical results for the model (11) for N⋆= 50 and N⋆= 60 (red and blue dots respectively). All values of r obtained in but ns ﬁts the PLANCK data only for N⋆≃60. 3.1. Saddle point with vanishing two derivatives If n is a natural number one cannot ﬁt the Planck data due to too small ns. In all of those cases a signiﬁcant contribution of the R2 term is needed in order to obtain ns ≳0.958. On the other hand for n −2 ≲10−2 one obtain ns ≃0.96. The case of n ≳2 seems to be especially interesting since it allows to reconstruct Starobinsky results it the presence of higher order terms. Fig. 3. Both panels present ns(n) for the model from Eq. (5) for N⋆= 50 and α2 = 0. If n is a natural number one cannot ﬁt the Planck data due to too small ns. In all of those cases a signiﬁcant contribution of the R2 term is needed in order to obtain ns ≳0.958. On the other hand for n −2 ≲10−2 one obtain ns ≃0.96. The case of n ≳2 seems to be especially interesting since it allows to reconstruct Starobinsky results it the presence of higher order terms. 3.2. Saddle point with vanishing k derivatives more terms to f (R) function (see also Figs. 2–5). Thus let us now consider 3.2. Saddle point with vanishing k derivatives more terms to f (R) function (see also Figs. 2–5). Thus let us now consider f (R) = R + α2 R2 M2 + l n=3 αn Rn M2(n−1) , (8) In general one can deﬁne the saddle point with ﬁrst k deriva- tives vanishing, which was analysed in Ref. [27]. In that case 1 −ns ≃ 2k N⋆(k−1) when freeze-out of primordial inhomogeneities happens close to the saddle point. Thus, for suﬃciently big k one can ﬁt the Planck data. In our case all dk V dφk = 0 at the saddle point are equivalent to dk f dRk = 0 for k > 2. The f (R) model from Eq. (5) cannot satisfy these equations, so in order to obtain a saddle point with vanishing higher order derivatives one needs to introduce (8) (8) where l > 4 is an even natural number. Again, without any loss of generality one can choose α3 to be any positive constant, so for simplicity we set α3 = 1. Then one can satisfy Eq. (4) and f (n) = 0 (for n = {3, 4, ..., l −2} and any value of α2) and the saddle point appears at M. Artymowski et al. 3.1. Saddle point with vanishing two derivatives In Subsection 3.2 we investigated The Einstein frame potential with zero value of the ﬁrst l −2 derivatives at the saddle point, where l ≥6 is an even natural number. To obtain such a saddle point we considered f (R) = R + α2R2 + l n=3 αnM2(1−n)Rn. We found analytical formulae for Rs and for all αn coeﬃcients, as well as the explicit value of f (R) after summation. Unfortunately the result is slightly disappointing, because the saddle point moves away from the scale of freeze-out of primordial inhomogeneities with growing l. Thus bringing us closer to the Starobinsky case as l gets bigger. We also obtained numerical results for ns, r and for the suppression scale M as a function of l. The ﬁnal result strongly depends on N⋆, and therefore on the thermal history of the uni- verse. One can ﬁt the PLANCK data for l ≳20 and N⋆≃60 even for α2 = 0. Again, for R slightly bigger than Rs one obtains an in- stability of potential, which for big l is orders of magnitude away from the freeze-out scale. Fig. 8. The minimal value of α2, which allows to avoid overshooting the meta-stable GR minimum. f (R) = R e − √ 2R M2o + √ 2 + α2 M2 o R . (12) (12) The α2 may be again used to stabilise the GR vacuum at φ = 0. The numerical results for N⋆= 60 are plotted in Figs. 6 and 7. As expected, for α ≫1 values of M/√α, r and ns obtain the limit of the Starobinsky theory. As shown in Fig. 7 the potentials have two branches, which split at some φ = φm, where φm is the minimal value of φ. The α2 term in necessary in order to stabilise the GR vacuum. For α2 = 0 one obtains two branches of potential which grow from φ = 0. Both of them exist only for φ > 0 with no min- imum. While increasing the value of α2 the splitting of branches moves towards φ < 0 and the inﬂationary branch obtains mini- mum at φ = 0. We investigated the stability of minimum from the perspective of classical evolution of the Einstein frame ﬁeld. 3.1. Saddle point with vanishing two derivatives (8) and (10) one obtains Numerical analysis shows that in order to obtain correct nor- malisation of primordial inhomogeneities one needs M = M(l). Nevertheless for l →∞one obtains M →Mo (where Mo ∼10−5 for α2 = 0), which implies Rs →∞for l →∞. Hence for l ≫1 one cannot obtain inﬂation close to saddle point. For l →∞one obtains f (R) = R + α2 M2 R2 M. Artymowski et al. / Physics Letters B 750 (2015) 595–600 599 Fig. 7. Left Panel: The Einstein frame potential as a function of the Ricci scalar. The GR minimum at R = 0 appears to be meta-stable, with a possibility of tunnelling to anti de Sitter vacuum. Right Panel: The Einstein frame potential V as a function of the Einstein frame ﬁeld φ for the model (12). Two branches of potential correspond to two solutions of ϕ = F(R). In order to avoid overshooting the minimum at R = 0 one requires α2 ≳0.7. Fig. 7. Left Panel: The Einstein frame potential as a function of the Ricci scalar. The GR minimum at R = 0 appears to be meta-stable, with a possibility of tunnelling to anti de Sitter vacuum. Right Panel: The Einstein frame potential V as a function of the Einstein frame ﬁeld φ for the model (12). Two branches of potential correspond to two solutions of ϕ = F(R). In order to avoid overshooting the minimum at R = 0 one requires α2 ≳0.7. Fig. 8. The minimal value of α2, which allows to avoid overshooting the meta-stable GR minimum. two additional terms proportional to Rn and Rn+1. We found an- alytical relation between their coeﬃcients and Rs, which is the value of the Ricci scalar at the saddle point. The potential becomes unstable for R slightly bigger than Rs – the second branch of the auxiliary ﬁeld equation ϕ = F(R) becomes physical, which leads to the second branch of potential and as a consequence to repulsive gravity. Signiﬁcant contribution of the R2 term extend the plateau before the saddle point and pushes away the instability from the inﬂationary region. For n ≥3 it is impossible to obtain correct ns, however for n slightly bigger than 2 one can ﬁt the PLANCK data. Acknowledgements This work was partially supported by the Foundation for Pol- ish Science International PhD Projects Programme co-ﬁnanced by the EU European Regional Development Fund and by National Science Centre under research grants DEC-2012/04/A/ST2/00099 and DEC-2014/13/N/ST2/02712. ML was supported by the Pol- ish National Science Centre under doctoral scholarship number 2015/16/T/ST2/00527. MA was supported by National Science Cen- tre grant FUGA UMO-2014/12/S/ST2/00243. 3.1. Saddle point with vanishing two derivatives Namely, we considered the slow-roll initial conditions at φ = φ⋆ for different values of α2 and checked whether the minimum is deep enough to stop the ﬁeld before it would reach φm. We post- pone the issue of quantum tunnelling to the anti-de Sitter vacuum for future work. In Subsection 3.3 we considered the limit l →∞, which re- sulted in f (R) = R(e− √ 2R/M2 o +( √ 2+α2)R/M2 o), which is basically Starobinsky model plus an exponentially suppressed correction. In such a case the saddle point (and therefore the instability for R > Rs) moves to inﬁnity and inﬂation happens far away from the saddle point. The α2 term is necessary to create the meta-stable minimum of the Einstein frame potential (Fig. 8). One can ﬁt the PLANCK data for 0.7 ≲α2 ≲1.4 and α2 ≳34. 4. Conclusions In this paper we considered several f (R) theories with sad- dle point in the Einstein frame potential. All models consist of GR term R, Starobinsky term α2R2 and higher order terms which are the source of the saddle point. In Subsection 3.1 we investigated M. Artymowski et al. / Physics Letters B 750 (2015) 595–600 600 References [15] H. Motohashi, arXiv:1411.2972 [astro-ph.CO]. [16] B.J. Broy, D. Roest, A. Westphal, arXiv:1408.5904 [hep-th]. [1] D.H. Lyth, A. Riotto, Phys. Rep. 314 (1999) 1, arXiv:hep-ph/9807278. [17] K. Kamada, J. Yokoyama, Phys. Rev. D 90 (10) (2014) 103520, arXiv:1405.6732 [hep-th]. [2] A.R. Liddle, New Astron. Rev. 45 (2001) 235, arXiv:astro-ph/0009491. [3] A. Mazumdar, J. Rocher, Phys. Rep. 497 (2011) 85, arXiv:1001.0993 [hep-ph]. [18] M. Artymowski, Z. Lalak, M. Lewicki, J. Cosmol. Astropart. Phys. 1506 (06) (2015) 032, arXiv:1502.01371 [hep-th]. [4] P.A.R. Ade, et al., Planck Collaboration, Astron. Astrophys. 571 (2014) A22, arXiv:1303.5082 [astro-ph.CO]. [19] V. Mukhanov, Univ. Pr., Cambridge, UK, 2005, 421 p. [5] A.A. Starobinsky, Phys. Lett. B 91 (1980) 99. 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https://openalex.org/W2067325342	https://zenodo.org/records/2517916/files/article.pdf	English	null	ORGANIZATION OF CONGRESS‘ IN SECONDARY VOCATIONAL MATHEMATICS	School science and mathematics	1,913	public-domain	6,403	VOCATIONAL MATHEMATICS VOCATIONAL MATHEMATICS 779 ORGANIZATION OF CONGRESS’ IN SECONDARY VOCA- TIONAL MATHEMATICS. BY W. T. STRATTON^ Kansas State Agricultural College, Manhattan, Kansas. During recent years there has been a demand that public education serve all the people in accordance with their several needs. Vocational schools are being established or courses ar- ranged in the already existing high schools to meet this demand. In such schools ’and courses the ’English, history, mathematics, and the sciences are taught with the view of furnishing the stu- dents the things they will need in their chosen vocations. Voca- tional mathematics, then, is that mathematics which will be of most help to the student in his every-day life in the store, fac- tory, on the farm, as a carpenter or in whatever line of work he may take up. It is my purpose in this paper to organize the material for courses in secondary vocational mathematics that will meet the demands of boys and girls who’ for various reasons are not looking forward to a college education. The discussion of this subject naturally falls into three divisions. I. The general conditions which make special courses in sec- ondary mathematics desirable. I. The general conditions which make special courses in sec- ondary mathematics desirable. II. The kind and extent of mathematics for mechanic arts students. III. The kind and extent of mathematics for agricultural students. I. GENERAL CONDITIONS WHICH MAKE SPECIAL COURSES DESIRABLE. Until recent years only two objects were kept in view in deter- mining what should make up the course in secondary mathematics preparation for college, and the general value of the subjects themselves. Later came in the consideration of preparing for business, or commercial life, which led to the introduction of bookkeeping and commercial arithmetic. Today the course of study must take into account the objects for which the boys and girls are being educated, their capabilities, and the opportunities open to young men and women demanding general and special educational qualifications. How to meet these requirements by giving the students just the kind of mathematics they need is a question of vital importance today. The American people have always stood for equality of op- portunity in every phase of life; and, as a result, a single type of SCHOOL SCIENCE AND MATHEMATICS 780 school has developed without reference to local conditions or per- sonal preference. Since mathematics is an exact science it has been one of the last to yield to the popular demand for a change. Time and experience have, however, brought clearly to view the fact that equality of educational opportunity can be provided only as different needs of the community and of particular groups of children are taken into consideration. The great num- ber of boys and girls do not enter professional work but go either from the high school or from the grades to the farm, store, or shop. In view of this fact their mathematics should be of such a nature as to prepare them best for the work they are likely to do. y y There is a demand today for finishing schools. A great many colleges and cities have already established schools with courses from two to four years in length in which the students may choose such work as appeals to them and will do them the most good after they get out. These schools are demanded by two different classes of students: (1.) The boys and girls of the towns and cities whose parents can not afford to send them through high school and then to college to get training in some industrial line. To this class also belong those who for various reasons" have left school on finishing the eighth grade and have gone into shops, or stores, or have taken up some trade. I. GENERAL CONDITIONS WHICH MAKE SPECIAL COURSES DESIRABLE. After a year or two they see the need of more training but can not spend the time necessary to complete a college course but will gladly take two or three years provided they can get a course that furnishes them proficiency in their chosen vocation. (2.) In the rural communities in almost every state in the Union there are a great many boys and girls who finish the country schools and would like to go to high school but the literary and classical education does not appeal to them in such a way as to lead them to believe that they will have greater earning capacity after spending two to four years there. They were raised on the farm and want to stay there; hence the, township or county high school should offer courses making for intelligence and prac- tical insight and give opportunity for them to gain such control of materials, tools, and scientific facts that their earning capacity is increased and they are better trained for citizenship. These’ schools should provide agricultural training for the boys and courses in home economics for the girls. I In such schools a new style of mathematics is demanded. The principles of mathematics are the same today as fifty years ago but that does not necessarily mean that the subject should be VOCATIONAL MATHEM ATICS 781 treated the same today as then. Students may be taught to add, subtract, multiply, and divide by using numbers in arithmetic or letters in algebra without reference to what they represent. To treat mathematics in this way would be to regard it as a science and would lead largely to a series of mental gymnastics, and the student would get very little insight into the subject. Mathe- matics should be treated as a science, to be sure, but as a science applied to practical living. From this point of view mathematics is changing from year to year. The graph in algebra was almost unheard of until recent years. Scientific investigators have de- manded a compact, accurate way to’ submit their data to those interested, hence came the use of the graph. It was found to be such a convenient way of presenting results of investigations that today it is used in almost every report sent out to the public. I. GENERAL CONDITIONS WHICH MAKE SPECIAL COURSES DESIRABLE. Let an important change occur in the mode of transacting busi- ness and the arithmetics will be changed accordingly. The old texts emphasized troy weight, apothecaries’ weight, circulating decimals, alligationmedial and alternatearithmetical and geometrical progression, all of which are antiquated or of ques- tionable value to the ordinary citizen. We are beginning to find in the new text-books such subjects as saving and loaning money, investments, banking, commission, and problems covering the practical phases of poultry husbandry, dairying, carpentry, farm mechanics, and every other subject that touches any considerable number of people in any community. The arithmetics used in a strictly agricultural district or by agricultural students in any secondary school should contain different material from texts used by students in the heart of a great city, or in a purely mining district, or a community where everybody is interested in a particular industry. Again, for students in secondary schools not all parts of the algebra, geometry, and trigonometry have applications. Stu- dents should not be required to take such parts, but topics should be selected which are fundamental in meeting the requirements of the technical studies or will be of practical value. If the selection is wisely made and the material properly arranged, logical training will not suffer. If we teach so that pupils really understand what they are doing then we are training their logical powers and giving them the help in their ethical and philosophi- cal ways of thinking which are so well developed by a study of Euclid. They will also have a ready, usable knowledge of the fundamental problems and processes of the subjects they have taken. SCHOOL SCIENCE AND MATHEMATICS 782 Mathematics has been too abstract and artificial. Aiming at training the pure reason it has got out of touch with facts and for many pupils has degenerated into mere jugglery of symbols without any thinking. It is no wonder then that mathematics fails to interest all but a very few. The theorem of limits usually stated, "If two variables are constantly equal as they approach a limit their limits are equal," which is of great interest to the grown mathematician is for the very best boys very difficult to grasp and if the complete mastery were necessary before taking up subjects that depend upon it most students would be barred .from the work entirely. I. GENERAL CONDITIONS WHICH MAKE SPECIAL COURSES DESIRABLE. During nine1 years of experience in teaching geometry in the Kansas State Agricultural College and other good schools to students, whose previous preparation is not questioned, I have not found a single student who could really grasp the meaning of limits. Occasionally one will get a pretty good notion of the meaning by the time he has completed plane and solid geometry. The teachers do not understand it as they should; however, it is not the fault of the teachers nor the stu- dents, the trouble lies in the fact that we are trying to do some- thing that can not be done. Mathematics which heaps up theorem after theorem without a thought of. how its results are to be turned to practical ac- count in the acquisition of knowledge in other fields can not possibly lead to’ any benefits to the- student. The value of math- ematical knowledge to students in these finishing schools lies in its applications. There is no reason why mathematicians should not devote themselves exclusively to pure mathematics, if they are so inclined, for without question a great many of the valuable discoveries in other sciences have been made with the help of fundamental formulae worked out by pure mathemati- cians. The trained mathematician has capital but it is useless so long as it is not turned to practical account in other sciences. We must learn to lay more stress upon the ability to use a theorem intelligently than to give a faultless demonstration. Farmers, mechanics, or engineers, do not care about non- Euclidean geometry nor for computations of plane triangles where the sum of the angles is less than 180°, nor any such problems. The engineer especially asks for the ability to use numbers rapidly and accurately in his particular field. Common sense demands that the mathematics of real life be coordinated with that taught in the schools. In order to accomplish this the teacher with the pupils should visit the VOCATIONAL MATHEMATICS 783 various industries of the vicinity. They will be brought face to face with real problems, not mere statements of problems. Visit stores, elevators, factories, cement workers where walks and foundations are being made. Visit a house or barn under construction and figure out with them the cost, using local prices, of the building from the excavation to the completion. I. GENERAL CONDITIONS WHICH MAKE SPECIAL COURSES DESIRABLE. In a large school the mathematics department should work with the shops, drafting room, and the domestic science and domestic art departments. Teachers of the technical subjects should also be in constant touch with the mathematics department antici- pating problems which will arise and reporting immediately any weakness shown by a pupil in problems and principle. We must come to a saner and more practically useful atti- tude in all our mathematics or we shall be pushed to the wall. Already there is a demand that mathematics be not required in the high schools and this will inevitably come unless the sub- ject is made to appeal more strongly to the ordinary high-school student. I believe if we are all absolutely frank we will admit that geometry as now taught in most schools is of all the sub- jects the one which we feel of least real use in advanced math- ematics; and as now taught, geometry is felt by students and by many teachers to be of least benefit to the student who does not pursue advanced studies. A great many mathematicians and text-book writers have been putting great stress on rigor in elementary geometry. Surely we should not be held to the old Euclidean style of geometry on that account. We should give up the meaningless pretense of teaching rigorous geometry to boys and girls of fourteen and fifteen years of age. That there is a demand for a change in our secondary mathematics is evidenced by its growing unpopularity and from the fact that it is not now considered so essential in every course offered. There is a demand then as indicated above for a new style of mathematics for students who do not go to college. II. MATHEMATICS FOR MECHANIC ARTS STUDENTS. What then should be the nature and extent o’f the mathematics for one of these finishing schools? The answer to this question will depend upon the work for which the student is preparing. The courses should be made to suit the needs of the two classes of students given above. We will first take up the course for the students of the first division. The student who is preparing for real leadership in the fac- SCHOOL SCIENCE AND MATHEMATICS 784 tory, machine shop, carpentry, contracting, or similar lines of .work, if he is to become a leader in his work, should have a rather extensive course in mathematics covering the,subjects of algebra, geometry and trigonometry. ALGEBRA: The elementary algebra making up the first yearns work §hould have for its immediate aim the mastery of the linear and quadratic equation. The equation can scarcely be over-emphasized in the early study of algebra. Real-life prob- lems can thus be introduced almost at the beginning of the study of the subject and long periods of drudgery on the abstract mathematics avoided. The students ought .to be made to feel that they are studying a real, live, practical subject and the abstract p’arts introduced for the sake of proficiency will simply give variety enough to make the study interesting. Where the interpretations of results of a practical nature are very im- portant care should be taken not to bring into the work long and complicated applications of principles. The student will not need to be able to’ work out long and complex expressions but needs a thorough working knowledge of the simple prin- ciples of algebra. A thorough course up to and including the quadratic equation and covering only simple radicals will be sufficient for any demands made upon the subject both in a practical way and as a foundation for work in geometry and trigonometry. The parts of the subject that should be em- phasized are: The four fundamental operations, simple paren- theses, special rules in multiplication and division, factoring of a simple type leading directly to the solution of the quadratic, simple fractions, ratio and proportion, simple simultaneous equa- tions of two unknowns, graphs, and simple radicals. The equation, as was indicated above, should be the great point of emphasis through6ut the course. The work should be more intensive and not so extensive. II. MATHEMATICS FOR MECHANIC ARTS STUDENTS. The theoretical considerations of the laws of signs, curious combinations of brackets, difficult factoring, complicated frac- tions, simultaneous quadratics, cube root, complex radicals, and imaginaries should be omitted. The time usually consumed in these subjects should be used in making the students more pro- ficient in what they do take, and in attention to problems they are meeting in their other subjects and will meet in their every- day life. Reasonable skill in straightforward manipulation is certainly expected and demanded but time can be spent to better advantage in making the student familiar with the few simple VOCATIONAL MATHEMATICS 785 devices at his disposal than by dealing with long tedious ex- amples in simplifications of fractions, or with "elegant devices" for solving problems especially constructed for the purpose, p y p p , GEOMETRY: In geometry a good many of the recent text- books writers have done a great deal toward vitalizing the work by arranging the material so as to set the student to work right at the start with ruler and compass. This plan gives the stu- dent something tangible to work with and if properly handled can be made of service to the teacher in presenting the prob- lems of superposition in triangles. This construction work should be accompanied by a great number of numerical exer- cises in which he will make use of his arithmetic and algebra. Excellent opportunities will also be offered here for the use of graphs in the solution of triangles. After several days of this kind of work they should be introduced to demonstrative geom- etry. In the demonstrative work more emphasis should be placed upon getting the students to think than upon requiring them to get the exact form of the demonstration given in the book. The construction work and the numerical calculations are not to be dropped as soon as the theorems are taken up but the three will go right along together.. Demonstrative geometry develops mathematical thinking better perhaps than any other phase of the work, yet to emphasize that alone makes of it merely a study of logic, for certainly no very great number of the theorems have direct application .to the problems of life. Geometry has its practical side which .must be emphasized if it is to hold its place in the. curriculum. II. MATHEMATICS FOR MECHANIC ARTS STUDENTS. A good many theorems are intuitionally evident and the proof may be omitted without destroying the sequence of thought. The "theory of limits" and "maxima and minima" should be left to the students of higher mathematics. These subjects are of no practical value here and are beyond the comprehension of the ordinary student. In solid geometry only a very few of the theorems should be proven in a formal way. It should be largely the mensuration of the simple solid. Solid geometry also offers an excellent field for construction work which will be of use to the student in other lines of work. The numerical prob- lems are of such a practical nature that the teacher will experi- ence no difficulty in keeping the students interested. By re- ducing the number of theorems almost all the time can be’spent at this kind of work. TRIGONOMETRY: No course in mathematics for a student SCHOOL SCIENCE AND MATHEMATICS 786 preparing for betterment in his vocation is entirely complete without a working knowledge of trigonometry. It opens up a new field of useful knowledge and furnishes powerful tools for the solution of problems involved. The course should con- sist o’f the study of the trigonometric functions with the de- velopment of the few fundamental formulae needed. Next should come a large number of problems on the right triangle making use of these formulae and the tables of natural trig- onometric functions. Logarithms should then be introduced and a large number of applied problems necessary to insure pro- ficiency in the use of logarithms should be based upon the right triangle and the general triangle. All work with complicated identities, multiple angles, and functions of angles greater than those found in the general triangle should be omitted. With the material of the course made up as above students will be intensely interested from start to finish and will never question the utility of the course. APPLIED MATHEMATICS : The student has now had one year in algebra, a year in geometry, and a third of a year in trig- onometry. At least another third of a year should be spent on the applications of the mathematics he has already learned along with perhaps one or two new features of direct value to him. During the entire course the teacher should constantly hold before the pupil the fact that he is studying mathematics. II. MATHEMATICS FOR MECHANIC ARTS STUDENTS. Dur- ing the first year the principles of algebra have received the greater part of the emphasis, however, when geometry is taken up the algebra and the arithmetic will not be forgotten but used at every place possible. This course should round up his whole mathematical knowledge and show him if he has not already found it out that what he has in his mathematics is some won- derful tools to be used in the solution of problems soon to con- front him. He should be given opportunity to make calculations and work problems that will come up in the particular trade for which he is preparing. The use of the slide rule is one of the new features introduced into this course. The whole term’s work should be crowded full of everyday problems bringing into play his formulae from arithmetic, his equation and the graph from algebra, his formulae and logical thinking from geometry, and the use of logarithms and the time-saving devices of trigonometry. III. MATHEMATICS FOR AGRICULTURAL STUDENTS. The agricultural high school is a finishing school with courses, VOCATIONAL MATHEMATICS 787 as indicated earlier in the discussion, fitted to the needs of boys and girls who find it almost impossible to obtain a college edu- cation. They are fitting themselves in the least possible time for the work on the farm. In view of this fact it is entirely proper that most of their time be spent along the line of prac- tical agriculture. A good part of their time will be spent in the laboratory, in the stock-judging pavilion, and in actual contact with the real farm work. If the student has had a pretty thor- ough course in arithmetic we should be able with one year’s work in mathematics to give him a working knowledge of the principles of mathematics sufficient to enable him to solve the problems of everyday life on the farm. The work for the girls should be essentially the same as that for the boys. The ma- jority of the applied problems will be the same. Only prob- lems pertaining to domestic science and domestic art will not be of value to the boys, and very little if any of the work for boys should be eliminated from the course for the girls. So what I shall say concerning the course will apply to the work for both. II. MATHEMATICS FOR MECHANIC ARTS STUDENTS. Assuming, then, that we have one year "for mathematics, what shall be the nature of this mathematics? A careful study of the field will convince one, I think, of the fact that arithmetic, algebra, and geometry all contain principles and formulae which will be of practical value in a course of this kind. We will now see what parts of each shall be utilized. ARITHMETIC : The field of arithmetic seems to be pretty well defined. Since the class will- likely be made up of students of different degrees of proficiency in the four fundamental opera- tions, a few days spent in review using as a basis problems taken from the experiences on the farm, will unify the class and insure better work throughout the term. The order to be followed in taking up the subjects has very little chance for variation. The chief point of difference from the old style arithmetic is in the choice of the problems. Agronomy with its problems in crops, soils, and fertilizers, dairying with its tests and feeding problems, constructions of barns, silos, and so on, animal husbandry with its suggestive problems, horti- culture, farm mechanics, botany and zoology, all offer prob- lems in common fractions, decimals, denominate numbers, per- centage, and quite a number of other subjects. In business transactions of interest to the farmer you will cover insurance, interest, mortgages, notes, discount, commission, banking, and so on. The study of the graph and its use in bulletins and SCHOOL SCIENCE AND MATHEMATICS 788 crop reports should be taken up here. Mensuration is another division of the subject for which problems are easily found. In fact there seems to be little difficulty in finding material in arithmetic sufficient to take the third of the year allotted to it. The main trouble has been to get the problems properly graded. They must be graded with ;a view of developing the subject of arithmetic logically, and also in such a way as to really make the subject-matter developed a unit and worth while to the student. There is at least one text that seems to have ac- complished both ends to a fair degree of success. Since this is true I will not attempt to go further into detail in the subject- matter to be considered. ALGEBRA : The field in vocational algebra for agricultural students has not been so fully explored. II. MATHEMATICS FOR MECHANIC ARTS STUDENTS. In fact I do not know of any text-book that attempts to give a brief course in algebra designed for such students. With one-third of a year at our disposal what should we attempt to do in the subject? If we follow the usual plan of devoting the time to the study of addition, subtraction, multiplication, division, and factoring we will have used all the time at our disposal and the students will not have anything of practical value. This knowledge will also be of very little use in the work in geometry. A course con- taining the practical things of the subject can be arranged and should contain* the following subjects in the order they are given here. I. The Equation. If not all the members of the class have had the equation in arithmetic develop the idea by use of the balances and by making use of many concrete examples from arithmetic. Just as soon as they have grasped the meaning of the equation start them in with a list of problems involving simple equations. The problems should be of a practical na- ture and should constantly draw on their fund of knowledge of arithmetic. Problems like the following kind should make up several days work in the course. (1.) To grow a ton each of corn and clover requires 813 tons of water. The clover requires twice as much water as the corn. How much water does each require ? (2.) A washing machine, linoleum, and range cost a farmer $91. The linoleum costs twice as much as the washing machine, and the range twice as much as the linoleum. What is the cost of each? (3.) A good ration for a cow is equal amounts of corn-and- 3.) A good ration for a cow is equal amounts of corn-and- VOCATIONAL MATHEMATICS 789 cob meal and cotton-seed meal, and five times as many pounds of hay as cotton-seed meal making 28 pounds in all. How many pounds of each were fed? Twelve or fifteen-problems of the kind given above drawn from a wide range of subjects will be sufficient here. Then introduce just as briefly as possible the axioms and by means of illustrative examples lead -to the matter of transposition in the equation. About fifteen good examples based upon scien- tific facts, the perimeters of rectangles, and other subjects of interest should be given here. (1.) The temperature at 7 a. m. was 10 and at noon -\|-9. How many degrees warmer was it at noon than at 7 a. m. ? (2.) A man’s income for the month was $140, rent $30, food $40, interest and incidental expenses $35. Using the positive and negative notation find the result at the end of the month. A long list of simple, well-graded problems will be sufficient to fix in. mind the four fundamental operations and the mean- ing of positive and negative numbers. I would conclude the II. MATHEMATICS FOR MECHANIC ARTS STUDENTS. Problems must be carefully se- lected so as not to involve the use of negative numbers. In every case the problems should be checked, thus developing the idea of substitution which the student needs in the solution of problems by the use of formulae. If the students are not able to handle the solution of the equations after they have gotten them from the given conditions a number of abstract equa- tions should be given sufficient to develop the desired skill. II. Positive and Negative Numbers. Positive and negative numbers should be introduced by the use of the thermometer, latitude and longitude, debts, and so on. From this study the students may be led almost unconsciously into’ the subjects of addition, subtraction, multiplication, and division of a simple kind, using as the basis hot abstract quantities alone but prob- lems bringing into play their knowledge of arithmetic. They will readily see that it is perfectly natural to count income, rising temperature, travel east or north, and so on, as positive while debts, falling temperature, sailing south or west are treated as negative quantities. After a clear understanding of the method of notation they should then take up a large number of exam- ples involving simple numbers which will give them the four fundamentals along with the. laws of signs. The discussion of the laws of signs from a theoretical point of view has no place here. Problems of the following will serve to bring about the desired result. (1.) The temperature at 7 a. m. was 10 and at noon -\|-9. How many degrees warmer was it at noon than at 7 a. m. ? A long list of simple, well-graded problems will be sufficient to fix in. mind the four fundamental operations and the mean- ing of positive and negative numbers. I would conclude the SCHOOL SCIENCE AND MATHEMATICS 790 chapter with a list of abstract problems covering the four opera- tions in order to insure that all the students have a ready work- ing knowledge and sufficient drill in handling positive and nega- tive numbers. III. The Fundamental Operations and Factoring. In the last chapter nothing was said about the exponent, power, or co- efficient. These definitions with illustrations should introduce this section. Some formal work in addition, subtraction, multi- plication, and division of polynomials should then follow. II. MATHEMATICS FOR MECHANIC ARTS STUDENTS. All the problems in multiplication should be simple in nature per- haps not higher than the third degree in the multiplicand and not more than three, usually two, terms in the multiplier. Out of the multiplication will come in natural order factoring and the solution of the equation by factoring. Here will be given an excellent opportunity to put in a long list of practical prob- lems involving addition, subtraction, multiplication, and divi- sion, and the solution of an entirely new kind of equation, the quadratic, by means of factoring. The problems should be care- fully graded and the quadratic type should be along the line of the following: (1.) A rectangle 3 feet longer than it is wide has an area of 28 sq. ft. What are its dimensions ? (2.) A field of grain is 32 rods long and 24 rods wide, how wide a strip must a farmer cut by going round and round the field to cut half the, grain ? IV. Fractions and their. Applications. Fractions should be taken up with a brief review of fractions in arithmetic. If the student has clearly in mind how to reduce fractions in arith- metic to their lowest terms, mixed numbers to improper frac- tions, improper fractions to mixed numbers and how to multiply, divide, and reduce fractions to a common denominator he will have only a brief transition to the same things in algebra. This transition can best be brought about by means of abstract prob- lems. After this has been done the rest of the work on frac- tions should be given to the solution of practical problems from the farm and the sciences. Problems bringing into use all the formulae he has heretofore had and will have soon in the differ- ent fields of work should be taken up here. The following are examples of a simple kind which may be used as practical problems in fractional equations. IV. Fractions and their. Applications. Fractions should be taken up with a brief review of fractions in arithmetic. If the student has clearly in mind how to reduce fractions in arith- metic to their lowest terms, mixed numbers to improper frac- tions, improper fractions to mixed numbers and how to multiply, divide, and reduce fractions to a common denominator he will have only a brief transition to the same things in algebra. II. MATHEMATICS FOR MECHANIC ARTS STUDENTS. This transition can best be brought about by means of abstract prob- lems. After this has been done the rest of the work on frac- tions should be given to the solution of practical problems from the farm and the sciences. Problems bringing into use all the formulae he has heretofore had and will have soon in the differ- ent fields of work should be taken up here. The following are examples of a simple kind which may be used as practical problems in fractional equations. (1.) According to the Arkansas Experiment Station 1 acre of corn pasture produces one-third as many pounds of pork as (1.) According to the Arkansas Experiment Station 1 acre of corn pasture produces one-third as many pounds of pork as VOCATIONAL MATHEMATICS 791 an acre of peanut pasture. If an acre of each together produces 416 pounds, what is the value of each as a pork producer? p , p p (2.) It was estimated that a good road built along a quarter section farm increased its value one tenth of itself. The farm sold for $16,280. What is the estimated value of good roads per acre? (2.) It was estimated that a good road built along a quarter section farm increased its value one tenth of itself. The farm sold for $16,280. What is the estimated value of good roads per acre? V. Graphs and their Applications. If the graph has not been taken up in arithmetic it should be taken up here in connection with the crop reports. For example let them represent by a diagram or graph the yield of wheat in Kansas for a certain number of years. This may be done by the blocking method at first then by the continuous line. After some work of this kind a typical graph of the variation of temperature for a certain period of time should be studied carefully. They will then be ready to take up the variable and the ordinary principles of graphing. The linear equation in two variables will then be readily graphed and the solution of simultaneous equations in two unknowns which require only approximate results can be solved. A large part of the work in graphs will be based upon observed data from the laboratory, from bulletins and reports of the work at the experiment station, and from government reports. II. MATHEMATICS FOR MECHANIC ARTS STUDENTS. The chapter will be concluded with a list of problems in simultaneous equations in two unknowns by means of elim- ination and substitution. There should also be a short list of abstract problems for drill but the majority of the sets of equa- tions will be obtained from the applied problems. VI. Miscellaneous Problems. For the ordinary class there has been enough material given under the five chapters above. If the class is particularly strong more problems may be needed, and I should give a short chapter to problems which will sup- plement the different subjects and at the same time furnish nothing but material of the most practical nature. It seems to me if the course is closely followed the students will get the things they will need in the course in geometry which is to fol- low and at the same time will get ,only the things that they can make use of after they are through school. APPLIED GEOMETRY: With only one third of a year to spend on geometry we must look only for the applications of the sub- ject and’not use any time for demonstration. The work should start with the definitions and illustrations of a straight line, angle, complement, supplement, and conjugate, then follow with a large number of equation problems. This work in equations SCHOOL SCIENCE AND MATHEMATICS 792 will fix clearly in mind the relation of the different angles and will also give a good drill and review of the equation work in algebra. Take up next the definitions of surface, triangle, rec- tangle, and all the common polygons. A great amount of valu- able work can be based upon the right triangle by making use of the carpenter’s square in getting the practical problems. Sim- ilar triangles and the different regular polygons offer excellent opportunities for applied problems which may be solved either by formulae, or by use of the graph. I should often require problems solved graphically so as always to keep before the students the fact that it is often the easiest and sometimes the only elementary method of solution for certain problems. The next division of the work should take up the circle and the many problems related to it. Start with the definition. II. MATHEMATICS FOR MECHANIC ARTS STUDENTS. If the idea of the polygon with a ’large number of sides has been taken up, the area of the sector and finally the area of the circle can be developed intuitionally and logically. From the area of the circle the area of the segment and the area of the circular ring will easily come. Problems on belts, pulleys, cutting speeds, and gears will furnish a list of the everyday problems of life. Occasionally a problem involving the area of an ellipse will oc- cur in practical work so it is well to give them the formula for the area and the meaning of the terms. Solid geometry should consist entirely of the mensuration of the prism, cylinder, pyramid, cone, frustums, sphere, and the anchor ring. Definitions of each of these should be given at the proper time and an intuitional development of the formulae given in each case. The last few days of the course in geom- etry should be spent in a general round up and review of the formulae learned, emphasizing the most important, so that the students will see that they have had something practical and will also have a clearer notion of how they may use what they have learned.
https://openalex.org/W4392761447	https://www.qeios.com/read/R8V5YI/pdf	English	null	Review of: "Pulse Amplitude Measurement Using Low Sampling ADC and Interpolation Technique"	null	2,024	cc-by	511	Qeios, CC-BY 4.0 · Review, March 13, 2024 Review of: "Pulse Amplitude Measurement Using Low Sampling ADC and Interpolation Technique" Chonghua Fang Potential competing interests: No potential competing interests to declare. Overall, this paper presents a valuable contribution to the field of signal processing and measurement techniques. The authors have provided a comprehensive comparison of various interpolation methods for measuring analog pulse amplitudes using low sampling rate ADCs. The paper is technically sound, and the subject matter is presented in a clear and comprehensive manner. The experimental setup and validation process are well-described, and the authors have provided a thorough analysis of the performance of each interpolation method based on relevant metrics such as relative error, mean square error, and mean absolute deviation. Additionally, the hardware resource utilization on the FPGA for each method is also evaluated, which is a valuable consideration for practical implementations. While the references provided are relevant, the authors could consider including a few additional references related to the specific application areas mentioned to provide more context. Additionally, there are a few minor grammatical and expression issues that could be addressed to improve the overall clarity and readability of the paper. In summary, this paper is a valuable contribution to the existing body of knowledge and is recommended for publication after addressing the minor issues mentioned above. In summary, this paper is a valuable contribution to the existing body of knowledge and is recommended for publication after addressing the minor issues mentioned above. In addition, the English language used in the paper is good. However, there are a few minor grammatical and expression issues that could be improved. Review of: "Pulse Amplitude Measurement Using Low Sampling ADC and Interpolation Technique" For example: In the abstract, "...were conducted for measurement of analog pulse peak height." could be rephrased as "...were conducted to measure the analog pulse peak height." on 2.1, "The greatest integer value that is less than or equal to x in this case is called the floor funct ection 2.1, "The greatest integer value that is less than or equal to x in this case is called the floor fu d be rephrased as "The greatest integer value less than or equal to x is called the floor function [x] " In Section 2.1, "The greatest integer value that is less than or equal to x in this case is called the floor function [x]." could be rephrased as "The greatest integer value less than or equal to x is called the floor function [x]." e rephrased as "The greatest integer value less than or equal to x is called the floor function [x]." In Section 3.2, "Each interpolation technique discussed in earlier section is implemented in FPGA separately." could be rephrased as "Each interpolation technique discussed in the earlier section is implemented separately on the FPGA." In Section 3.2, "Each interpolation technique discussed in earlier section is implemented in FPGA separately." could be rephrased as "Each interpolation technique discussed in the earlier section is implemented separately on the FPGA." Qeios ID: R8V5YI · https://doi.org/10.32388/R8V5YI 1/1
https://openalex.org/W2565762286	https://www.frontiersin.org/articles/10.3389/fpsyg.2016.02032/pdf	English	null	Commentary: From ‘sense of number’ to ‘sense of magnitude’ – The role of continuous magnitudes in numerical cognition	Frontiers in psychology	2,017	cc-by	1,862	GENERAL COMMENTARY published: 04 January 2017 doi: 10.3389/fpsyg.2016.02032 Peter Kramer* and Paola Bressan Keywords: sense of number, sense of magnitude, numerosity estimation, occupancy, statistical learning A commentary on From ‘sense of number’ to ‘sense of magnitude’ – The role of continuous magnitudes in numerical cognition by Leibovich, T., Katzin, N., Harel, M., and Henik, A. (2016). Behav. Brain. Sci. [Epub ahead of print]. doi: 10.1017/S0140525X16000960 From ‘sense of number’ to ‘sense of magnitude’ – The role of continuous magnitudes in numerical cognition by Leibovich, T., Katzin, N., Harel, M., and Henik, A. (2016). Behav. Brain. Sci. [Epub ahead of print]. doi: 10.1017/S0140525X16000960 Unlike abstract ones in mathematics, concrete sets of elements in the real world have continuous physical properties, such as overall area and density. The dominant view has it that humans can estimate the discrete numerosities of such sets independently of the co-varying continuous magnitudes; i.e., that humans have a “sense of number.” It has indeed been claimed that various animals, ranging from monkeys to tiny ﬁsh, have this sense too. A recent paper by Leibovich et al. (2016) questions all of this (see also Morgan et al., 2014; Gebuis et al., 2016) and argues convincingly that numerosity estimation is not independent from continuous magnitudes but relies on them; that we have not a “sense of number” but a “sense of magnitude.” Edited by: Bert Reynvoet, KU Leuven, Belgium Reviewed by: Marcus Lindskog, Uppsala University, Sweden Correspondence: Peter Kramer peter.kramer@unipd.it Edited by: Bert Reynvoet, KU Leuven, Belgium Reviewed by: Marcus Lindskog, Uppsala University, Sweden Correspondence: Peter Kramer peter.kramer@unipd.it Edited by: Bert Reynvoet, KU Leuven, Belgium Reviewed by: Marcus Lindskog, Uppsala University, Sweden Yet the authors fail to cite a classic article that made the very same argument 25 years ago, and—unlike Leibovich et al.—supported it with a quantitative model (Allik and Tuulmets, 1991). Although neither density, nor overall area, nor any other single continuous magnitude can provide reliable information about numerosity, Leibovich et al. imply that all of them together can; they suggest that “statistical learning” will take care of extracting this information and turn numerosity estimates out of it. How statistical learning achieves this feat and whether the resulting numerosity estimates will ﬁt observed ones remains, unfortunately, unclear. Allik and Tuulmets’s alternative “occupancy” model has its limits (e.g., Kramer et al., 2011; Bertamini et al., 2016) but it is speciﬁc, it is quantitative, and it predicts observed numerosity estimation surprisingly well with just a single free parameter. *Correspondence: Peter Kramer peter.kramer@unipd.it Specialty section: This article was submitted to Cognition, a section of the journal Frontiers in Psychology To understand the occupancy model, consider a set of identical dots and imagine that each of them is covered with a larger disk, as in Figure 1. The model posits that the total area occupied by the disks (occupancy) will be linearly related to the estimated numerosity of the dots. As long as dot densities remain relatively low (Durgin, 1995), the model accounts for nearly 90% of the variance in human data (Allik and Tuulmets, 1991). The closer together the dots are and thus the more the disks overlap, the smaller occupancy is and thus the smaller the estimated numerosity of the dots is predicted to be—as indeed observed (e.g., DeWind et al., 2015). Notably, because large numerosities tend to be dense, the occupancy model predicts they will tend to be underestimated. This prediction has been corroborated repeatedly too (e.g., Izard and Dehaene, 2008). How Leibovich et al.’s statistical learning could make the same prediction is hard to tell. Received: 31 October 2016 Accepted: 14 December 2016 Published: 04 January 2017 FIGURE 1 \| Effect of item spacing on occupancy. The “occupancy” of a dot may be represented as a larger, concentric virtual disk that covers it, shown here in gray. (The radius of the disk is a free parameter that, during model ﬁtting, Allik and Tuulmets estimated to be 0.33◦of visual angle.) The three black dots in the bottom row are closer together than those in the top row. As a result, the virtual disks in the bottom row overlap, making for a smaller occupancy (total gray area) and therefore a smaller predicted numerosity estimate. For illustration purposes only a few dots are shown; note, however, that numerosity estimation concerns numerosities outside the “subitizing” range of one to four items. The occupancy model—even in Durgin’s (1995) variant—is too simple to work under all conditions (Kramer et al., 2011). Still, if the degree of overlap of the virtual disks covering the dots is interpreted as an inverse measure of the mutual discriminability of the dots, the model makes an intuitive point. The point is that the less discriminable from one another items are, the lower their estimated numerosity ought to be. If so, the occupancy model could be expanded and improved (Kramer et al., 2011) by taking into account anything that aﬀects the items’ (1) discriminability (their distance from one another and from ﬁxation, for example), (2) retention across saccades (if presentation times allow eye movements), and (3) retention in memory (if the stimulus is presented or inspected sequentially or compared to another one). The occupancy model deserves at the very least discussion— if not revival and development. It has the potential to challenge not only “sense of number” models but also Leibovich et al.’s statistical-learning alternative to them. Unlike the latter, the occupancy model is a “sense of magnitude” model that does not require learning, is deterministic, quantitative, perception- rather than cognition-based, and not inconsistent with the idea that numerosity estimation might be innate and so simple that even ﬁsh could do it. AUTHOR CONTRIBUTIONS Although people are under the impression they are blessed with a high-resolution image of the world around them, careful experimentation has revealed that this is largely an illusion (e.g., Durgin, 1995). In numerosity-estimation experiments the PK wrote the ﬁrst draft of the manuscript; both authors discussed it, critically revised it, and agreed on the ﬁnal version. dispersion. Atten. Percept. Psychophys. 78, 1460–1471. doi: 10.3758/s13414-01 6-1100-0 DeWind, N. K., Adams, G. K., Platt, M. L., and Brannon, E. M. (2015). Modeling the approximate number system to quantify the contribution of visual stimulus features. Cognition 142, 247–265. doi: 10.1016/j.cognition.2015.05.016 Durgin, F. H. (1995). Texture density adaptation and the perceived numerosity and distribution of texture. J. Exp. Psychol. Hum. Percept. Perform. 21, 149–169. Gebuis, T., Cohen Kadosh, R., and Gevers, W. (2016). Sensory-integration system rather than approximate number system underlies numerosity processing: Citation: Kramer P and Bressan P (2017) Commentary: From ‘sense of number’ to ‘sense of magnitude’ – The role of continuous magnitudes in numerical cognition. Front. Psychol. 7:2032. doi: 10.3389/fpsyg.2016.02032 January 2017 \| Volume 7 \| Article 2032 Frontiers in Psychology \| www.frontiersin.org Item Discriminability and Numerosity Estimation Kramer and Bressan stimuli tend to be randomly placed items, usually dots, and some of them end up clumped together. These items may be impossible to discriminate from one another, even when they can all be perceptually detected. This crowding eﬀect (Whitney and Levi, 2011) is a serious concern, because the opportunities for crowding necessarily grow with numerosity and have been shown to aﬀect its estimation (Valsecchi et al., 2013; Anobile et al., 2015). So far, studies that claim that numerosity estimation does not fully rely on continuous magnitudes, or that it is more cognitive than perceptual in nature, have not been very speciﬁc about how the perceptual system extracts numerosity from the stimulus (see also Morgan et al., 2014). These studies have considered a wide range of perceptual factors such as density, overall area, and item size. None of these factors, however, has ever been claimed to be crucial in numerosity estimation. The one factor that has for as long as 25 years (i.e., occupancy) is instead consistently ignored, including now by Leibovich et al. (2016). Izard and Dehaene (2008), for example, do cite Allik and Tuulmets (1991) and control for various factors including “occupied area,” but they use the term to mean overall area (which nobody considers critical to numerosity estimation) and not occupancy (which Allik and Tuulmets themselves consider critical). Izard and Dehaene dismiss occupancy as “a complex combination” of two parameters (density and occupied area). Occupancy does not strike us as complex, but even if it were, the fact that it reﬂects well-established perceptual discriminability limitations that have been demonstrated to aﬀect numerosity estimation—rather than hypothetical cognitive ones that continue to be debated—should give the occupancy model an edge over any “sense of number” model. In defense of their own “sense of magnitude” model, which Leibovich et al. fail to cite too, Morgan et al. (2014) make a similar point. dispersion. Atten. Percept. Psychophys. 78, 1460–1471. doi: 10.3758/s13414-01 6-1100-0 DeWind, N. K., Adams, G. K., Platt, M. L., and Brannon, E. M. (2015). Modeling the approximate number system to quantify the contribution of visual stimulus features. Cognition 142, 247–265. doi: 10.1016/j.cognition.2015.05.016 Frontiers in Psychology \| www.frontiersin.org January 2017 \| Volume 7 \| Article 2032 REFERENCES Allik, J., and Tuulmets, T. (1991). Occupancy model of perceived numerosity. Percept. Psychophys. 49, 303–314. doi: 10.3758/BF03205986 Anobile, G., Turi, M., Cicchini, G. M., and Burr, D. C. (2015). Mechanisms for perception of numerosity or texture-density are governed by crowding-like eﬀects. J. Vis. 15, 1–12. doi: 10.1167/15.5.4 Bertamini, M., Zito, M., Scott-Samuel, N. E., and Hulleman, J. (2016). Spatial clustering and its eﬀect on perceived clustering, numerosity, and January 2017 \| Volume 7 \| Article 2032 Frontiers in Psychology \| www.frontiersin.org 2 Item Discriminability and Numerosity Estimation Kramer and Bressan Whitney, D., and Levi, D. M. (2011). Visual crowding: a fundamental limit on conscious perception and object recognition. Trends Cogn. Sci. 15, 160–168. doi: 10.1016/j.tics.2011. 02.005 a critical review. Acta Psychol. 171, 17–35. doi: 10.1016/j.actpsy.2016. 09.003 Izard, V., and Dehaene, S. (2008). Calibrating the mental number line. Cognition 106, 1221–1247. doi: 10.1016/j.cognition.2007.06.004 Kramer, P., Di Bono, M. G., and Zorzi, M. (2011). Numerosity estimation in visual stimuli in the absence of luminance-based cues. PLoS ONE 6:e17378. doi: 10.1371/journal.pone.0017378 Conﬂict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Leibovich, T., Katzin, N., Harel, M., and Henik, A. (2016). From ‘sense of number’ to ‘sense of magnitude’ - the role of continuous magnitudes in numerical cognition. Behav. Brain. Sci. doi: 10.1017/S0140525X16000960. [Epub ahead of print]. Copyright © 2017 Kramer and Bressan. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Morgan, M. J., Raphael, S., Tibber, M. S., and Dakin, S. C. (2014). A texture- processing model of the ‘visual sense of number’. Proc. Biol. Sci. 281:20141137. doi: 10.1098/rspb.2014.1137 Valsecchi, M., Toscani, M., and Gegenfurtner, K. R. (2013). Perceived numerosity is reduced in peripheral vision. J. Vis. 13, 1–16. doi: 10.1167/13.13.7 January 2017 \| Volume 7 \| Article 2032 Frontiers in Psychology \| www.frontiersin.org 3
https://openalex.org/W3189911179	https://link.springer.com/content/pdf/10.1007/s10530-021-02609-1.pdf	English	null	Has the introduction of two subspecies generated dispersal barriers among invasive possums in New Zealand?	Biological invasions	2,021	cc-by	9,466	Catriona D. Campbell . Phil Cowan . Bernd Gruber . Anna J. MacDonald . Clare E. Holleley . Stephen D. Sarre Received: 20 October 2020 / Accepted: 23 July 2021 / Published online: 11 August 2021 The Author(s) 2021 Abstract The introduction of species into new environments provides the opportunity for the evolu- tion of new forms through admixture and novel selection pressures. The common brushtail possum, Trichosurus vulpecula vulpecula from the Australian mainland and T.v.fuliginosus from Tasmania, were introduced multiple times to New Zealand from Australia to become one of New Zealand’s most signiﬁcant pests. Although derived from two sub- species, possums in New Zealand are generally considered to be a single entity. In a previous analysis, we showed that possums in the Hawkes Bay region of New Zealand appeared to consist of at least two overlapping populations. Here, we extend that analysis using a genotype-by-sequencing approach to examine the origins and population structure of those possums and compare their genetic diversity to animals sam- pled from Australia. We identify two populations of each subspecies in Hawkes Bay and provide clear evidence of a contact zone between them in which a hybrid form is evident. Our analysis of private alleles shows higher rates of dispersal into the contact zone than away from it, suggesting that the contact zone functions as a sink (and hence as a barrier) between the two subspecies. Given the widespread and overlap- ping distribution of the two subspecies across both large islands in New Zealand, it is possible that many such contact zones exist. These results suggest an opportunity for a more targeted approach to control- ling this pest by recognising sub-speciﬁc differences and identifying the contact zones that may form between them. Supplementary Information The online version contains supplementary material available at https://doi.org/10.1007/ s10530-021-02609-1. Keywords Contact zone Dispersal barrier Genetic admixture Private alleles C. D. Campbell B. Gruber A. J. MacDonald C. D. Campbell B. Gruber A. J. MacDonald S. D. Sarre (&) Centre for Conservation Ecology and Genomics, Institute for Applied Ecology, University of Canberra, Bruce ACT 2617, Australia e-mail: stephen.sarre@canberra.edu.au Biol Invasions (2021) 23:3831–3845 https://doi.org/10.1007/s10530-021-02609-1 (0123456789().,-volV)( 01234567 89().,-volV) ORIGINAL PAPER Has the introduction of two subspecies generated dispersal barriers among invasive possums in New Zealand? Catriona D. Campbell . Phil Cowan . Bernd Gruber . Anna J. MacDonald . Clare E. Holleley . Stephen D. Sarre Introduction Identifying the role that such factors can play in successful establishment, may offer novel perspectives or pathways for the identiﬁ- cation of high-risk invaders or provide opportunities for enhanced control of invasive pests (Sz}ucs et al. 2017). The introduction of common brushtail possums (Trichosurus vulpecula; hereafter called possums) to New Zealand from Australia is one well documented instance where different forms of the same species have been brought together into a new environment. Introduced to establish a fur trade (Montague, 2000), possums were shipped to New Zealand from Tasmania and several other undocumented locations on main- land Australia, most likely in New South Wales and Victoria, between 1858 and the 1920s. All introduc- tions ceased after that time but the transfer of possums within New Zealand continued into the 1940s. Possums are endemic to Australia with ﬁve currently recognised subspecies distributed across the continent and on several offshore islands (How, Hillcox 2000; Kerle et al. 1991). New Zealand has no naturally occurring marsupials and possums are now one of the most serious threats to biodiversity and agriculture in New Zealand damaging vegetation (Cowan 1991; Nugent et al. 2001); preying on birds and their eggs (Brown et al. 1993); and spreading bovine tuberculosis (Innes, Barker 1999). New Zealand management agencies have conducted extensive possum control for decades (Donnell 1995; Morgan 1990; Nugent, Cousins 2014; Nugent, Morriss 2013) and now aim, with the support of conservation groups, to eradicate brushtail possums as part of the ‘‘Predator-free New Zealand’’ campaign (Russell et al. 2015). Resolving complex introduction histories and pat- terns of admixture in invasive species can be difﬁcult, but access to population level genomic data can reveal how species integrate into new environments and whether diverse origins have enhanced or inhibited their invasion success. Here, we use genotype-by- sequencing to examine the origins and contemporary population structure of possums in Hawkes Bay, and compare their genetic diversity to animals sampled from across their native range in Australia. We test the proposition that the sub-populations, as previously deﬁned by the distribution of microsatellite DNA variation (Sarre et al. 2014), are representative of the ancestral mainland and ancestral Tasmanian origins, and that the Tasmanian and mainland forms have established a hybrid swarm in the Hawkes Bay region that affects the direction and rate of gene ﬂow. Introduction Introductions of species into novel environments can provide the opportunity for the evolution of new forms as they naturalise (Sz}ucs et al. 2017). In some cases, alien species have been introduced to the same location on multiple occasions from different sources, P. Cowan Landcare Research, Lincoln 7640, New Zealand C. E. Holleley Australian National Wildlife Collection, CSIRO, National Research Collections Australia, Canberra ACT 2601, Australia 12 3 3 C. D. Campbell et al. 3832 extent of their different ancestry (Kean 1971; Pracy 1974; Triggs, Green 1989), but it is generally assumed (although seldom stated) that New Zealand possums breed indiscriminately with respect to those origins (Montague 2000). However, a recent microsatellite DNA analysis of possums in Hawkes Bay, on the North Island of New Zealand, identiﬁed at least two genetically distinct groups of possums, separated by a zone of putatively introgressed animals (Sarre et al. 2014). That work implied that there were at least two breeding forms in the region, probably representing the Tasmanian subspecies and the south-eastern mainland subspecies (T.v.fuliginosus and T.v.vulpec- ula respectively), rather than one continuous popula- tion (Sarre et al. 2014). That proposition is supported by historical records which show that there were three recorded introductions of possums into the area (Fig. 1): possums taken directly from Tasmania and released in the Lake Waikaremoana area in 1898; ‘‘black furred’’ possums (also presumed to be Tasma- nian in origin but translocated from elsewhere in New Zealand) released on the Mahia Peninsula a short time after the ﬁrst introduction; and possums from an unknown location on mainland Australia released in the Mohaka river area in the early 1900s (Pracy 1974; Sarre et al. 2014). creating genetic combinations of individuals that have not previously existed (Kolbe et al. 2004). Such admixture can enhance ﬁtness through hybrid vigour or increased genetic diversity (Barker et al. 2019; Dlugosch, Parker 2008; Facon et al. 2008; Hirsch et al. 2017; Rius, Darling 2014) or reduce ﬁtness through outbreeding depression (Rhymer, Simberloff 1996). It is unclear how frequently genetic factors are intrinsic to the success or otherwise of invasive species (Barker et al. 2019; Rius, Darling 2014) although propagule pressure (something that will affect the levels of genetic diversity within an invader) is one of the few acknowledged general characteristics of successful invaders (Bock et al. 2015). Introduction We also use asymmetries in the number of private alleles between pairs of sub-populations to determine the direction of that geneﬂow. A symmetrical distribution of private alleles among sub-populations indicates symmetry in the rates of gene ﬂow between sub- populations and subspecies and would argue against It is well known that the different colour forms seen in New Zealand possums are representative to some 123 123 Introduction of two subspecies generated dispersal barriers 3833 Fig. 1 Brushtail possum introduction sites (Black: Lake Waikaremoana; Mahia Peninsula; Grey: Mohaka River) and sample collection sites (numbered from 1 to 35) in the Hawkes Bay study site in New Zealand. See Sarre et al. (2014) for further information Fig. 1 Brushtail possum introduction sites (Black: Lake Waikaremoana; Mahia Peninsula; Grey: Mohaka River) and sample collection sites (numbered from 1 to 35) in the Hawkes Bay study site in New Zealand. See Sarre et al. (2014) for further information Methods the formation of genetic barriers between the two subspecies. We demonstrate that one sub-population is genetically identiﬁable as the Tasmanian subspecies T.v. fuliginosus, that possums from that sub-popula- tion are distinct from animals that are most likely mainland in origin, and that they have maintained that distinctiveness even after more than 80 years of contact. We also show that the sub-population believed to originate from mainland Australia is indeed largely T.v.vulpecula in origin, although we could not identify the mainland region from which those possums were obtained. Our data support the proposition that brushtail possums in Hawkes Bay do not behave as a single panmictic population, but rather as two subspecies with a narrow zone of contact. Gene ﬂow into and out of that contact zone is asymmetrical and indicative of a genetic barrier between the two subspecies. The formation of similar hybrid barriers elsewhere in New Zealand could have important implications for the control of this invasive species. the formation of genetic barriers between the two subspecies. We demonstrate that one sub-population is genetically identiﬁable as the Tasmanian subspecies T.v. fuliginosus, that possums from that sub-popula- tion are distinct from animals that are most likely mainland in origin, and that they have maintained that distinctiveness even after more than 80 years of contact. We also show that the sub-population believed to originate from mainland Australia is indeed largely T.v.vulpecula in origin, although we could not identify the mainland region from which those possums were obtained. Our data support the proposition that brushtail possums in Hawkes Bay do not behave as a single panmictic population, but rather as two subspecies with a narrow zone of contact. Gene ﬂow into and out of that contact zone is asymmetrical and indicative of a genetic barrier between the two subspecies. The formation of similar hybrid barriers elsewhere in New Zealand could have important implications for the control of this invasive species. Genotype-by-sequencing DNA was extracted from each 2mm2 piece of ear tissue sample by Diversity Arrays Technology Pty Ltd (DArT) using a salting out method as reported by (Couch et al. 2016). We used the DArTseqTM geno- type-by-sequencing approach, which uses a combina- tion of restriction enzyme complexity reduction and high throughput sequencing (Courtois et al. 2013; Cruz et al. 2013; Kilian et al. 2012) to simultaneously identify and genotype SNP markers in the absence of a reference genome. A double digest approach with the restriction enzymes PstI and SphI was used for complexity reduction before the addition of custom adapters (Kilian et al. 2012) to restriction site over- hangs. Fragments that included both a PstI and an SphI adapter were ampliﬁed using primers complementary to the adapters. These also incorporated molecular identiﬁer barcode tags, to allow multiplexing of up to 96 samples per sequencing run. PCR conditions consisted of: denaturation at 94 0C for 1 min; 30 cycles of 94 0C for 20 s, 58 0C for 30 s and 72 0C for 45 s; and a ﬁnal extension period of 72 0C for 7 min. PCR products were pooled for sequencing on an Illumina HiSeq 2000 platform using 77 cycles of single end sequencing. Raw sequence reads were ﬁltered and processed using a proprietary DArT analytical pipeline. Poor quality sequences were removed, with more stringent criteria being placed upon the barcode region than the rest of the sequence. Approximately 2,000,000 sequences per barcode were identiﬁed and used in marker calling, during which identical sequences were collapsed and ﬁltered before screening to identify variable markers using DArT proprietary SNP and SilicoDArT algorithms (DArTsoft14) To further deﬁne and visualise the population genetics of animals in Hawkes Bay, we ran a STRUCTURE analysis (Pritchard et al. 2000) using all loci with identical ﬁlters (n = 502) for each value of K (assumed number of ancestral populations) between 2 and 5 with 10 repeats each of 100,000 iterations. The ﬁrst 50,000 iterations were disregarded as burnin and then each of the 10 repeats were summarised using the programs Evanno (Evanno et al. 2005) and Clumpp (Jakobsson, Rosenberg 2007). We ran STRUCTURE using both ancestral models (No Admixture and Admixture) because they differ in their underlying assumptions about the origins of the population under study (Porras-Hurtado et al. 2013). The No Admixture scenario assumes no prior knowl- edge about the origin of populations under study. Analysis of genotype-by-sequencing data Analysis of genotype-by-sequencing data of F1, F2 and ‘pure’ ancestry, as well as an even number of males and females, and used the NewHy- brid analysis to sort all 1,605 individuals into three genetic groups: (1) largely Tasmanian in origin, (2) largely Australian mainland in origin, and (3) of mixed ancestry. We then stratiﬁed for collection site within those three groups, focussing most on samples from the sites closest to the three known sites of introduc- tion and the previously identiﬁed contact zone (Fig. 2), and then randomly chose individuals across those strata for inclusion in the present study using genotyping-by-sequencing. The R package dartR (Gruber et al. 2018) was used for population genomic analyses of the DArTseq SNP data, including F-statistics, principal coordinates analysis (PCoA) and ﬁxed difference analysis. SNPs were ﬁltered using the RepAvg function (Repro- ducibility C 0.95), CallRate (0.95), with lower and upper read depth thresholds of 5 and 90 respectively, and to exclude monomorphic loci and secondary SNPs. We estimated ancestry proportions using SNP data and geographic location in tess3r (Caye et al. 2016), for K values of between two and eight with the default settings (code supplied on request). Fixed differences were identiﬁed between the New Zealand and Aus- tralian (mainland and Tasmanian combined) possum populations and through a second analysis in which we considered only the sub-populations at the Hawkes Bay study site identiﬁed using PCoA and tess3r (Caye et al. 2016). Sample selection Sample selection In order to determine the Australian origins of the invasive possums at our study site in Hawkes Bay, New Zealand, we obtained samples from a broad range of locations in eastern mainland Australia (n = 19) and Tasmania (n = 11). Speciﬁcally, we sampled tissue (mainly ear tissue) from museum specimens and road killed animals, from six states and territories (Northern Territory, Australian Capital Territory, Queensland, New South Wales, Victoria and Tasma- nia) and Flinders Island (located in the Bass Strait between Tasmania and mainland Australia; Fig. 2). These samples encompass the four commonly recog- nised subspecies of Trichosurus vulpecula in eastern and northern Australia (Kerle et al. 1991): T.v vulpecula (south-eastern Australia), T.v. arnhemensis (Northern Territory), T.v. johnstonii (Queensland) and 12 12 3 3 3834 C. D. Campbell et al. matic of collection locations of samples used in this study from Australia including Tasmania and Flinders I chematic of collection locations of samples used in this study from Australia including Tasmania and Flinders Island Fig. 2 Schematic of collection locations of samples used in this study from Australia including Tasmania a collection locations of samples used in this study from Australia including Tasmania and Flinders Island Speciﬁcally, we applied New Hybrid analysis (An- derson, Thompson 2002) to the microsatellite data previously reported by Sarre et al. (2014) to estimate the percentage of ancestry for each individual possum and indicate whether an individual is an F1 or F2 cross between subspecies. We then selected individual possums that represented all possible combinations T.v.fuliginosus (Tasmania and Flinders Island) and represent the subspecies most likely to include the source populations for New Zealand possums (Fig. 2). Possums from the Hawkes Bay region of New Zealand were sampled from 24 sites (n = 253) and were selected as a subset of the samples previously geno- typed using microsatellite DNA (Sarre et al. 2014). 12 123 Introduction of two subspecies generated dispersal barriers 3835 Genotype-by-sequencing In our case, we know something about the origins of the populations drawn from the introduction history and we have an hypothesis arising from a previous microsatellite DNA study (mainland, Tasmanian and Hybrid) so for this analysis we ignored that knowledge and checked to see if the three groups still emerged. We then ran the Admixture model using our prior knowledge of the origin of the populations to provide insight into the proportion of individuals that can be attributed to each of the presumed ancestral popula- tions. In order to do this, we combined individuals from those sites closest to the mainland introduction 12 3 3 3836 C. D. Campbell et al. sites, those from the previously identiﬁed hybrid zone (Sarre et al. 2014) and those closest to the sites populated by possums from Tasmania into sub- populations A, B and C respectively. We expected that A and C (Mainland and Tasmanian origin) would each have a unique signature, while individuals from population B should show a mixture of both A and C. microsatellite DNA markers (Kennington et al. 2003; Sundqvist et al. 2016) by using the raw counts of private alleles rather than comparisons between observed and simulated asymmetries. Under our approach, we consider that a detectable asymmetry in the number of private alleles between neighbouring sub-populations is indicative of asymmetric geneﬂow between those two sub-populations. Conversely, sym- metry in the numbers of private alleles is indicative of equal rates of gene ﬂow in both directions between two sub-populations. p p We also used a NewHybrids analysis to examine population structure among Hawkes Bay possums. While broadly similar to that provided by STRUC- TURE, NewHybrids analysis differs in that it uses a detailed inheritance model to check to see if individ- uals are indeed hybrids when two potential source populations mainly comprising of pure individuals can be identiﬁed (Anderson, Thompson 2002). As NewHybrids can only be applied to a maximum of 200 loci, we reduced the available loci to 15,523 by ﬁltering on call rate (C 0.95), and then selected the most informative (based on their polymorphic infor- mation content as implemented in dartR) 200 SNPs for NewHybrids analysis, using 30,000 sweeps with a burn in of approximately 100 sweeps, and using the Jefferies prior for both Pi and Theta. Genotype-by-sequencing To further validate the result and its independence from the 200 loci identiﬁed as most informative, we re-ran the analysis with 200 randomly selected loci 30 times. To study the gene ﬂow into and out of the contact zone using private alleles we clustered adjacent sample sites with the aim of obtaining an approxi- mately even number of individuals per cluster. We used the proximity of individuals as the main criterion for clustering, while also considering the genetic similarity of individuals as indicated by the NewHy- brid approach (see above) using microsatellite DNA data gathered previously by Sarre et al. (2014). Using this approach, we generated six clusters (A1, B1, B2, B3, C1, C2) within the three sub-populations and conﬁrmed their validity by running a Discriminant Analysis of Principal Component (Jombart et al. 2010). We then counted the number of private alleles for each pairwise comparison among the 6 sub- populations. Sample sizes of individuals in each sub- population varied between 20 and 36. To account for the uneven number of individuals, we sampled all sub- populations to 20 individuals (with replacement) and counted the number of private alleles in each pair of sub-samples. To reduce noise in the data set, we ﬁltered the identiﬁed markers by minor allele fre- quency with a threshold of [ 0.1 (based on 40*0.1 [ 4 alleles) so that a cluster contained at least 5 alleles before an allele could be called private. This resulted in a data set of 506 SNPs, with a minimum minor allele frequency of 0.1 and mean minor allele frequency of 0.23. The mean number of private alleles between each resampled pair of sub-populations was then calculated from a thousand repetitions of the procedure. Finally, we calculated the mean and 95% conﬁdence intervals of the number of private alleles and the pairwise ratio of private alleles. Sub-popula- tion pairs that had a private allele ratio larger than one, in more than 975 out of 1000 cases, were considered signiﬁcantly asymmetric and hence regarded as exhibiting asymmetric gene ﬂow. Directionality of gene ﬂow through asymmetry in private alleles Results 9,000 years ago (Veevers 1991). There was also good support for differentiation between T. v. vulpecula and two other mainland subspecies (T. v. arnhemensis and T. v. johnsoni) although there was overlap between the latter two subspecies (Fig. 3). We genotyped 283 individual possums from Australia and New Zealand using the DArTseq approach, generating 27,339 SNPs, 6,596 of which were retained after ﬁltering. Ten individuals were also removed by ﬁltering because of high numbers of missing data leaving a total of 273 possum genotypes for analysis. We identiﬁed far fewer private alleles in the T.v.fulig- inosus samples from Tasmania (mean = 25.3; n = 11) than in T.v.vulpecula sampled from mainland Aus- tralia (mean = 296.6; n = 12). Possums sampled in Tasmania also exhibited lower levels of expected (0.04) and observed (0.03) heterozygosity relative to their mainland counterparts (0.13 and 0.11 respec- tively). There was strong support (17.3% weight for PCoA Axis 1) for differentiation between Tasmanian individuals of T.v.fuliginosus (including those from Flinders Island) and all other individuals sampled from mainland Australia (Fig. 3). These ﬁndings are indicative of the Tasmanian history of geographical isolation, following separation of the island from mainland Australia by rising sea levels more than Within the Hawkes Bay study area, we identiﬁed the three distinct sub-populations (A, B, and C) seen previously using microsatellite DNA analysis (Sarre et al. 2014). Sub-population A, genetically the closest to the introduction site of animals from mainland Australia, is discrete from sub-populations B and C, but does not cluster with any mainland Australian animals sampled here (Fig. 4). Sub-populations B and C cluster most closely together and have a lower pairwise FST (0.03; Table 1) than the other pairwise comparisons (A-B: FST = 0.06; A-C: FST = 0.15). The latter FST is equivalent to that observed for T. v. vulpecula and T. v. fuliginosus in their native ranges (FST = 0.16). The highest pairwise FST values were recorded between mainland T. v. vulpecula and groups B and C (FST = 0.25 and 0.40 respectively; Table 1). No ﬁxed allelic differences were observed between any pairwise combination of the Hawkes Bay sub- Fig. 3 PCoA of SNP genotypes from Australian brushtail possums, showing clear differences between T.v.fuliginosus (blue–Tasmania and Flinders Island) and T.v.vulpecula (purple– Australian mainland) along axis 1 but with some overlap between the other two subspecies T.v. johnsoni (green) and T.v. arnhemensis (red). Directionality of gene ﬂow through asymmetry in private alleles To further determine the direction of interactions within the Hawkes Bay study site, we estimated the levels of asymmetry of gene ﬂow between sub- populations using a novel approach based on the numbers of private alleles present in each sub- population. Speciﬁcally, we used the idea that a sub- population that has a net loss of individuals towards another sub-population through emigration will lose private alleles over time as it shares them with the recipient sub-population. Under this process, the sub- population that has fewer emigrants will lose private alleles (through sharing) at a slower rate that its paired sub-population while also accumulating private alleles through genetic drift. We used this logic to explore the direction of gene ﬂow by counting the number of private alleles between sub-populations across the study area while accounting for differences in sample size. This approach differs from those developed for 12 123 123 Introduction of two subspecies generated dispersal barriers 3837 Results Mainland possums show greater diversity than the tightly clustered Tasmanian possums 1 3 Fig. 3 PCoA of SNP genotypes from Australian brushtail possums, showing clear differences between T.v.fuliginosus (blue–Tasmania and Flinders Island) and T.v.vulpecula (purple– Australian mainland) along axis 1 but with some overlap between the other two subspecies T.v. johnsoni (green) and T.v. arnhemensis (red). Mainland possums show greater diversity than the tightly clustered Tasmanian possums 12 3838 C. D. Campbell et al. i d b h Th ll b f Fig. 4 PCoA analysis of SNP genotypes from brushtail possums sampled from the Hawkes Bay region of New Zealand (n = 253), mainland Australia (n = 19) and Tasmania and Flinders Island (n = 11). Australian animals: blue = T.v. john- soni; aqua = T.v. arnhemensis; pink = T.v. vulpecula (all mainland); purple = T.v. fuliginosus (Tasmania). New Zealand animals: red (A) = samples closest to the introduction site for possums of mainland origin; brown (B) = sites previously identiﬁed by Sarre et al. (2014) as likely to contain animals of mixed Tasmanian and mainland ancestry; and olive green (C) = samples closest to the ﬁrst release site of Tasmanian animals animals: red (A) = samples closest to the introduction site for possums of mainland origin; brown (B) = sites previously identiﬁed by Sarre et al. (2014) as likely to contain animals of mixed Tasmanian and mainland ancestry; and olive green (C) = samples closest to the ﬁrst release site of Tasmanian animals Fig. 4 PCoA analysis of SNP genotypes from brushtail possums sampled from the Hawkes Bay region of New Zealand (n = 253), mainland Australia (n = 19) and Tasmania and Flinders Island (n = 11). Australian animals: blue = T.v. john- soni; aqua = T.v. arnhemensis; pink = T.v. vulpecula (all mainland); purple = T.v. fuliginosus (Tasmania). New Zealand Table 1 Pairwise Fst estimates among three groups of pos- sums in the Hawkes Bay region of New Zealand (A = main- land ancestry, B = mixed ancestry, C = Tasmanian ancestry) and two subspecies of Australian common brushtail possums (T.v. fuliginosus and T.v. vulpecula) restricted between them. The smallest number of private alleles were seen between sub-populations B and C, suggesting that gene ﬂow is higher between those two than between sub-populations A and B or between A and C. A B C T. v. Results fuliginosus 25 11 0 58.5 13.6 A T. v. vulpecula 25 12 0 17.3 102.6 B T. v. fuliginosus 60 11 0 24.8 4.7 B T. v. vulpecula 60 12 0 8.0 42.2 C T. v. fuliginosus 165 11 0 9.5 1.5 C T. v. vulpecula 165 12 0 3.3 15.0 T.v.fuliginosus T.v.vulpecula 11 12 0 25.3 296.6 using the STRUCTURE analysis (Fig. 6) which showed that the most appropriate number of groups to emerge from both No Admixture and Admixture models was three (Fig. S2) and, as predicted, demon- strated that the sub-population designated as ‘‘B’’ most closely resembled a combination of sub-populations A and C. Similarly, the NewHybrid plots (Fig. S3; S4) show that sub-population B consists largely of F2 hybrid individuals along. Territory, that cluster closest to the Hawkes Bay individuals of putatively mainland ancestry. We also used SNP genotype data combined with geographic information on each sample location to generate a visual representation of population genetic structure among the Hawkes Bay possums only using tess3r (Fig. 5; Fig. S1). Here, we examined three scenarios for the ancestral makeup of the possums. (1) Two ancestral sub-populations (mainland Australia and Tasmania origins; k = 2); (2) Three ancestral sub- populations (T.v.vulpecula–mainland, T. v. fuligi- nosus–Tasmania, and the contact zone population; k = 3); and (3) Four ancestral sub-populations (T.v.vulpecul–mainland, two different introductions of T.v.fuliginosus–Tasmania, and the contact zone population; k = 4). Our estimates of ancestry propor- tions and ancestral allele frequencies show a clear disjunction between the two subspecies at their contact zone (Fig. 5; Panel 1), with the contact zone being genetically closer to the populations descended from T.v.fuliginosus than to those descended from T.v.vulpecula. The contact zone itself seems to run to the west of the Mohaka River that divides the two release points (Fig. 5; Panel 2). We ﬁnd no evidence of two distinct ancestral groups of T.v.fuliginosus arising from the dual introductions of possums from Tasmania (Fig. 5; Panel 3) and it appears that this subspecies is relatively homogenous at its source within Tasmania (Fig. 4). These results were similar to those obtained with small numbers of both parental genotypes while sub-populations A and C are comprised largely of parental individuals with mainland and Tasmanian origins respectively. There is little evidence of F1 individuals in any of the Hawkes Bay animals genotyped. Results vulpecula B 0.06 – – – C 0.15 0.03 – – T. v. vulpecula 0.12 0.25 0.40 – T. v. fuliginosus 0.08 0.07 0.10 0.16 The PCoA analysis including all mainland Aus- tralian, Tasmanian, and Hawkes Bay samples (Fig. 4) shows that the majority of mainland Australian possums are genetically different from both New Zealand and Tasmanian individuals. In contrast, the native Tasmanian possums cluster very closely with the Hawkes Bay possums of putative Tasmanian ancestry (sub-populations B and C), providing addi- tional genetic evidence for Tasmanian ancestry for those Hawkes Bay animals. The possums from sub- population A appear to be distinct from both Hawkes Bay sub-populations B and C and from the native Tasmanian and mainland Australian samples. We identiﬁed two mainland Australian individuals, one sampled from around the Sydney area of New South Wales and the other from the Australian Capital populations A, B, or C, or between any of those three groups and the two Australian subspecies (Table 2). Private alleles were observed in all pairwise combi- nations of groups. Overall, these data suggest that that both B and C are composed largely of descendants of Tasmanian animals. All three Hawkes Bay genetic sub-populations exhibit private alleles when compared to each other (Table 2) suggesting that gene ﬂow is 12 123 Introduction of two subspecies generated dispersal barriers 3839 Table 2 Fixed differences, number of private alleles, and corrected number of private alleles between Hawkes Bay, New Zealand groups of possums (groups A, B and C) and Australian common brushtail possums sampled from Tasmania (T.v. fuliginosus) and the south eastern mainland (T.v. vulpecula). The accumulation of ﬁxed differences between populations indicates low levels of gene ﬂow. The ‘‘corrected’’ number of private alleles has been divided by the larger of the two samples sizes to account for the difference between groups The accumulation of ﬁxed differences between populations indicates low levels of gene ﬂow. The ‘‘corrected’’ number of private alleles has been divided by the larger of the two samples sizes to account for the difference between groups Pop 1 Pop 2 Sample size Pop 1 Sample size Pop 2 Fixed differences between populations No. Private alleles (corrected) Pop 1 No. Private alleles (corrected) Pop 2 A B 25 60 0 5.6 7.0 A C 25 165 0 4.0 6.9 B C 60 165 0 2.8 3.5 A T. v. Results Finally, asymmetric geneﬂow was identiﬁed between several pairs of the six genetic clusters using our private allele approach (Fig. 7, Table S1). The number of private alleles was consistently lower in sub-population A1 (mainland origin individuals) than in the three sub-populations within the contact zone (B1, B2 and B3). The same was true for sub- population C1 (Tasmanian origin individuals) relative to sub-populations within the contact zone (B1, B2 and B3). We also identiﬁed a signiﬁcant asymmetry in the geneﬂow between sub-populations C1 and C2. There was no apparent effect of distance on the asymmetries between clusters (Fig. S6). In summary, the asymme- try in private alleles indicates that the direction of 12 3 3 C. D. Campbell et al. 3840 Fig. 5 Estimates of ancestry proportions and ancestral allele frequencies in the Hawkes Bay region of New Zealand identiﬁed using tess3r (Caye et al., 2016). This ﬁgure is generated using SNP genotype data and geographic information on sample locations. Three ancestral groupings were tested to visualise the likely ancestry of the Hawkes Bay possums: Two ancestral populations (left-hand panel: mainland Australia and Tasmania; k = 2); Three ancestral populations (middle panel: T.v.vulpec- ula—mainland, T. v. fuliginosus–Tasmania, and the contact zone population; k = 3); and Four ancestral populations (right- hand panel: T.v.vulpecula—mainland, two different introduc- tions of T.v.fuliginosus—Tasmania, and the contact zone population; k = 4). See Fig. S1 for a graphical representation of the cross-validation scores Fig. 5 Estimates of ancestry proportions and ancestral allele frequencies in the Hawkes Bay region of New Zealand identiﬁed using tess3r (Caye et al., 2016). This ﬁgure is generated using SNP genotype data and geographic information on sample locations. Three ancestral groupings were tested to visualise the likely ancestry of the Hawkes Bay possums: Two ancestral populations (left-hand panel: mainland Australia and Tasmania; k = 2); Three ancestral populations (middle panel: T.v.vulpec- ula—mainland, T. v. fuliginosus–Tasmania, and the contact zone population; k = 3); and Four ancestral populations (right- hand panel: T.v.vulpecula—mainland, two different introduc- tions of T.v.fuliginosus—Tasmania, and the contact zone population; k = 4). See Fig. S1 for a graphical representation of the cross-validation scores geneﬂow is into the contact zone, with only minor geneﬂow outwards. geneﬂow is into the contact zone, with only minor geneﬂow outwards. Results are derived from two Australian subspecies are more closely resembling their two ancestral entities sepa- rated by a contact zone than one integrated population. This is despite having ample opportunity to disperse, mingle, and interbreed for over 80 years (between 40 and 80 generations). We found that new genetic forms, representing individuals of mixed ancestry (F2 ?) between the two subspecies, occur mainly in the intermediate contact zone between the sites of the original introductions. We also observe that gene ﬂow tends to move inwards, into that contact zone more commonly than outwards and found no evidence of parental genotypes moving across the contact zone and into the opposite parental sub-population. As such, the contact zone that has formed appears to act as a sink and limits dispersal. Discussion New Zealand possums are the subject of enormous national pest animal control effort and considerable scientiﬁc endeavour (Byrom et al. 2016; Clout, Sarre 1997; Eason et al. 2017; Forsyth et al. 2018; Goldson et al. 2015; Livingstone et al. 2015; Montague 2000; Rouco et al. 2018; Rouco et al. 2017). While the history of their introduction from Tasmania and the Australian mainland is extremely well documented, New Zealand possums are considered almost univer- sally to be representatives of a single lineage, albeit one with colour variants. This view is consistent with the prevailing taxonomy that discriminates between them only at the subspeciﬁc level (Kerle et al. 1991). Here, we demonstrate that this expectation of possums mixing indiscriminately with respect to subspecies, does not hold in practice in the introduced New Zealand population, at least within the Hawkes Bay study area. Speciﬁcally, possums in Hawkes Bay that Our results provide clear evidence that possums in Hawkes Bay behave as three distinct groups rather than a single panmictic population—a result that reﬂects previous ﬁndings of genetic structure at the site (Etherington et al. 2014; Sarre et al. 2014). We show that Hawkes Bay possums with putative Tas- manian ancestry have high genetic similarity to modern Tasmanian populations (T.v.fuliginosus), with 123 Fig. 6 STRUCTURE analysis plot of Hawkes Bay brushtail possums with admixture for all loci (following ﬁltering). The blue vertical lines in the structure plots represent the putative boundaries between the three groups with the left-hand side including samples from sites closest to the introduction sites for mainland origin animals and those on the right including animals closest to the introduction sites for animals from Tasmania Fig. 7 Asymmetry in geneﬂow estimated using the number of private alleles. Black lines indicate signiﬁcant asymmetries in geneﬂow based on 95% conﬁdence intervals obtained through 1000 replicates of resamples of 20 individuals; the level of signiﬁcance is indicated by the thickness of the lines; arrows indicate the predominant direction of gene ﬂow. Grey arrows indicate non-signiﬁcant asymmetries between pairs of sub- populations 123 Introduction of two subspecies generated dispersal barriers 3841 Introduction of two subspecies generated dispersal barriers 3841 Fig. 6 STRUCTURE analysis plot of Hawkes Bay brushtail possums with admixture for all loci (following ﬁltering). Discussion Alternatively, the possums that derive from mainland Australia may be descended from a mixture of animals from several mainland sources, that were brought together in Hawkes Bay, and therefore do not resemble any single extant Australian population. Another possibil- ity is that genetic drift—through founder effects and 80 years of isolation and/or selection imposed by a New Zealand environment—may have altered the genetic makeup of the Hawkes Bay population of T.v.vulpecula sufﬁciently to diverge from mainland Australian possums. This may even have resulted in the retention of genetic variants that are now rare (or even lost) in mainland Australia—a phenomenon seen in stoats introduced to New Zealand from England (Veale et al. 2015). Each of these three factors may contribute to our observations, although the fact that possums from sub-population A are genetically more similar to T.v.fuliginosus and to sub-populations B and C (Fig. 4), than they are to T.v.vulpecula, suggests that there has been leakage of T.v.fuliginosus alleles into sub-population A. A more detailed genomic haplo- typic study, combined with mitochondrial DNA analysis, may resolve this issue. Additional sampling closer to the original release site for T.v.vulpecula in the Hawkes Bay may also help to identify the mainland location of origin for these possums. The distribution of private alleles among the possum sites suggests that there is more immigration into the contact zone than out of it, from both the T.v.fuliginosus dominated population in the east and north of the zone, and from the T.v.vulpecula domi- nated population to the south west. The net result is that very few individuals with predominantly T.v.vulpecula ancestry (sub-population A) migrate completely through the contact zone (sub-population B) and integrate into the predominantly T.v.fuligi- nosus population (sub-population C), or vice versa. The contact zone thus appears to act as a sink for the two subspecies and, as such, represents a non- topographical (invisible) barrier to dispersal. The existence of non-topographical or invisible barriers have implications for management, in this case particularly for the national program aimed at a predator free New Zealand (Russell et al. 2015). Populations with a mix of both grey (T.v.vulpecula) and black (T.v.fuliginosus) individuals occur in many parts of New Zealand (Cowan 2001; Kean 1971; Triggs, Green 1989) and hybrid animals have been reported previously in the Orongorongo valley on the North Island (Kean 1971). Discussion These genome-level observations from the Hawkes Bay region mirror genetic patterns found more broadly in New Zealand possums using allozyme electrophoresis (Triggs, Green 1989). In that study, populations consisting predominantly of black possums were shown to be genetically similar to possums in Tasma- nia, while predominantly grey populations were genetically closer to Victorian and New South Wales possums. Our results provide strong evidence that animals in sub-population B are a hybrid form of T.v. vulpecula and T.v. fuliginosus—the product of interbreeding between sub-populations A and C. The formation of a stable contact zone between two colonisers can arise when competition between the two is stronger than intraspeciﬁc competition or when hybrids are inviable or less ﬁt than the parent taxa (Goldberg, Lande 2007). To our knowledge, interactions between the two subspecies or the viability of their offspring in nature have not been reported. We cannot therefore distin- guish between these two possibilities. The positioning of the contact zone between two colonisers is likely to be attracted to, but not necessarily centred upon, a partial dispersal barrier (Goldberg, Lande 2007). In the case of the possums of Hawkes Bay, the presence of the Mohaka River between the release sites of the mainland Australian origin T.v.vulpecula and the Tasmanian origin T.v. fuliginosus provides such a partial barrier, and may have inﬂuenced the position- ing of the contact zone near the eastern edge of this river. only modest levels of introgression of alleles from T.v.vulpecula. In contrast, Hawkes Bay possums with putatively Australian mainland origin are genetically distinct from all Australian mainland individuals genotyped. As such, we were unable to identify the mainland Australian source for this group. These genome-level observations from the Hawkes Bay region mirror genetic patterns found more broadly in New Zealand possums using allozyme electrophoresis (Triggs, Green 1989). In that study, populations consisting predominantly of black possums were shown to be genetically similar to possums in Tasma- nia, while predominantly grey populations were genetically closer to Victorian and New South Wales possums. There are several possible explanations for the genetic differences observed between the putative T.v.vulpecula animals in Hawkes Bay and the T.v.vulpecula animals sampled from mainland Aus- tralia. One possibility is that hybridisation between individuals of mainland Australian and Tasmanian origin masks the genetic signature of the mainland origins of New Zealand possums. Discussion The blue vertical lines in the structure plots represent the putative boundaries between the three groups with the left-hand side including samples from sites closest to the introduction sites for mainland origin animals and those on the right including animals closest to the introduction sites for animals from Tasmania Fig. 6 STRUCTURE analysis plot of Hawkes Bay brushtail possums with admixture for all loci (following ﬁltering). The blue vertical lines in the structure plots represent the putative boundaries between the three groups with the left-hand side including samples from sites closest to the introduction sites for mainland origin animals and those on the right including animals closest to the introduction sites for animals from Tasmania including samples from sites closest to the introduction sites for mainland origin animals and those on the right including animals closest to the introduction sites for animals from Tasmania including samples from sites closest to the introduction sites for mainland origin animals and those on the right including animals closest to the introduction sites for animals from Tasmania Fig. 6 STRUCTURE analysis plot of Hawkes Bay brushtail possums with admixture for all loci (following ﬁltering). The blue vertical lines in the structure plots represent the putative boundaries between the three groups with the left-hand side Fig. 7 Asymmetry in geneﬂow estimated using the number of private alleles. Black lines indicate signiﬁcant asymmetries in geneﬂow based on 95% conﬁdence intervals obtained through 1000 replicates of resamples of 20 individuals; the level of signiﬁcance is indicated by the thickness of the lines; arrows indicate the predominant direction of gene ﬂow. Grey arrows indicate non-signiﬁcant asymmetries between pairs of sub- populations Fig. 7 Asymmetry in geneﬂow estimated using the number of private alleles. Black lines indicate signiﬁcant asymmetries in geneﬂow based on 95% conﬁdence intervals obtained through 1000 replicates of resamples of 20 individuals; the level of signiﬁcance is indicated by the thickness of the lines; arrows indicate the predominant direction of gene ﬂow. Grey arrows indicate non-signiﬁcant asymmetries between pairs of sub- populations 12 123 3842 C. D. Campbell et al. only modest levels of introgression of alleles from T.v.vulpecula. In contrast, Hawkes Bay possums with putatively Australian mainland origin are genetically distinct from all Australian mainland individuals genotyped. As such, we were unable to identify the mainland Australian source for this group. Discussion It is therefore possible that many such contact zones occur on the main islands of New Zealand. These zones could inhibit local disper- sal and provide barriers that were previously invisible, 123 Introduction of two subspecies generated dispersal barriers 3843 but which could now be exploited for control. Conversely, possums can disperse quickly into depop- ulated habitats (Efford et al. 2000; Ji et al. 2001), so poisoning or trapping across contact zones could inadvertently break down existing subspeciﬁc barriers and increase local rates of dispersal. Documentation of the existence of additional contact zones on the main islands of New Zealand would provide greater oppor- tunities to take advantage of these invisible barriers, with potential to produce faster localised eradication results. could function as barriers to dispersal across New Zealand. These hybrid zones could provide opportu- nities for managers to adopt a more nuanced approach to control of this introduced pest species, by account- ing for differences between the subspecies and by taking advantage of previously unrecognised borders at contact zones. Acknowledgements We thank Arthur Georges and Andrew O’Reilly Nugent for advice on data analysis and Matt Young, Sam Venables, and Llara Weaver for lab assistance. Samples were kindly contributed by Robert Palmer and Leo Joseph (Australian National Wildlife Collection), Belinda Bauer and Kathryn Medlock (Tasmanian Museum and Art Gallery), Tammy Gordon and David Maynard (Queen Victoria Museum and Art Gallery), Joanne Sumner (Museum Victoria), Sandy Ingleby (Australia Museum), Gavin Dally (Museum and Art Gallery of the Northern Territory) and Elise Dewar (Department of Primary Industries, Parks, Water and Environment). The New Zealand samples were originally curated by Nicola Aitken. Possums are the principal wildlife host of bovine tuberculosis in New Zealand (Morgan 1990), and the main cause of Tb persistence in cattle and deer herds in New Zealand, although the disease has not been recorded in Australian possums (Nugent, Cousins 2014; Tweddle, Livingstone 1994). If hybrid possums are less ﬁt than either of the parental subspecies then the risk that possums are susceptible to bovine tuberculosis may be exacerbated. Moreover, there is some evidence that black coated animals, presumably T.v.fuliginosus, exhibit higher resistance to 1080 poison (Henderson et al. 1999; McIlroy 1982) than T.v.vulpecula–a phenomenon that has previously been hypothesised for marsupials more generally (Deakin et al. 2013). Discussion Thus, baiting with 1080 (a common approach used to control possums in New Zealand) could affect disproportionately the viability of one subspecies over the other, or even the viability of certain hybrids over others. Further research in this area, particularly through genomic analyses (Eldridge et al. 2020) will be particularly important if total eradication of possums is to be a key management objective. Authors’ contribution Conceived project: SDS, CDC, BG, AJM, PC; Collected samples: PC (direct trapping of New Zealand samples) with SDS; CDC coordination of collection of Australian samples from roadkill and museum specimens; Conducted lab work: CDC; Conducted analyses and generated ﬁgures: CDC and BG; Wrote the manuscript: CDC and SDS with contributions from AJM, CEH, BG, PC. All authors read and approved the ﬁnal manuscript. Funding Project funding was provided to SDS from the Invasive Animals CRC (1.L.21). CDC was supported by an Australian Postgraduate Award and an Invasive Animals CRC postgraduate scholarship. Code availability All genetic data are available on https:// ﬁgshare.com/; https://doi.org/10.6084/m9.ﬁgshare.13040522 (embargoed for 12 months). Declarations In conclusion, we have identiﬁed clear evidence of a contact zone or border between two subspecies of introduced brushtail possums in Hawkes Bay, New Zealand, that produces a hybrid form. More work is required to determine whether this zone has formed because of inter-sub-speciﬁc competition or reduced hybrid ﬁtness. The contact zone functions as a sink, with an asymmetry of gene ﬂow tending towards the contact zone and away from both subspecies. It is therefore likely that the zone functions as a barrier to dispersal between the subspecies. Greater sampling would be required to determine the extent of the contact zone. Given the widespread overlapping distribution of the two subspecies in New Zealand, it is possible that there are many such hybrid zones that Conﬂict of interest There were no conﬂicts of interest or competing interests identiﬁed. Ethics approval Possum tissues were obtained from muse- ums, the UC tissue collection (reported previously in Sarre et al. 2014), or road killed animals in Tasmania. 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https://openalex.org/W4233042046	https://spaces.at.internet2.edu/spaces/flyingpdf/pdfpageexport.action?pageId=190358348	English	null	A Brief History	null	2,021	cc-by	132	TI.158.1 A Brief History PDF: A Brief History Text: A Brief History More Information Repository ID TI.158.1 Persistent URL http://doi.org/10.26869/TI.158.1 Title A Brief History Authors Ken Klingenstein Sponsor Internet2 Trust and Identity executive management Review Status preserve Publish Date June 22, 2021 DOI Signature Deprecated No Future Review Supersedes Format pdf, text Related Docs Development Location IP Framework CC BY 4.0 Subject Tags Notes TI.158.1 A Brief History PDF: A Brief History Text: A Brief History More Information Repository ID TI.158.1 Persistent URL http://doi.org/10.26869/TI.158.1 Title A Brief History Authors Ken Klingenstein Sponsor Internet2 Trust and Identity executive management Review Status preserve Publish Date June 22, 2021 DOI Signature Deprecated No Future Review Supersedes Format pdf, text Related Docs Development Location IP Framework CC BY 4.0 Subject Tags Notes TI.158.1 A Brief History
https://openalex.org/W1971027009	https://hal-amu.archives-ouvertes.fr/hal-01218062/file/fetchObject.pdf	English	null	Innate Immune Response to Streptococcus pyogenes Depends on the Combined Activation of TLR13 and TLR2	PloS one	2,015	cc-by	12,118	To cite this version: To cite this version: Christina Fieber, Marton Janos, Tina Koestler, Nina Gratz, Xiao-Dong Li, et al.. Innate Immune Response to Streptococcus pyogenes Depends on the Combined Activation of TLR13 and TLR2. PLoS ONE, 2015, 10 (e0119727), 10.1371/journal.pone.0119727.s001. hal-01218062 Christina Fieber, Marton Janos, Tina Koestler, Nina Gratz, Xiao-Dong Li, et al.. Innate Immune Response to Streptococcus pyogenes Depends on the Combined Activation of TLR13 and TLR2. PLoS ONE, 2015, 10 (e0119727), 10.1371/journal.pone.0119727.s001. hal-01218062 HAL Id: hal-01218062 https://amu.hal.science/hal-01218062v1 Submitted on 20 Oct 2015 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. RESEARCH ARTICLE Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by the Austrian Science Fund (FWF) grants P24540-B21 and W1220-B09 DP ‘Molecular Mechanisms of Cell Signaling’ to PK, by funding from the European Union Seventh Framework Programme Marie Curie Initial Training Networks (FP7-PEOPLE-2012-ITN) for the project INBIONET under grant agreement PITN-GA- 2012-316682, and by a joint research cluster initiative of the University of Vienna and the Medical University of Vienna. The funders had no role in study design, OPEN ACCESS Citation: Fieber C, Janos M, Koestler T, Gratz N, Li X-D, Castiglia V, et al. (2015) Innate Immune Response to Streptococcus pyogenes Depends on the Combined Activation of TLR13 and TLR2. PLoS ONE 10(3): e0119727. doi:10.1371/journal. pone.0119727 Innate Immune Response to Streptococcus pyogenes Depends on the Combined Activation of TLR13 and TLR2 Christina Fieber1, Marton Janos1, Tina Koestler2, Nina Gratz1¤, Xiao-Dong Li6, Virginia Castiglia1, Marion Aberle1, Martina Sauert1, Mareike Wegner3, Lena Alexopoulou4, Carsten J. Kirschning5, Zhijian J. Chen6, Arndt von Haeseler2,7, Pavel Kovarik1* 1 Max F. Perutz Laboratories, University of Vienna, Vienna, Austria, 2 Center for Integrative Bioinformatics Vienna, Max F. Perutz Laboratories, University of Vienna, Medical University of Vienna, Vienna, Austria, 3 Universitätsklinikum Freiburg, Universitäts-Hautklinik, Freiburg, Germany, 4 Centre d'Immunologie de Marseille-Luminy (CIML), Aix-Marseille Université UM 2, Marseille, France, 5 Institute of Medical Microbiology, University of Duisburg-Essen, Essen, Germany, 6 Howard Hughes Medical Institute, Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, Texas, United States of America, 7 Bioinformatics and Computational Biology, Faculty of Computer Science, University of Vienna, Vienna, Austria ¤ Current address: Department of Infectious Diseases, St. Jude Children's Research Hospital, Memphis, Tennessee, United States of America * pavel.kovarik@univie.ac.at Academic Editor: Jörg Hermann Fritz, McGill University, CANADA Academic Editor: Jörg Hermann Fritz, McGill University, CANADA Copyright: © 2015 Fieber et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Abstract Innate immune recognition of the major human-specific Gram-positive pathogen Strepto- coccus pyogenes is not understood. Here we show that mice employ Toll-like receptor (TLR) 2- and TLR13-mediated recognition of S. pyogenes. These TLR pathways are non- redundant in the in vivo context of animal infection, but are largely redundant in vitro, as only inactivation of both of them abolishes inflammatory cytokine production by macrophages and dendritic cells infected with S. pyogenes. Mechanistically, S. pyogenes is initially recog- nized in a phagocytosis-independent manner by TLR2 and subsequently by TLR13 upon in- ternalization. We show that the TLR13 response is specifically triggered by S. pyogenes rRNA and that Tlr13−/−cells respond to S. pyogenes infection solely by engagement of TLR2. TLR13 is absent from humans and, remarkably, we find no equivalent route for S. pyogenes RNA recognition in human macrophages. Phylogenetic analysis reveals that TLR13 occurs in all kingdoms but only in few mammals, including mice and rats, which are naturally resistant against S. pyogenes. Our study establishes that the dissimilar expression of TLR13 in mice and humans has functional consequences for recognition of S. pyogenes in these organisms. Academic Editor: Jörg Hermann Fritz, McGill University, CANADA Received: June 16, 2014 Accepted: January 30, 2015 Published: March 10, 2015 Copyright: © 2015 Fieber 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. Innate Immunity to Streptococcus pyogenes Depends on TLR2 and TLR13 which have most likely evolved by gene duplication and exon shuffling from an ancestral gene early in metazoan evolution [1]. TLRs recognize specific pathogen-associated molecular pat- terns (PAMPs) which are common to different pathogen species. Streptococcus pyogenes, also called Group A Streptococcus, is an important Gram-positive human pathogen yet its recogni- tion by innate immune cells remains unknown [2,3]. S. pyogenes causes a broad range of mostly self-limiting diseases including pharyngitis (strep throat), scarlet fever or impetigo [4,5]. It may also cause invasive and life-threatening infections such as necrotizing fasciitis and toxic shock with 30% mortality rate. S. pyogenes accounts for over 700 million mild and more than 650,000 severe invasive infections worldwide annually [6]. Together with S. pneumoniae, S. pyogenes is one of the most frequently found co-infecting bacteria in specimens of the 1918 flu pandemics and in patients of the recent H1N1 flu outbreak [7]. data collection and analysis, decision to publish, or preparation of the manuscript. data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors declare no financial or commercial conflict of interest. The exceptionally large variety of S. pyogenes-related infectious diseases is caused in part by variations of virulence factor armament of S. pyogenes strains and in part by the genetic make- up of the host [8,9]. On the host site, animal studies demonstrated that innate immune cells, most notably macrophages, dendritic cells and neutrophils, play an essential role in defense during subcutaneous infection, a model of invasive S. pyogenes infection [10–13]. Despite the importance of the innate immune system for host defense, the TLRs and PAMPs involved in functional recognition of S. pyogenes are not defined. We and others have shown that S. pyogenes-induced production of inflammatory cytokines, including TNF and IL- 6, by murine bone marrow-derived macrophages (BMDMs; bone marrow cells differentiated in the presence of CSF1) and conventional dendritic cells (cDCs; bone marrow cells differenti- ated in the presence of GM-CSF) is entirely dependent on the signaling adaptor MyD88. Con- sistently, MyD88 is required for survival of mice during S. pyogenes infection [14]. The TLRs triggering the protective innate immune response are not known. Studies by us and others demonstrated that S. pyogenes induces cytokine production in the absence of the MyD88-de- pendent TLR2, TLR4 and TLR9 [13,15]. Introduction Several receptor families are employed by innate immune cells to detect infecting agents, Toll- like receptors (TLRs) being the most prominent members. Mammals possess several TLRs 1 / 20 PLOS ONE \| DOI:10.1371/journal.pone.0119727 March 10, 2015 PLOS ONE \| DOI:10.1371/journal.pone.0119727 March 10, 2015 Mice Tlr2−/−, Tlr3−/−, Tlr7−/−, Tlr8−/−, Tlr379 triple knockout, Trif−/−, Tlr13−/−, Unc93b1−/−, and Unc93b1Tlr2 double knockout, MyD88−/−mice were on C57BL/6 background and housed under specific pathogen-free conditions. C57BL/6 wild type (WT) mice were purchased from Charles River Laboratories. Age- and sex-matched 7–12 weeks old mice were used for experiments. Ethics statement All animal experiments were carried out in accordance with the Austrian law for animal care (GZ 680 205/67-BrGt/2003). Animal experiments were authorized through the licenses BMWF-66.009/0031-II/10b/2008 and BMWF-66.006/0006-II/3b/2013, issued by the Austrian Ministry of Science to PK. Bacterial cultures The Streptococcus pyogenes serotype M1 strains ISS3348 (provided by Roberta Creti, Instituto Superiore di Sanita, Italy) was used as described previously [21]. As exemplified by our study, the lack of TLR13 in humans results in the lack of TLR-mediated recognition of S. pyo- genes rRNA. A 13 nucleotide long sequence of bacterial 23S rRNA has been recently demonstrated to act as PAMP recognized by the TLR13 in murine cells [16– 18]. TLR13, whose ligand has long remained unknown, is located in endosomes and similarly to other endosomal TLRs (TLR3, TLR7, TLR9) requires Unc93b1, a COPII vesicle membrane protein, for trafficking to endosomes [19,20]. Deletion of Unc93b1 abolishes responses of cells to ligands sensed by endosomal TLRs including TLR13 [19,20]. It remains unknown whether recognition of bacterial rRNA by TLR13 regulates the host defense in mice. Importantly, it is not understood how human immune cells recognize S. pyogenes and whether S. pyogenes RNA plays a role in this process. Here we report that S. pyogenes infection of mouse BMDMs and cDCs triggers both TLR2 and TLR13 pathways. Both pathways are to large part redundant in vitro: the TLR2 pathway becomes apparent only in Tlr13−/−cells thereby explaining the lack of evidence for a role of TLR2 in previous studies. The TLR13 pathway is activated by S. pyogenes rRNA and is depen- dent on phagocytosis and endosomal recognition. Consistently, we find that Unc93b1 plays an essential role in cytokine induction by S. pyogenes RNA. Unexpectedly, the TLR2 and the endo- somal TLR recognition pathway are not redundant in vivo: mice deficient in either of these pathways display an increased susceptibility to S. pyogenes infection. These data show that a protective immune reaction is mounted only by triggering both pathways. Humans lack TLR13 and we find that human innate immune cells are not capable of inducing TNF and IL-8 in response to S. pyogenes RNA. We demonstrate that human cells sense S. pyogenes through TLR2. However, human primary macrophages produce TNF and IL-8 upon S. pyogenes infec- tion also under conditions of antibody-mediated TLR2 inhibition indicating that a TLR2-inde- pendent sensing pathway operates in these cells. Phylogenetic analysis reveals a remarkable lack of TLR13 in primates including humans as opposed to mice and rats, which possess highly 2 / 20 Innate Immunity to Streptococcus pyogenes Depends on TLR2 and TLR13 conserved Tlr13 genes and are naturally resistant against S. pyogenes. As exemplified by our study, the lack of TLR13 in humans results in the lack of TLR-mediated recognition of S. pyo- genes rRNA. conserved Tlr13 genes and are naturally resistant against S. pyogenes. PLOS ONE \| DOI:10.1371/journal.pone.0119727 March 10, 2015 Cell culture Primary bone marrow derived macrophages (BMDMs) and conventional dendritic cells (cDCs) were obtained from the femur and tibia bone marrow of 7–10 weeks old mice and stimulated as described previously [15,21]. Human Embryonic Kidney 293 cells (HEK) cells were cultivated in DMEM with 10% FCS. HEK293XL cells stably expressing either human TLR7 or TLR8 were obtained from Invivogen, and HEK cells stably expressing human TLR3 were generated by stable transfection with the vector pUNO-HA bearing human TLR3 (Invivo- gen). THP-1 cells were cultivated in Roswell Park Memorial Institute medium (RPMI) with 10% FCS. THP-1 cells were differentiated using 10 nM PMA in RPMI with 10% FCS for 24 hours followed by cultivation for additional 48 hours in RPMI with 10% FCS without PMA. THP-1 cells stably expressing TLR13 were generated by lentiviral transduction. HA-tagged mouse TLR13 was cloned from pUNO1-mTLR13-HA3x (Invivogen) into pCDH-CMV- MCS-EF1-puro (System Biosciences) and packaged in HEK293T cells using pMDLg/pRRE (Addgene#12251), pCMV-VSV-G (Addgene #8454) and pRSV-Rev (Addgene #12253). Trans- duced cells were selected with 1.5 μg/ml puromycin followed by clonal expansion and screen- ing for clones positive for expression of the HA epitope-tagged TLR13. Human peripheral blood mononuclear cells (PBMC) were isolated by Ficoll-Paque (GE Healthcare) density cen- trifugation from buffy coats (purchased from Red Cross, Vienna). To differentiate PBMC into monocyte-derived macrophages approximately 1–2x108 cells were plated in RPMI 1640 media with 2 mM L-glutamine containing 7.5% heat-inactivated (HI) autologous human fibrin-de- pleted plasma and antibiotics (MoAM; monocyte adhesion media) in 15 cm round dishes for 45–90 min. Non-adherent cells were removed by gently washing the dish with warm media, fresh MoAM was added and the cells were left in an incubator at 37°C with 5% CO2 overnight. The next day, the monocytes were detached with PBS containing 5 mM EDTA, counted and 3 / 20 PLOS ONE \| DOI:10.1371/journal.pone.0119727 March 10, 2015 Innate Immunity to Streptococcus pyogenes Depends on TLR2 and TLR13 plated in X-Vivo 10 (Lonza, 04-743Q) supplemented with 1% HI autologous human fibrin-de- pleted plasma, 2 mM glutamine and antibiotics. The experiment was performed on day 7 to 8. S. pyogenes infection in vitro For infection assays, cells were seeded on 6 cm dishes (1x106 cells/dish) or 12-well plates (2– 3x105 cells/well for human primary macrophages) in media without antibiotics. Cells were then infected as described previously with some minor changes [21]. Briefly, cells were infected with S. pyogenes at a multiplicity of infection (MOI) of 50 or 100. After 30 min of incubation at 37°C, bacteria were killed by adding 60 μg/ml penicillin to the media. Infections under condi- tions of blocked phagocytosis were performed by pre-treatment of cells with cytochalasin D (50 μM; Sigma) 30 min prior to infection with S. pyogenes. For TLR neutralization, antibodies against human TLR2 (5 μg/ml; Invivogen, maba2-htlr2) and control IgG antibodies (5 μg/ml; Invivogen, maba2-ctrl), or antibodies against mouse TLR2 (5 μg/ml; eBioscience, 16-9024-83) and control IgG (5 μg/ml; eBioscience, 14-4714) were used 30 min prior to infection. Experimental model of S. pyogenes infection Standard model of subcutaneous S. pyogenes infection was used as described previously [21]. Survival curves were analyzed by the Logrank Test using GraphPad Prism 4 (GraphPad soft- ware, San Diego, USA). All data are presented as mean SD. Comparison between two groups was performed using t-test. P values 0.05 were considered as significant. S. pyogenes extracts S. pyogenes extracts treated with proteinase K, DNase or RNase were generated as described previously [21]. PLOS ONE \| DOI:10.1371/journal.pone.0119727 March 10, 2015 S. pyogenes RNA and DNA fractions Isolation of bacterial RNA and DNA was performed according to standard techniques, with some modifications [22]. Briefly, for RNA isolation bacteria were grown to mid-log phase and lysed using 2% SDS and 1 mg/ml proteinase K (Sigma-Aldrich). For isolation of RNA, Trizol LS reagent (Invitrogen) was used. RNA was treated with DNase (TURBO DNA-free; Ambion). For RNA-free samples RNA was treated with RNase A (Roche, 10 mg/ml, heat-inactivated) for30 min at 37°C followed by addition of EDTA (pH 8, 2.5 mM) and further incubation for 10 min at 70°C. For control, sample containing only H2O (RNase control) was treated in the same way. mRNA fraction was obtained using the MICROBExpress Kit (Ambion). Briefly, after the Trizol step of the total bacterial RNA isolation fragments shorter than 200 nucleotides were removed from total RNA using miRNeasy Mini Kit (Qiagen). mRNA was then isolated ac- cording to the MICROBExpress Kit protocol. Bacterial rRNA was eluted from the MICROBExpress Kit beads by adding TE buffer and incubating for 10 min at 70°C. Bacterial mRNA isolated with the MICROBExpress Kit contained 0.3% bacterial rRNA as assessed with qPCR analysis. Pure bacterial mRNA was obtained from total RNA using the Ribo-Zero rRNA Removal Kit (Epicenter) which yields mRNA without contaminating rRNA, but does not allow elution of rRNA. For DNA preparation, over-night cultures of bacteria were harvested, resuspended in TE buffer (10 mM Tris-HCl pH 8, 1 mM EDTA) and treated with lysozyme (Sigma, 2.5 mg/ ml) for 30 min at 37°C. Subsequently, RNase A (10 mg/ml), 20% SDS and 0.5 M EDTA were added. After incubation for 40 min at 37°C, 20 mg/ml proteinase K was added, and the samples were further incubated for 30 min at 37°C. DNA was isolated using phenol:chloroform:isoamy- lalcohol (Sigma-Aldrich). Isolated bacterial DNA was treated with RNase A (10 mg/ml) for PLOS ONE \| DOI:10.1371/journal.pone.0119727 March 10, 2015 4 / 20 Innate Immunity to Streptococcus pyogenes Depends on TLR2 and TLR13 30 min at 37°C. Adherent macrophages and HEK cells were seeded (1x106 cells per 6 cm dish) the day prior to transfection, non-adherent cDCs and THP-1 cells (1x106 cells per 6 cm dish) were seeded 2 hours prior to transfection, PMA-stimulated THP-1 cells were seeded 3 days prior to transfection (1x106 cells per 6 cm dish) with 1 to 5 μg RNA or DNA using DOTAP. S. pyogenes RNA and DNA fractions The transfection mixture was added to dishes containing 1.5 ml media without antibiotics. Quantitative RT-PCR (qRT-PCR) qRT-PCR was performed as described previously [21] using Quantitec Primer Assays (QIA- GEN) and the SYBR Green (Promega) detection on the Realplex Mastercycler (Eppendorf). For normalization, the mouse Hprt primers mHPRT-fwd 50-GGATTTGAATCACGTTTGTG TCAT-30, and mHPRT-rev 50-ACACCTGCTAATTTTACTGGCAA-30; and the human HPRT primers hHPRT- fwd 5- TGT GTG CTC AAG GGG GGC-3’ and hHPRT-rev 5’-CGT GGG GTC CTT TTC ACC-3’ were used. Cytokine measurement Cytokines in supernatants of stimulated cells were measured using DuoSET ELISA kits (R&D Systems) for mouse and human TNF, mouse IL-6 and human IL-8. Identification of predicted TLR13 orthologs Mouse TLR13 protein sequence was used for searching the OMA Browser (http://omabrowser. org) [23] which contains about 1,320 species. To account for species not present in the OMA Browser we additionally performed a standard NCBI Blast search (http://blast.ncbi.nlm.nih. gov) [24] against the non-redundant protein sequences [25]. The OMA Browse hits amended with NCBI Blast hits were further analyzed using FACT (Feature Architecture Comparison Tool; http://www.cibiv.at/FACT) [26] to identify those sequences which exhibit similar feature architecture as the query protein (mouse TLR13). If the architecture was similar, then the table entry was regarded as predicted ortholog. Thus, the predicted orthologs are similar in sequence and in their feature architecture and can be regarded to a high degree of confidence as true orthologs. Fluorescence microscopy Fluorescence microscopy Fluorescence microscopy was performed as described previously [21]. Transfection and stimulation of cells For one dish or well, 10 μl of bacterial extracts (containing 5 μg of nucleic acids), or 1 to 5 μg of purified RNA or DNA were used to form complexes with 30 μl DOTAP (Roche) as described previously [21]. The TLR3 ligand poly(I:C) (5 μg, Amersham), the TLR13 ligand (RNA oligori- bonucleotide SA19 with the sequence 5’-GGACGGAAAGACCCCGUGG-3’ [16], 1 μg or 5 μg) and the poor TLR13 ligand (RNA oligoribonucleotide mut-SA19 with the sequence 5’-GGAC GGGAAGACCCCGUGG-3’ [16], 1 μg) were transfected into cells using DOTAP. The TLR7 and TLR8 ligand R848 (Invivogen) was used at 5 μg/ml, LPS (from E.coli, Sigma) was used at 10 ng/ml and LTA (Invivogen) was used at 5 μg/ml. S. pyogenes rRNA activates mouse BMDMs and cDCs by triggering TLR13 S. pyogenes infection of BMDMs and cDCs induces a cytokine burst in a MyD88-dependent but TLR2-, TLR4- and TLR9-independent way [13,15]. We tested the role of other TLRs known to signal in MyD88-dependent manner. We included TLR3/TLR7/TLR9 (TLR379) tri- ple-deficient cells to account for potential redundancy of these three nucleic acids-sensing TLRs, Trif-deficient cells to directly assess the downstream signaling adaptor of TLR3, and TLR8-deficient cells. BMDMs and cDCs lacking TLR8 or Trif as well as cells TLR379 triple-de- ficient cells responded to S. pyogenes infection by producing TNF and IL-6 in comparable amounts to wild type (WT) control cells (Fig. 1A and C). Similarly, induction of IL-6 was not impaired in TLR379 triple-deficient BMDMs and cDCs (Fig. 1B and D). We have recently shown that S. pyogenes RNA induced IFN-β in cDCs, but not BMDMs [21]. S. pyogenes DNA was able to stimulate IFN-β in BMDMs but not cDCs [21]. Further, S. pyogenes RNA was shown to induce proinflammatory cytokines in cDCs [18]. To directly com- pare the stimulatory activity S. pyogenes nucleic acids in BMDMs and cDCs, we transfected S. pyogenes extracts treated with DNase I, RNase A, or proteinase K into cells using DOTAP which mediates delivery into endosomes but not cytosol [29]. With the exception of the RNase A-treated fraction, all were able to activate TNF production in both cell types (Fig. 1E and F) showing that S. pyogenes RNA but not DNA was functionally recognized. DNA purified from S. pyogenes did not stimulate TNF production in BMDMs and cDCs (Fig. 1G). Purified S. pyo- genes RNA elicited responses in TLR8-, Trif-, and TLR379-deficient BMDMs and cDCs but not in MyD88−/−cells (Fig. 1H and I). Thus, stimulation of mouse innate immune cells with S. pyogenes RNA recapitulated responses to infection of cells with live S. pyogenes. rRNA of several bacterial species was shown to stimulate TLR13 [16,17]. Total S. pyogenes RNA could stimulate TLR13 in cDCs [18]. To determine the stimulatory function of S. pyo- genes RNA in more detail, we separated total S. pyogenes RNA into mRNA, 16S and 23S rRNA, and into a fraction containing RNA smaller than 200 nucleotides including tRNA and 5S rRNA. Stimulation of BMDMs with rRNA resulted in TNF induction similar to stimulation with total RNA (Fig. 1J). In contrast, RNA <200 nucleotides was not stimulatory and the mRNA fraction exhibited a strongly decreased ability to induce TNF (Fig. 1J). Phylogenetic analysis The evolutionary relationships among TLR13 proteins in species and TLR repertoires in hu- mans and mice were constructed using alignment with mafft with the linsi option [27], and the tree was reconstructed with iqtree (-m TEST −b 100) [28]. 5 / 20 PLOS ONE \| DOI:10.1371/journal.pone.0119727 March 10, 2015 Innate Immunity to Streptococcus pyogenes Depends on TLR2 and TLR13 PLOS ONE \| DOI:10.1371/journal.pone.0119727 March 10, 2015 GenBank accession numbers Tnf: NM_013693, TNF: NM_000594, Il6: NM_031168, Il8: NM_000584, MyD88: NM_010851, Tlr2: NM_011905, Tlr3: NM_126166, TLR3: NM_003265, Tlr4: NM_021297, Tlr7: NM_133211, TLR7: NM_016562, Tlr8: NM_133212, TLR8: NM_138636, Tlr13: NM_205820, Trif: NM_174989, Unc93b1: NM_019449, Hprt: NM_013556, HPRT: NM_000194 GenBank accession numbers Tnf: NM_013693, TNF: NM_000594, Il6: NM_031168, Il8: NM_000584, MyD88: NM_010851, Tlr2: NM_011905, Tlr3: NM_126166, TLR3: NM_003265, Tlr4: NM_021297, Tlr7: NM_133211, TLR7: NM_016562, Tlr8: NM_133212, TLR8: NM_138636, Tlr13: NM_205820, Trif: NM_174989, Unc93b1: NM_019449, Hprt: NM_013556, HPRT: NM_000194 S. pyogenes rRNA activates mouse BMDMs and cDCs by triggering TLR13 Detailed analysis revealed that the mRNA fraction contained 0.3% rRNA. To evaluate whether the contaminat- ing rRNA in the mRNA fraction was responsible for the residual TNF-inducing activity we em- ployed a different RNA separation method which allowed isolation of pure mRNA but not other RNA fractions. The rRNA-free mRNA was no longer able to induce TNF (Fig. 1K). Thus, rRNA is the sole cytokine-inducing component of S. pyogenes RNA in both BMDMs and cDCs suggesting TLR13-mediated recognition, as reported for 23S rRNA from other bacterial species [16,17]. We confirmed the role of TLR13 by using Tlr13−/−BMDMs and cDCs: Tlr13−/− cells were not responding to S. pyogenes RNA (Fig. 1L-O). In conclusion, TLR13 is a non-redun- dant immune receptor for sensing S. pyogenes rRNA by both BMDMs and cDCs. 6 / 20 PLOS ONE \| DOI:10.1371/journal.pone.0119727 March 10, 2015 S. pyogenes infection triggers TLR2 and Unc93b1-dependent TLR13 responses TLR13 has been localized to endosomes suggesting that the recognition of S. pyogenes RNA oc- curs after phagocytosis [30]. In agreement, exposure of cells to S. pyogenes RNA in the absence of DOTAP did not result in TNF induction (Fig. 2A). The endosomal TLR sorting chaperon Unc93b1 is required for signaling through endosomal TLRs [19,20,31]. Unexpectedly, S. pyo- genes infection of Unc93b1-deficient BMDMs resulted in comparable TNF production as infec- tion of WT cells (Fig. 2B). These findings implied that during infection with live bacteria, the endosomal pathway recognizing S. pyogenes RNA was redundant with another mode of S. pyo- genes recognition. Deficiency in TLR2, which is involved in endocytosis-independent recogni- tion of cell wall components from Gram-positive bacteria, resulted in slightly reduced cytokine production early after infection but at later time points the responses of TLR2-deficient and WT BMDMs were comparable (Fig. 2B), in agreement with previous studies [13,15]. The con- tribution of TLR2-depedent pathway was more apparent in cDCs but it became less visible with increasing time of infection (Fig. 2C). Lack of TLR2 strongly impaired cytokine produc- tion by cDCs in the very early phase (i.e. 2.5 h post infection), whereas this phase appeared nor- mal in Unc93b1-deficient cells. Moreover, the TLR2-mediated TNF accumulation (i.e. TNF produced in Unc93b1-deficient cells) was increasing over time suggesting that cells were pro- ducing cytokines also at later time points, i.e. at the time when the Unc93b1 pathway was also becoming active. To test this, we stimulated solely the TLR2 pathway using LTA, and replaced the LTA-containing medium with medium without LTA 1 or 2 h after stimulation followed by additional incubation for 5 or 4 h, respectively. The supernatants of these cells contained comparable TNF amounts, but the TNF levels were lower than those produced by cells with permanent presence of LTA (i.e. cells without media change) (Fig. 2D). This experiment dem- onstrated that the cells were continuously producing TNF during the incubation period. The temporal profile of TNF production by S. pyogenes-infected cells (Fig. 2B and D) was consistent with a largely host cell surface-localized TLR2-dependent S. pyogenes recognition early in the infection and an endosome-dominated recognition in the later phase. The endosomal recogni- tion was triggered by S. pyogenes RNA, as revealed by the lack of responses in Unc93b1-defi- cient cells stimulated with RNA (Fig. 2E). To confirm that in the absence of phagocytosis the recognition of S. Innate Immunity to Streptococcus pyogenes Depends on TLR2 and TLR13 into BMDMs (E) or cDCs (F) using DOTAP. After stimulation for 6 h, supernatants were collected and TNF release was measured by ELISA. (G) RNA or DNA isolated from S. pyogenes was transfected into WT BMDMs or cDCs and after 6 h of stimulation supernatants were collected and TNF release was measured by ELISA. (H, I) BMDMs (H) and cDCs (I) from Tlr8−/−, Trif−/−, MyD88−/−and Tlr379 triple-deficient mice as well as control mice (WT) were transfected with S. pyogenes or mammalian RNA or left untreated. Supernatants were collected after 6 h and TNF release was measured by ELISA. (J) S. pyogenes total RNA as well as the mRNA (isolated with the MICROBExpress Bacterial mRNA Enrichment Kit, contains 0.3% rRNA), rRNA and nt>200 fractions were used for transfection of BMDMs. TNF release was determined in supernatants of cells after 6 h of stimulation using ELISA. (K) S. pyogenes total RNA as well as the mRNA (isolated with the Ribo-Zero rRNA Removal Kit) were transfected into BMDMs. TNF release was measured 6 h after stimulation using ELISA. (L— O) BMDMs (L, M) and cDCs (N, O) from Tlr13−/−mice as well as control mice (WT) were transfected with S. pyogenes or mammalian RNA or left untreated. Supernatants were collected after 6 h and TNF (L, N) or IL-6 (M, O) release was measured by ELISA. Error bars in all panels represent SDs (n3). doi:10.1371/journal.pone.0119727.g001 doi:10.1371/journal.pone.0119727.g001 Fig 1. S. pyogenes rRNA induces cytokine production in a TLR13-dependent way and independently of TRIF, TLR3, TLR7, Innate Immunity to Streptococcus pyogenes Dep Innate Immunity to Streptococcus pyogenes Depends on TLR2 and TLR13 Fig 1. S. pyogenes rRNA induces cytokine production in a TLR13-dependent way and independently of TRIF, TLR3, TLR7, TLR8 and TLR9. BMDMs (A, B) or cDCs (C, D) from Tlr8−/−, Trif−/−or Tlr379 triple-deficient mice as well as control mice (WT) were infected with S. pyogenes (MOI = 50) or left uninfected. At indicated time points, supernatants were collected and TNF (A, C) or IL-6 (B, D) release was measured by ELISA. (E, F) S. pyogenes cells were sonicated and the extracts were treated with either DNase I, RNase A, proteinase K, or left untreated (control extract). These extracts were delivered Fig 1. S. pyogenes rRNA induces cytokine production in a TLR13-dependent way and independently of TRIF, TLR3, TLR7, TLR8 and TLR9. BMDMs (A, B) or cDCs (C, D) from Tlr8−/−, Trif−/−or Tlr379 triple-deficient mice as well as control mice (WT) were infected with S. pyogenes (MOI = 50) or left uninfected. At indicated time points, supernatants were collected and TNF (A, C) or IL-6 (B, D) release was measured by ELISA. (E, F) S. pyogenes cells were sonicated and the extracts were treated with either DNase I, RNase A, proteinase K, or left untreated (control extract). These extracts were delivered PLOS ONE \| DOI:10.1371/journal.pone.0119727 March 10, 2015 NE \| DOI:10.1371/journal.pone.0119727 March 10, 2015 7 / 20 PLOS ONE \| DOI:10.1371/journal.pone.0119727 March 10, 2015 S. pyogenes infection triggers TLR2 and Unc93b1-dependent TLR13 responses pyogenes proceeds ex- clusively in a TLR2-dependent way, we infected WT and TLR2-deficient BMDMs in the pres- ence of the phagocytosis inhibitor cytochalasin D. The blockage of phagocytosis almost abolished responses of TLR2-deficient cells to S. pyogenes (Fig. 2F) and prevented S. pyogenes internalization (Fig. 2G). To further substantiate the essential role of TLR2 and the endosomal TLRs in sensing S. pyogenes we infected TLR2 and Unc93b1 double-deficient cells.Responses of these cells to S. pyogenes infection were lost (Fig. 2H-J). Since we showed that TLR13 was the only endosomal TLR capable of sensing S. pyogenes (Fig. 1), the results obtained using TLR2 and Unc93b1 double-deficient cells further strengthened the exclusive roles of TLR2 and 8 / 20 PLOS ONE \| DOI:10.1371/journal.pone.0119727 March 10, 2015 Innate Immunity to Streptococcus pyogenes Depends on TLR2 and TLR13 and after medium change cells were incubated without LTA for additional 5 or 4 h, respectively, As a control, BMDMs were treated with LTA for 6 h. TNF was determined in the supernatants by ELISA. (E) BMDMs from Unc93b1−/−mice as well as control mice (WT) were transfected with streptococcal- or mammalian RNA. Supernatants were collected after 6 h and TNF release was measured by ELISA. (F) BMDMs from Tlr2−/−as well as control mice (WT) were pre-treated for 30 min with cytochalasin D or left untreated prior infection with S. pyogenes (MOI = 50). At indicated time points, supernatants were collected and TNF release was measured by ELISA. (G) BMDMs were pre-treated with cytochalasin D (right panels) or left untreated (left panels) before infection with CFSE-labeled (green) S. pyogenes (MOI = 50). After 4 h cells were fixed and stained with anti S. pyogenes antibody (anti-S.p.) for extracellular bacteria (red). Immunofluorescence image depicts CFSE labeled bacteria (both extra- and intracellular) in green, extracellular antibody-stained bacteria in red, and a merge of the two channels in showing extracellular bacteria in yellow and intracellular bacteria in green. (H—J) BMDMs (H, I) and cDCs (J) from Unc93b1Tlr2 double-deficient mice as well as control mice (WT) were infected with S. pyogenes (MOI = 50) or left uninfected. At indicated time points, supernatants were collected and TNF (H, J) or IL-6 (I) release was measured by ELISA. LPS treatment (6 h) served as specificity control. (K—M) BMDMs (K, L) and cDCs (M) from Tlr13−/−as well as control mice (WT) were incubated for 30–45 min with anti-TLR2, control IgG or left untreated prior to infection with S. pyogenes (MOI = 100) or stimulation with LTA. Supernatants were collected 4 h after infection and TNF (K, M) or IL-6 (L) release was measured by ELISA. Error bars in all panels represent SDs (n3). and after medium change cells were incubated without LTA for additional 5 or 4 h, respectively, As a control, BMDMs were treated with LTA for 6 h. TNF was determined in the supernatants by ELISA. (E) BMDMs from Unc93b1−/−mice as well as control mice (WT) were transfected with streptococcal- or mammalian RNA. Supernatants were collected after 6 h and TNF release was measured by ELISA. (F) BMDMs from Tlr2−/−as well as control mice (WT) were pre-treated for 30 min with cytochalasin D or left untreated prior infection with S. pyogenes (MOI = 50). At indicated time points, supernatants were collected and TNF release was measured by ELISA. (G) BMDMs were pre-treated with cytochalasin D (right panels) or left untreated (left panels) before infection with CFSE-labeled (green) S. pyogenes (MOI = 50). After 4 h cells were fixed and stained with anti S. pyogenes antibody (anti-S.p.) for extracellular bacteria (red). Immunofluorescence image depicts CFSE labeled bacteria (both extra- and intracellular) in green, extracellular antibody-stained bacteria in red, and a merge of the two channels in showing extracellular bacteria in yellow and intracellular bacteria in green. (H—J) BMDMs (H, I) and cDCs (J) from Unc93b1Tlr2 double-deficient mice as well as control mice (WT) were infected with S. pyogenes (MOI = 50) or left uninfected. At indicated time points, supernatants were collected and TNF (H, J) or IL-6 (I) release was measured by ELISA. LPS treatment (6 h) served as specificity control. (K—M) BMDMs (K, L) and cDCs (M) from Tlr13−/−as well as control mice (WT) were incubated for 30–45 min with anti-TLR2, control IgG or left untreated prior to infection with S. pyogenes (MOI = 100) or stimulation with LTA. Supernatants were collected 4 h after infection and TNF (K, M) or IL-6 (L) release was measured by ELISA. Error bars in all panels represent SDs (n3). doi:10.1371/journal.pone.0119727.g002 TLR13 in responses to S. pyogenes. Finally, antibody-mediated blocking of TLR2 in TLR13-de- ficient cells resulted in strong reduction of cytokine production 4 h after infection, at the time of predominantly TLR2-mediated cell surface-localized S. pyogenes recognition (Fig. 2K-M). Together, these results demonstrate that TLR2 and TLR13 pathways play a leading role in rec- ognition of live S. pyogenes by murine cells. These two pathways are largely redundant and only the inactivation of both of them abolishes sensing of S. pyogenes infection. Innate Immunity to Streptococcus pyogenes Depends on TLR2 and TLR13 Fig 2. S. pyogenes is recognized by a combination of Tlr2- and Tlr13-mediated sensing. (A) S. pyogenes RNA was either or transfected into BMDMs using DOTAP. Supernatants were collected after 6 h and TNF release was measured by ELISA. (B, from Tlr2−/−and Unc93b1−/−mice as well as control mice (WT) were infected with S. pyogenes (MOI = 50) or left uninfected, o control. At indicated time points, supernatants were collected and TNF release was measured by ELISA. (D) BMDMs were stim Innate Immunity to Streptococcus pyogenes Fig 2. S. pyogenes is recognized by a combination of Tlr2- and Tlr13-mediated sensing. (A) S. pyogenes RNA was either added directly to the BMDMs or transfected into BMDMs using DOTAP. Supernatants were collected after 6 h and TNF release was measured by ELISA. (B, C) BMDMs (B) or cDCs (C) from Tlr2−/−and Unc93b1−/−mice as well as control mice (WT) were infected with S. pyogenes (MOI = 50) or left uninfected, or treated with LTA or LPS as a control. At indicated time points, supernatants were collected and TNF release was measured by ELISA. (D) BMDMs were stimulated for 1 or 2 h with LTA, Fig 2. S. pyogenes is recognized by a combination of Tlr2- and Tlr13-mediated sensing. (A) S. pyogenes RNA was either added directly to the BMDMs or transfected into BMDMs using DOTAP. Supernatants were collected after 6 h and TNF release was measured by ELISA. (B, C) BMDMs (B) or cDCs (C) from Tlr2−/−and Unc93b1−/−mice as well as control mice (WT) were infected with S. pyogenes (MOI = 50) or left uninfected, or treated with LTA or LPS as a control. At indicated time points, supernatants were collected and TNF release was measured by ELISA. (D) BMDMs were stimulated for 1 or 2 h with LTA, PLOS ONE \| DOI:10.1371/journal.pone.0119727 March 10, 2015 9 / 20 NE \| DOI:10.1371/journal.pone.0119727 March 10, 2015 Human macrophages sense S. pyogenes through TLR2 but they also employ TLR2-independent pathways that differ from TLR13-mediated recognition Human macrophages sense S. pyogenes through TLR2 but they also employ TLR2-independent pathways that differ from TLR13-mediated recognition S. pyogenes is not considered to be pathogenic in mice outside the laboratory [32,33]. Our data demonstrate that mice launch protective immune responses by recognizing S. pyogenes RNA by TLR13. TLR13 occurs in mice but not humans raising the question whether S. pyogenes RNA is recognized in human innate immune cells by a different TLR. To address S. pyogenes RNA recognition by human cells, we employed HEK293 cells stably expressing human TLR3, TLR7 or TLR8, the only known RNA-recognizing TLRs in humans [34]. S. pyogenes RNA and the reported TLR13 ligand oligoribonucleotide SA19 [16,17] were not stimulatory, regardless of the TLR tested, as assessed by IL-8 protein and mRNA expression (Fig. 4). The TLR3 ligand polyinosinic:polycytidylic acid (poly(I:C)), as well as the TLR7 and TLR8 agonist R848, elicited responses in HEK293 cells expressing the cognate TLR, and as ex- pected, TNF stimulated HEK293 cells independently of TLRs (Fig. 4). In conclusion, these re- sults show that none of the RNA-recognizing human TLRs is capable of substituting for the missing TLR13. To explore the recognition of live S. pyogenes by human innate immune cells, we employed the human monocytic cell line THP-1. THP-1 cells respond only poorly to TLR ligands, unless differentiated into more mature macrophage-like cells [35]. THP-1 cells differentiated into macrophages using phorbol-12-myristate-13-acetate (PMA) exhibited increased responsive- ness to LPS, lipoteichoic acid (LTA) and R848, as compared to undifferentiated THP-1 cells (Fig. 5A-D). Importantly, TNF and IL-8 measurements revealed that PMA-differentiated THP-1 cells were responding neither to the canonical TLR13 ligand oligoribonucleotide SA19 [16,17] nor to S. pyogenes RNA (Fig. 5A-D and 6A). To test whether expression of murine TLR13 renders THP-1 cells responsive to bacterial RNA we generated THP-1 cells stably ex- pressing TLR13. Stimulation of these cells with oligoribonucleotide SA19 or S. pyogenes RNA resulted in a strong TNF induction whereas stimulation with a poor TLR13 ligand (mutated oligoribonucleotide SA19, mut-SA19 [16,17]) caused a low TNF induction. These data demon- strate that human THP-1 macrophages were not able to sense bacterial RNA corroborating the observed inability of HEK293 cells expressing human TLR3, TLR7 or TLR8, to respond to bac- terial RNA. However, exogenous TLR13 allows THP-1 cells to sense bacterial RNA in a similar way as the endogenous TLR13 in mouse cells. Unc93b1 and TLR2 are required for defense against S. pyogenes in mice Considering the highly redundant functions of the TLR2- and Unc93b1-mediated sensing pathways in vitro (Fig. 2B-D), we asked whether these pathways were redundant during infec- tion of mice. TLR2- or Unc93b1-deficient mice were subcutaneously inoculated with the S. pyogenes strain ISS-3348, an M1 serotype strain that is virulent in mice [21]. We observed an increased susceptibility of both TLR2- and Unc93b1-deficient animals to infection compared to WT controls (Fig. 3A-B). Thus, both the TLR2 and Unc93b1 pathways play essential and non-redundant roles in mouse defense against S. pyogenes. Consistent with the inability of S. pyogenes DNA to elicit TNF production in vitro (Fig. 1G), mice lacking the DNA receptor TLR9 were not significantly more susceptible to S. pyogenes infection than control mice (Fig. 3C). The requirement for both TLR2 and Unc93b1 pathways in mouse defense against S. pyogenes implied that a strong activation of innate responses is favorable for the host. In con- clusion, the findings demonstrate that a complete dual TLR activation of innate immune cells is of critical importance for defense against S. pyogenes in mice. The data also provide the Fig 3. Entire activation of the immune responses through both Tlr2- and Unc93b1-dependent pathways is required for protective defense against S. pyogenes in mice. (A—C) Kaplan-Meier survival curves of C57BL/6 and Tlr2−/−, Unc93b1−/−(8 mice per genotype) or Tlr9−/−mice (14 mice per genotype) after subcutaneous infection with 1×108 CFU of S. pyogenes ISS3348. Survival was monitored for 6 days. Significance: = p<0.05; ** = p<0.01. doi:10.1371/journal.pone.0119727.g003 Fig 3. Entire activation of the immune responses through both Tlr2- and Unc93b1-dependent pathways is required for protective defense against S. pyogenes in mice. (A—C) Kaplan-Meier survival curves of C57BL/6 and Tlr2−/−, Unc93b1−/−(8 mice per genotype) or Tlr9−/−mice (14 mice per genotype) after subcutaneous infection with 1×108 CFU of S. pyogenes ISS3348. Survival was monitored for 6 days. Significance: * = p<0.05; ** = p<0.01. doi:10.1371/journal.pone.0119727.g003 doi:10.1371/journal.pone.0119727.g003 PLOS ONE \| DOI:10.1371/journal.pone.0119727 March 10, 2015 10 / 20 Innate Immunity to Streptococcus pyogenes Depends on TLR2 and TLR13 strongest evidence yet for a crucial contribution of pathogen-derived RNA recognition to the initiation of protective host response. PLOS ONE \| DOI:10.1371/journal.pone.0119727 March 10, 2015 Human macrophages sense S. pyogenes through TLR2 but they also employ TLR2-independent pathways that differ from TLR13-mediated recognition LTA treatment of THP-1 cells resulted in TNF induction similar to infection with S. pyo- genes (Fig. 6A) suggesting that S. pyogenes was recognized through TLR2. After blocking TLR2, THP-1 cells were no longer responding to infection with S. pyogenes (Fig. 6B-C) indicating that TLR2 was the key receptor in these cells. We then examined TNF and IL-8 production by pri- mary human macrophages treated with S. pyogenes RNA or live bacteria. Similar to THP-1 cells, primary human macrophages were barely responding to S. pyogenes RNA but reacted well to infection (Fig. 6D-E). Interestingly, DOTAP-mediated stimulation of primary macro- phages with bacterial extracts revealed that these cells were activated by bacterial components delivered into the cells (Fig. 6F-G). Extracts treated with RNase were also capable of inducing TNF and IL-8 although the activity was lower than that of untreated extract (Fig. 6F-G). Thus, experiments using isolated S. pyogenes RNA (Fig. 6D-E) and bacterial extracts (Fig. 6F-G) indi- cated that primary human macrophages were stimulated by bacterial products delivered into the cells, with bacterial RNA contributing to the stimulatory activity. However, the delivery of 11 / 20 purified S. pyogenes RNA was not capable of activating primary human macrophages (Fig. 6D- Fig 4. Human RNA-recognizing TLRs are not capable of sensing S. pyogenes RNA. (A, D) HEK293 stably expressing human TLR3 (HEK293-hTLR3) or control HEK293 cells (HEK ctl) were transfected with the TLR3 ligand poly(I:C) (5 μg), the Tlr13 ligand oligoribonucleotide SA19 (5 μg), S. pyogenes RNA (5 μg) using DOTAP or stimulated with human TNF (10 ng/ml). IL-8 release was measured in supernatants 24 h post stimulation by ELISA (A) or the levels of IL8 mRNA were determined by qRT-PCR (normalized to HPRT) (D). Mean values ± SD are shown (n 3). (B, C, E, F) HEK293XL cells stably expressing human TLR7 (B, E) or TLR8 (C, F) or control HEK293XL cells were transfected with SA19 (5 μg), S. pyogenes RNA (5 μg) or stimulated with the TLR7 and TLR8 ligand R848 (5 μg/ml) or human TNF (10 ng/ml). IL-8 release was measured in supernatants 24 h post stimulation by ELISA (B, C) or the levels of IL8 mRNA were determined by qRT-PCR (normalized to HPRT) (E, F). Mean values ± SD are shown (n 3). doi:10.1371/journal.pone.0119727.g004 Innate Immunity to Streptococcus pyogenes Depends on TLR2 and TLR13 Innate Immunity to Streptococcus pyogenes Depends on TLR2 and TLR13 A-recognizing TLRs are not capable of sensing S. pyogenes RNA. (A, D) HEK293 stably expressing human TLR Fig 4. Human RNA-recognizing TLRs are not capable of sensing S. pyogenes RNA. (A, D) HEK293 stably exp Fig 4. Human RNA-recognizing TLRs are not capable of sensing S. pyogenes RNA. (A, D) HEK293 stably expressing human TLR3 (HEK293-hTLR3) or control HEK293 cells (HEK ctl) were transfected with the TLR3 ligand poly(I:C) (5 μg), the Tlr13 ligand oligoribonucleotide SA19 (5 μg), S. pyogenes RNA (5 μg) using DOTAP or stimulated with human TNF (10 ng/ml). IL-8 release was measured in supernatants 24 h post stimulation by ELISA (A) or the levels of IL8 mRNA were determined by qRT-PCR (normalized to HPRT) (D). Mean values ± SD are shown (n 3). (B, C, E, F) HEK293XL cells stably expressing human TLR7 (B, E) or TLR8 (C, F) or control HEK293XL cells were transfected with SA19 (5 μg), S. pyogenes RNA (5 μg) or stimulated with the TLR7 and TLR8 ligand R848 (5 μg/ml) or human TNF (10 ng/ml). IL-8 release was measured in supernatants 24 h post stimulation by ELISA (B, C) or the levels of IL8 mRNA were determined by qRT-PCR (normalized to HPRT) (E, F). Mean values ± SD are shown (n 3). purified S. pyogenes RNA was not capable of activating primary human macrophages (Fig. 6D- E), which is in marked contrast to mouse cells (Fig. 1H-I). Finally, infection of primary human purified S. pyogenes RNA was not capable of activating primary human macrophages (Fig. 6D- E), which is in marked contrast to mouse cells (Fig. 1H-I). Finally, infection of primary human PLOS ONE \| DOI:10.1371/journal.pone.0119727 March 10, 2015 12 / 20 Fig 5. Macrophage-like differentiated THP-1 cells respond to endosomal TLR ligands but not to the canonical TLR13 ligand. (A, B) THP-1 cells were either treated with 10 nM PMA or left untreated (as described in Material and Methods) before stimulation with R848 (5 μg/ml), LTA (5 μg/ml), LPS (10 ng/ml) or DOTAP-mediated delivery of poly(I:C) (5 μg). Supernatants were collected after 8 h and IL-8 (A) or TNF (B) release was determined by ELISA. Error bars represent SDs (n3). (C, D) RNA was isolated from differentiated or undifferentiated THP-1 cells treated as described in (A) and (B) for 4 or 8 h. IL8 (C) and TNF (D) mRNA levels were determined by qRT-PCR (normalized to HPRT). Mean values ± SD are shown (n 3). (E) THP-1 cells stably expressing TLR13 (TLR13 THP-1) were stimulated with TLR13 ligand SA19, poor TLR13 ligand (mut-SA19), S. pyogenes RNA or LTA for 4 h. TNF was determined in supernatants by ELISA. Error bars in represent SDs (n3). doi:10.1371/journal.pone.0119727.g005 Innate Immunity to Streptococcus pyogenes Depends on TLR2 and TLR13 Innate Immunity to Streptococcus pyogenes Depends on TLR2 and TLR13 Fig 6. Human macrophage-like cells respond to S. pyogenes infection by TLR2-dependent signaling but fail to recognize S. p PMA-differentiated THP-1 macrophages were stimulated with S. pyogenes RNA (5 μg, using DOTAP), LTA (5 μg/ml), infected with S. p or left untreated. Supernatants were collected 6 h after treatment and TNF release was measured by ELSA. (B, C) PMA-differentiated with an anti-TLR2 (αTLR2) or control IgG antibody for 30 min or left untreated prior to infection with S. pyogenes (MOI = 100), supernat post infection and TNF (B) or IL-8 (C) release was measured by ELISA. (D, E) Primary human macrophages were either transfected w mammalian RNA using DOTAP, infected with S. pyogenes (MOI = 100), stimulated with LTA (5 μg/ml) or LPS (10 ng/ml) or left untreat collected after 24 h and TNF (D) or IL-8 (E) release was measured by using ELSA. (F, G) Primary human macrophages were stimulate extracts, S. pyogenes extracts treated with RNase (+RNase), or left unstimulated. Supernatants were collected after 6 h and TNF (F) o measured by ELSA. (H, I) Primary human macrophages were incubated with an anti-TLR2 (αTLR2) or control IgG antibody for 30 min infection with S. pyogenes (MOI = 100 and MOI = 5) or stimulation with LTS. Supernatants were collected 6 h post infection and TNF (H was measured by ELISA. Error bars in all panels represent SDs (n3). Innate Immunity to Streptococcus pyogenes Depen Fig 6. Human macrophage-like cells respond to S. pyogenes infection by TLR2-dependent signaling but fail to recognize S. pyogenes RNA. (A) PMA-differentiated THP-1 macrophages were stimulated with S. pyogenes RNA (5 μg, using DOTAP), LTA (5 μg/ml), infected with S. pyogenes (MOI = 100) or left untreated. Supernatants were collected 6 h after treatment and TNF release was measured by ELSA. (B, C) PMA-differentiated THP-1 were incubated with an anti-TLR2 (αTLR2) or control IgG antibody for 30 min or left untreated prior to infection with S. pyogenes (MOI = 100), supernatants were collected 6 h post infection and TNF (B) or IL-8 (C) release was measured by ELISA. (D, E) Primary human macrophages were either transfected with 5 μg S. pyogenes or mammalian RNA using DOTAP, infected with S. pyogenes (MOI = 100), stimulated with LTA (5 μg/ml) or LPS (10 ng/ml) or left untreated. Innate Immunity to Streptococcus pyogenes Depends on TLR2 and TLR13 Fig 5. Macrophage-like differentiated THP-1 cells respond to endosomal TLR ligands but not to the canonical TLR13 ligand. (A, B) THP-1 cells were either treated with 10 nM PMA or left untreated (as described in Material and Methods) before stimulation with R848 (5 μg/ml), LTA (5 μg/ml), LPS (10 ng/ml) or DOTAP-mediated delivery of poly(I:C) (5 μg). Supernatants were collected after 8 h and IL-8 (A) or TNF (B) release was determined by ELISA. Error bars represent SDs (n3). (C, D) RNA was isolated from differentiated or undifferentiated THP-1 cells treated as described in (A) and (B) for 4 or 8 h. IL8 (C) and TNF (D) mRNA levels were determined by qRT-PCR (normalized to HPRT). Mean values ± SD are shown (n 3). (E) THP-1 cells stably expressing TLR13 (TLR13 THP-1) were stimulated with TLR13 ligand SA19, poor TLR13 ligand (mut-SA19), S. pyogenes RNA or LTA for 4 h. TNF was determined in supernatants by ELISA. Error bars in represent SDs (n3). doi:10.1371/journal.pone.0119727.g005 macrophages with S. pyogenes in the presence of a TLR2-blocking antibody revealed that the cells were responding under conditions of TLR2 inhibition (Fig. 6H-I). Reduction of MOI from 100 to 5 did not change the inability of the TLR2-blocking antibody to prevent TNF and IL-8 production although the antibody efficiently inhibited responses to LTA (Fig. 6H-I). PLOS ONE \| DOI:10.1371/journal.pone.0119727 March 10, 2015 13 / 20 Supernatants were collected after 24 h and TNF (D) or IL-8 (E) release was measured by using ELSA. (F, G) Primary human macrophages were stimulated with S. pyogenes extracts, S. pyogenes extracts treated with RNase (+RNase), or left unstimulated. Supernatants were collected after 6 h and TNF (F) or IL-8 (G) release was measured by ELSA. (H, I) Primary human macrophages were incubated with an anti-TLR2 (αTLR2) or control IgG antibody for 30 min or left untreated prior to infection with S. pyogenes (MOI = 100 and MOI = 5) or stimulation with LTS. Supernatants were collected 6 h post infection and TNF (H) or IL-8 (I) release was measured by ELISA. Error bars in all panels represent SDs (n3). doi:10.1371/journal.pone.0119727.g006 Together, these data demonstrate that primary human macrophages differ from THP-1 cells in that they are able to sense S. pyogenes independently of TLR2. Neither THP-1 nor PLOS ONE \| DOI:10.1371/journal.pone.0119727 March 10, 2015 14 / 20 Innate Immunity to Streptococcus pyogenes Depends on TLR2 and TLR13 primary human macrophages are able to respond to bacterial RNA although bacterial RNA might contribute to responses of primary human macrophages if delivered together with bacterial extracts. TLR13 is absent from most mammals but occurs in other vertebrates, insects, Annelida and plants To obtain a more comprehensive picture of the presence of TLR13 and the bacterial rRNA rec- ognition pathway various organisms we performed a phylogenetic analysis of the predicted TLR13 protein sequences in all kingdoms (Fig. 7A and S1 Table). TLR13 is absent in primates and most other mammals. TLR13 occurs in mice and rats, but is missing in other rodents in- cluding guinea pig and squirrel. However, TLR13 is found in non-mammalian vertebrates as well as in other kingdoms including insects, Annelids and plants. The mouse and rat TLR13 proteins are highly conserved but distant from TLR13 found in other non-mammalian species (Fig. 7A). Thus, TLR13 appears to have been lost in primates and most other mammals, except for e.g. mice and rats. We directly compared the entire TLR repertoires of mice and humans by generating a TLR tree for these two species (Fig. 7B). The tree revealed that TLR11, TLR12 and TLR13, which occur in mice but not humans, are located in one branch. Interestingly, these three endosomal TLRs do not group together with other known endosomal TLRs, most notably the nucleic acid-sensing TLR7, TLR8 and TLR9, which cluster in a separate branch. The re- maining nucleic acid-sensing TLR3 groups with the flagellin-recognizing TLR5 in yet another branch, however this branch is only weakly supported (bootstrap value 60%). The distribution of nucleic sensing TLRs in three different branches indicates that the ability of sensing nucleic acids has been acquired before the final diversification of TLRs. PLOS ONE \| DOI:10.1371/journal.pone.0119727 March 10, 2015 Discussion Microbes colonizing surface tissues of complex multicellular organisms consist of harmless species as well as of species capable of converting into invading pathogens, when the surface barrier function is disabled. In this study we establish that the human pathogen S. pyogenes, which asymptomatically colonizes one-third of the human population and as such regarded as commensal microbe [36,37], elicits innate immune responses by two recognition modes in mice: 1) Host cell surface-localized sensing of S. pyogenes through TLR2; 2) Endosome-local- ized sensing of S. pyogenes rRNA through TLR13 upon phagocytosis of the pathogen. Activa- tion of both pathways is required for successful defense in mice showing that only a dual TLR stimulation launches an adequate immune response. Our results further reveal that the recog- nition of S. pyogenes RNA by the TLR system is missing in human cells. We find that TLR13 occurs in mammals only in exceptional cases, with mice and rats being prominent examples. Notably, S. pyogenes is not a natural pathogen in these two mammalian species [32,33]. Previous studies revealed a critical role of TLR-mediated innate immune response in pro- tecting mice against S. pyogenes: mice deficient in MyD88 exhibited decreased survival in a sub- cutaneous infection model [14], the model we employed in this study. IL-1β, which also signals via MyD88, has been proposed to be dispensable for host defense as mice deficient in the Nlrp3 inflammasome did not exhibit increased susceptibility to S. pyogenes in an intraperitoneal in- fection model [38]. Despite the well-defined role of MyD88 in host protection, in vitro infec- tion experiments have so far failed to identify an essential role for any TLR tested [13,15]. In this study, we observe that only the absence of both TLR2 and Unc93b1 abolishes cytokine pro- duction. These TLR pathways are largely redundant in vitro explaining the lack of measurable effects of TLR2 deficiency described in previous reports [13,15]. The less redundant response in cDCs compared to BMDMs may result from differences in signal processing modules in PLOS ONE \| DOI:10.1371/journal.pone.0119727 March 10, 2015 15 / 20 Innate Immunity to Streptococcus pyogenes Depends on TLR2 and TLR13 Fig 7. Phylogenetic analysis of TLR13 in species and of the TLR repertoires in humans and mice. (A) The phylogenetic tree displa species where we identified an ortholog to TLR13 using the OMA browser and NCBI Blast search. Innate Immunity to Streptococcus pyogenes Depends on TLR2 and TLR13 branch than TLR3, and the mouse-specific TLR13 clusters yet in another branch together non-nucleic acid sensors TLR11 and TLR12. Numbers at the branch points indicate bootstrap values (cut-off > 50). branch than TLR3, and the mouse-specific TLR13 clusters yet in another branch together non-nucleic acid sensors TLR11 and TLR12. Numbers at the branch points indicate bootstrap values (cut-off > 50). branch than TLR3, and the mouse-specific TLR13 clusters yet in another branch together non-nucleic acid sensors branch points indicate bootstrap values (cut-off > 50). doi:10.1371/journal.pone.0119727.g007 doi:10.1371/journal.pone.0119727.g007 these two cell types. Indeed, S. pyogenes RNA induces IFN-β production in cDCs but not in BMDMs [21]. Similar to TLR2/Unc93b1 double-deficient cells, S. pyogenes-induced cytokine production is abolished also in TLR13-deficient cells under conditions of blocked TLR2 signal- ing revealing the key functions of TLR2 and TLR13 in S. pyogenes sensing. TLR9 has been re- ported to enhance survival of mice in an intraperitoneal model of S. pyogenes infection [39]. Thus, in that infection model TLR9 may contribute to mouse protection by sensing S. pyogenes DNA by cells other than BMDMs and cDCs, or indirectly, by its known ability to sense self- DNA liberated from dead cells [40]. TLR13 recognizes with a remarkable specificity a highly conserved bacterial rRNA sequence [16,17]. Given the ubiquitous presence of the TLR13 ligand it is surprising that the contribu- tion of this receptor to immune responses against bacteria has remained unknown until very recently. The major reason for the long unrecognized function of TLR13 is the dominant role of other TLRs, most notably TLR2 and TLR4, in sensing of many bacteria. In contrast to this, sensing of S. pyogenes occurs through a partially redundant engagement of TLR2 and TLR13 in mouse cells. It is at present unclear why stimulation of TLR2 by S. pyogenes peptidoglycan and LTA, which in purified form are capable of triggering TLR2 [41], does not play an equally dom- inant role in recognition as reported for other Gram-positive pathogens including Staphylococ- cus aureus or Listeria monocytogenes [42,43]. S. pyogenes has no robust life cycle once internalized by host cells which employ both phagolysosomal killing and autophagy to eradi- cate this pathogen [44]. Thus, it is conceivable that the efficient multilayered processing of S. Discussion The hits were confirmed with FACT to predicted orthologs have similar feature architecture as the query protein. Thus, the orthologs are firstly similar in sequence and second feature architecture. Numbers at the branch points indicate bootstrap values. (B) The phylogenetic tree displays evolutionary relationsh mouse TLR proteins. The tree was constructed as described in (A). Note that if a particular TLR is found in both species then both orthol evolutionary relationship (bootstrap value 100). The nucleic acid-sensing TLRs do not cluster in one branch: TLR7, TLR8 and TLR9 are PLOS ONE \| DOI:10.1371/journal.pone.0119727 March 10, 2015 Fig 7. Phylogenetic analysis of TLR13 in species and of the TLR repertoires in humans and mice. (A) The phylogenetic tree displays a choice of species where we identified an ortholog to TLR13 using the OMA browser and NCBI Blast search. The hits were confirmed with FACT to test whether the predicted orthologs have similar feature architecture as the query protein. Thus, the orthologs are firstly similar in sequence and secondly similar in their feature architecture. Numbers at the branch points indicate bootstrap values. (B) The phylogenetic tree displays evolutionary relationship of human and mouse TLR proteins. The tree was constructed as described in (A). Note that if a particular TLR is found in both species then both orthologs exhibit very close evolutionary relationship (bootstrap value 100). The nucleic acid-sensing TLRs do not cluster in one branch: TLR7, TLR8 and TLR9 are found in a different PLOS ONE \| DOI:10.1371/journal.pone.0119727 March 10, 2015 16 / 20 PLOS ONE \| DOI:10.1371/journal.pone.0119727 March 10, 2015 pyogenes within host cells allows a better exposure of TLR13 to bacterial rRNA when compared to other Gram-positive bacteria, particularly those capable of intracellular growth. A similarly important role of TLR13 is likely to be common in the recognition of other streptococci as shown e.g. by TLR13-dependent recognition of S. pneumoniae RNA and by Unc93b1-depen- dent recognition of Group B Streptococcus (GBS) RNA [16,45]. Notably, a recent study re- vealed that GBS rRNA but not rRNA devoid of the TLR13 recognition sequence was able to stimulate mouse macrophages implying that this pathogen is sensed through TLR13 [46]. The lack of TLR13 in humans is intriguing given the absolute level of conservation of the TLR13 ligand in both Gram-positive and -negative bacteria [16,17]. Our findings establish that human RNA-recognizing TLRs cannot compensate for the lack of TLR13. Consistently, human innate immune cells do not respond to S. pyogenes RNA by production of TNF and IL- 8. Although human THP-1 macrophages employ solely TLR2 for recognition of S. pyogenes, this simple mode of sensing is not operational in primary human macrophages: Primary cells recognize products present in bacterial extracts delivered into the cells. Bacterial RNA, if deliv- ered in combination with other components of the bacterial extract, might contribute to this way of host cell stimulation. Future studies should elucidate the nature of intracellular recogni- tion receptors involved in recognition of S. pyogenes in primary human cells. Such receptors may be different from TLRs since a recent study revealed that the human but not the mouse NLRP3 inflammasome is activated by all three bacterial RNA species (mRNA, rRNA and tRNA) [47]. The lack of TLR13 is fully penetrant in primates and widespread among mammals. The presence of predicted TLR13 in non-mammalian vertebrates, insects and plants indicates that primates have lost this TLR early in their evolution, whereas mice and rats preserved the gene. The selective maintenance of TLR13 in a very limited number of mammalian species poses the elementary question of the evolutionary pressure imposed on most mammals to lose TLR13, 17 / 20 PLOS ONE \| DOI:10.1371/journal.pone.0119727 March 10, 2015 Innate Immunity to Streptococcus pyogenes Depends on TLR2 and TLR13 and of the evolutionary advantage gained in mice and rats by keeping this gene. Primates might have eliminated TLR13 in order to limit the lifelong burden of continuous sensing of rRNA de- rived from e.g. Supporting Information S1 Table. Tlr13 is missing in primates but occurs in mice and rarely in other mammals while it can be found in non-mammalian vertebrates, insects, Annelida and plants. Tlr13 S1 Table. Tlr13 is missing in primates but occurs in mice and rarely in other mammals while it can be found in non-mammalian vertebrates, insects, Annelida and plants. Tlr13 orthologs were searched for and identified using the OMA browser (light blue) or a standard NCBI Blast search against non-redundant protein sequences (dark blue). Putative hits were tested for occurrence of similar feature architecture as in the query protein, i.e. mouse Tlr13, using FACT (Feature Architecture Comparison Tool; http://www.cibiv.at/FACT). Hits exhibiting similar pro- tein architecture as mouse Tlr13 were defined as predicted orthologs and are depicted in light blue (hits obtained using the OMA browser) or dark blue (hits obtained using NCBI Blast). Shown is a choice of mammalian species as well as Tlr13-containing representatives from other kingdomes. (PDF) Acknowledgments We thank Shizuo Akira for the permission to use MyD88−/−, Tlr2−/−, Tlr3−/−, Tlr7−/−and Trif−/− mice. We are grateful to Giuseppe Teti for his advice on infection experiments. environmental microbiota or to increase tolerance to bacterial infections [48,49]. Alternatively, the existence of pathways not present in mice, for example a more promiscuous human NLRP3 inflammasome [47], might provide human cells with immune mechanisms, which can compensate for the evolutionary loss of TLR13. The resistance of mice and the suscep- tibility of humans to S. pyogenes infection involves specific differences in host-pathogen interac- tions including components of the complement system, antimicrobial peptides and activation of T cells by streptococcal superantigens, but the full complexity of the divergences is not well un- derstood [8,50,51]. Our study suggests that the differences in recognition of S. pyogenes by mouse compared to human cells might play an important role in susceptibility to infection. The data presented here establish S. pyogenes RNA as critical player in successful immune defense of mice and uncover a previously unrecognized role of TLR2 in mouse and man in rec- ognition of this pathogen. Author Contributions Conceived and designed the experiments: CF PK. Performed the experiments: CF NG TK VC MJ MA MS. Analyzed the data: CF NG TK VC MJ AvH PK. Contributed reagents/materials/ analysis tools: CK MW LA XL ZJC. Wrote the paper: CF AvH PK. 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https://openalex.org/W4249347072	https://mr.copernicus.org/articles/2/629/2021/mr-2-629-2021.pdf	English	null	Reply on RC2	null	2,021	cc-by	14,191	n. Reson., 2, 629–642, 2021 s://doi.org/10.5194/mr-2-629-2021 uthor(s) 2021. This work is distributed under Creative Commons Attribution 4.0 License. Open Access High-afﬁnity tamoxifen analogues retain extensive positional disorder when bound to calmodulin Lilia Milanesi1,2, Clare R. Trevitt1, Brian Whitehead1, Andrea M. Hounslow1, Salvador Tomas2,6, Laszlo L. P. Hosszu1,3, Christopher A. Hunter4, and Jonathan P. Waltho1,5 1Department of Molecular Biology and Biotechnology, University of Shefﬁeld, Shefﬁeld S10 2TN, UK 2Department of Biological Sciences, School of Science, Birkbeck University of London, London WC1E 7HX, UK 3Medical Research Council Prion Unit, University College of London Institute of Neurology, Queen Square, London WCN1 3BG, UK 4Department of Chemistry, University of Cambridge, Lensﬁeld Road, Cambridge CB2 1EW, UK 5Manchester Institute of Biotechnology, University of Manchester, 131 Princess Street, Manchester M1 7DN, UK 6Departament de Química, Universitat de les Illes Balears, Cra. de Valldemossa, km 7.5. 07122 Palma de Mallorca, Spain Correspondence: Jonathan P. Waltho (j.waltho@shefﬁeld.ac.uk) Received: 14 January 2021 – Discussion started: 5 February 2021 Revised: 29 March 2021 – Accepted: 3 April 2021 – Published: 13 August 2021 Abstract. Using a combination of NMR and ﬂuorescence measurements, we have investigated the structure and dynamics of the complexes formed between calcium-loaded calmodulin (CaM) and the potent breast can- Magn. Reson., 2, 629–642, 2021 https://doi.org/10.5194/mr-2-629-2021 © Author(s) 2021. This work is distributed under the Creative Commons Attribution 4.0 License. High-afﬁnity tamoxifen analogues retain extensive positional disorder when bound to calmodulin Lilia Milanesi1,2, Clare R. Trevitt1, Brian Whitehead1, Andrea M. Hounslow1, Salvador Tomas2,6, Laszlo L. P. Hosszu1,3, Christopher A. Hunter4, and Jonathan P. Waltho1,5 1Department of Molecular Biology and Biotechnology, University of Shefﬁeld, Shefﬁeld S10 2TN, UK 2Department of Biological Sciences, School of Science, Birkbeck University of London, London WC1E 7HX, UK 3Medical Research Council Prion Unit, University College of London Institute of Neurology, Queen Square, London WCN1 3BG, UK 4Department of Chemistry, University of Cambridge, Lensﬁeld Road, Cambridge CB2 1EW, UK 5Manchester Institute of Biotechnology, University of Manchester, 131 Princess Street, Manchester M1 7DN, UK 6Departament de Química, Universitat de les Illes Balears, Cra. de Valldemossa, km 7.5. 07122 Palma de Mallorca, Spain Correspondence: Jonathan P. Waltho (j.waltho@shefﬁeld.ac.uk) Received: 14 January 2021 – Discussion started: 5 February 2021 Revised: 29 March 2021 – Accepted: 3 April 2021 – Published: 13 August 2021 High-afﬁnity tamoxifen analogues retain extensive positional disorder when bound to calmodulin Lilia Milanesi1,2, Clare R. Trevitt1, Brian Whitehead1, Andrea M. Hounslow1, Salvador Tomas2,6, Laszlo L. P. Hosszu1,3, Christopher A. Hunter4, and Jonathan P. Waltho1,5 a Milanesi1,2, Clare R. Trevitt1, Brian Whitehead1, Andrea M. Hounslow1, Salvador Tomas2,6 Laszlo L. P. Hosszu1,3, Christopher A. Hunter4, and Jonathan P. Waltho1,5 Laszlo L. P. Hosszu1,3, Christopher A. Hunter4, and Jonathan P. Waltho1,5 1Department of Molecular Biology and Biotechnology, University of Shefﬁeld, Shefﬁeld S10 2TN, UK 2Department of Biological Sciences, School of Science, Birkbeck University of London, London WC1E 7HX, UK 3Medical Research Council Prion Unit, University College of London Institute of Neurology, Queen Square, London WCN1 3BG, UK 4Department of Chemistry, University of Cambridge, Lensﬁeld Road, Cambridge CB2 1EW, UK 5Manchester Institute of Biotechnology, University of Manchester, 131 Princess Street, Manchester M1 7DN, UK 6Departament de Química, Universitat de les Illes Balears, Cra. de Valldemossa, km 7.5. 07122 Palma de Mallorca, Spain Correspondence: Jonathan P. Waltho (j.waltho@shefﬁeld.ac.uk) Correspondence: Jonathan P. Waltho (j.waltho@shefﬁeld.ac.uk) Correspondence: Jonathan P. Waltho (j.waltho@shefﬁeld.ac.uk) Received: 14 January 2021 – Discussion started: 5 February 2021 Revised: 29 March 2021 – Accepted: 3 April 2021 – Published: 13 August 2021 Abstract. Using a combination of NMR and ﬂuorescence measurements, we have investigated the structure and dynamics of the complexes formed between calcium-loaded calmodulin (CaM) and the potent breast can- cer inhibitor idoxifene, a derivative of tamoxifen. High-afﬁnity binding (Kd ∼300 nM) saturates with a 2 : 1 idoxifene : CaM complex. The complex is an ensemble where each idoxifene molecule is predominantly in the vicinity of one of the two hydrophobic patches of CaM but, in contrast with the lower-afﬁnity antagonists TFP, J-8, and W-7, does not substantially occupy the hydrophobic pocket. At least four idoxifene orientations per domain of CaM are necessary to satisfy the intermolecular nuclear Overhauser effect (NOE) restraints, and this requires that the idoxifene molecules switch rapidly between positions. The CaM molecule is predominantly in the form where the N and C-terminal domains are in close proximity, allowing for the idoxifene molecules to contact both domains simultaneously. Hence, the 2 : 1 idoxifene : CaM complex illustrates how high-afﬁnity binding occurs without the loss of extensive positional dynamics. In vitro, idoxifene showed an inhibition potency for CaM of some 4–5 times that of tamox- ifen, a higher toxicity towards ER-positive MCF-7 human breast cancer cells, and higher in vivo clinical activity com- pared to tamoxifen (MacGregor and Jordan, 1998; Dowsett et al., 2000). These properties of idoxifene make it an in- teresting target for a structural study of its binding to CaM. Here we use heteronuclear multidimensional NMR and ﬂuo- rescence spectroscopy to determine the binding afﬁnity, stoi- chiometry, and solution-binding mode of idoxifene to Ca2+- CaM. A comparison with a previously determined molecular model of the same complex and the structures of CaM pep- tides and CaM–ligand complexes reveals an unusual binding mode that broadens the repertoire of recognition processes involving CaM. antagonism in all cases (Cifuentes et al., 2004; Dolan et al., 2009; Pawar et al., 2009; Byer et al., 2011). Hence, an under- standing of the structural determinants of tamoxifen binding to CaM may help in the development of yet more effective therapies. To date there have been few such studies: insights into the nature of the complex come mainly from molecular modelling and structure activity relationship studies (SARs) (Edwards et al., 1992; Hardcastle et al., 1995, 1996). These studies led to the synthesis of idoxifene, a derivative of ta- moxifen in which the basic dimethyl amino side chain has been replaced by a pyrrolidine and iodine has been placed in one of the phenyl rings (Fig. 1). In vitro, idoxifene showed an inhibition potency for CaM of some 4–5 times that of tamox- ifen, a higher toxicity towards ER-positive MCF-7 human breast cancer cells, and higher in vivo clinical activity com- pared to tamoxifen (MacGregor and Jordan, 1998; Dowsett et al., 2000). These properties of idoxifene make it an in- teresting target for a structural study of its binding to CaM. Here we use heteronuclear multidimensional NMR and ﬂuo- rescence spectroscopy to determine the binding afﬁnity, stoi- chiometry, and solution-binding mode of idoxifene to Ca2+- CaM. A comparison with a previously determined molecular model of the same complex and the structures of CaM pep- tides and CaM–ligand complexes reveals an unusual binding mode that broadens the repertoire of recognition processes involving CaM. In the present study we show that the complex between CaM and the high-afﬁnity antagonist idoxifene represents a still different binding mode of a CaM antagonist. 1 Introduction In some of these complexes, CaM adopts an extended structure more similar to that of uncomplexed CaM (Elshorst et al., 1999; Samal et al., 2011), and the hydrophobic pockets of CaM do not bind to the hydrophobic residues of the target peptide, al- though nanomolar binding afﬁnity is retained (Yamauchi et al., 2003; Izumi et al., 2008). zymes. On binding of most of these peptides to CaM, the ﬂexible tether between the two globular domains bends such that the N-terminal domain comes close to the C-terminal domain and the α helices that usually form in the bound peptides stabilize and ﬁx the position of the two CaM do- mains (Maximciuc et al., 2006; Frederick et al., 2007). In this mode of binding, also called the wrap-around mode, the hy- drophobic pockets in the globular domains become occupied by side chains of hydrophobic residues within the peptides and the complex adopts a compact, globular structure. How- ever, more recent structural studies show alternative modes of CaM binding to proteins and peptides. In some of these complexes, CaM adopts an extended structure more similar to that of uncomplexed CaM (Elshorst et al., 1999; Samal et al., 2011), and the hydrophobic pockets of CaM do not bind to the hydrophobic residues of the target peptide, al- though nanomolar binding afﬁnity is retained (Yamauchi et al., 2003; Izumi et al., 2008). Figure 1. The chemical structures of tamoxifen and idoxifene. The numbering scheme for idoxifene used here is shown: phenyl group: H7–H11; ethyl group: H12–H13; p-iodo-phenyl group: H15–H19; p-phenoxy group: H21–H25; pyrrolidine group: H28–H31. Figure 1. The chemical structures of tamoxifen and idoxifene. The numbering scheme for idoxifene used here is shown: phenyl group: H7–H11; ethyl group: H12–H13; p-iodo-phenyl group: H15–H19; p-phenoxy group: H21–H25; pyrrolidine group: H28–H31. A similar diversity of binding modes has been observed in CaM bound to small-molecule antagonists that share features of the target peptides. They have hydrophobic regions and basic functional groups but have greater mobility in the CaM- bound state, making it more difﬁcult to determine the extent of domain closure in these complexes relative to those with peptides (Prozialeck and Weiss, 1982; Craven et al., 1996; Osawa et al., 1998). Some of these antagonists, such as the antipsychotic drug triﬂuoperazine (TFP), the highly selective inhibitors of CaM-mediated processes W-7 and J-8, calmida- zolium, the arylalkylamine derivative DPD, and certain bi- functional ligands, bind CaM with afﬁnities in the nanomo- lar to low micromolar range. These ligands form complexes that are often characterized by a higher degree of proximity of the two CaM domains compared with the complexes be- tween the protein and low-afﬁnity ligands (Reid et al., 1990; Osawa et al., 1998, 1999; Trevitt et al., 2005; Kovesi et al., 2008). In contrast, an alternative mode of binding has also been reported for the high-afﬁnity antagonist Kar-2 that does not involve the hydrophobic pockets of CaM (Horvath et al., 2005). antagonism in all cases (Cifuentes et al., 2004; Dolan et al., 2009; Pawar et al., 2009; Byer et al., 2011). Hence, an under- standing of the structural determinants of tamoxifen binding to CaM may help in the development of yet more effective therapies. To date there have been few such studies: insights into the nature of the complex come mainly from molecular modelling and structure activity relationship studies (SARs) (Edwards et al., 1992; Hardcastle et al., 1995, 1996). These studies led to the synthesis of idoxifene, a derivative of ta- moxifen in which the basic dimethyl amino side chain has been replaced by a pyrrolidine and iodine has been placed in one of the phenyl rings (Fig. 1). 1 Introduction undergo a large conformational change, where the domains become less compact and a hydrophobic pocket is opened (Finn et al., 1995; Kuboniwa et al., 1995; Zhang et al., 1995). These hydrophobic pockets play a central role in the bind- ing of various CaM targets (Meador et al., 1992; Ikura et al., 1992; Craven et al., 1996; Osawa et al., 1998; Harmat et al., 2000; Kovesi et al., 2008). Calmodulin (CaM) is an important intracellular calcium re- ceptor found in all eukaryotic cells. Calcium-loaded CaM binds to more than 300 target enzymes that modulate vari- ous cellular functions (Ikura and Ames, 2006; Swulius and Waxham, 2008). CaM consists of two globular domains sep- arated by a solvent-exposed helical region that is not con- tinuous in solution (the so-called ﬂexible tether), allowing the two domains to be independently mobile (Barbato et al., 1992; Chou et al., 2001; Trevitt et al., 2005). On binding cal- cium, the four helices in each of the two globular domains The proposed mechanism of action of CaM-mediated en- zyme regulation is based largely on structural studies of com- plexes between CaM and peptides of 20–30 residues corre- sponding to CaM interaction domains rather than intact en- Published by Copernicus Publications on behalf of the Groupement AMPERE. L. Milanesi et al.: High-afﬁnity tamoxifen Figure 1. The chemical structures of tamoxifen and idoxifene. The numbering scheme for idoxifene used here is shown: phenyl group: H7–H11; ethyl group: H12–H13; p-iodo-phenyl group: H15–H19; p-phenoxy group: H21–H25; pyrrolidine group: H28–H31. L. Milanesi et al.: High-afﬁnity tamoxifen 630 zymes. On binding of most of these peptides to CaM, the ﬂexible tether between the two globular domains bends such that the N-terminal domain comes close to the C-terminal domain and the α helices that usually form in the bound peptides stabilize and ﬁx the position of the two CaM do- mains (Maximciuc et al., 2006; Frederick et al., 2007). In this mode of binding, also called the wrap-around mode, the hy- drophobic pockets in the globular domains become occupied by side chains of hydrophobic residues within the peptides and the complex adopts a compact, globular structure. How- ever, more recent structural studies show alternative modes of CaM binding to proteins and peptides. Idox- ifene is a triphenylethylene-derivative analogue of tamoxifen (Fig. 1), one of the ﬁrst agents of choice for the treatment and prevention of breast cancer (Marshall, 1998; Powles, 2013). The classical view of the mechanism of action of tamoxifen is that it competes with estradiol for binding to the estrogen receptor (ER) (Shiau et al., 1998). However, several studies indicate that tamoxifen inhibition of breast cancer growth is the result of a complex interplay involving both ER bind- ing and CaM antagonism (Gulino et al., 1986; Cifuentes et al., 2004; Li and Sacks, 2007; Gallo et al., 2008). In addi- tion to its therapeutic efﬁcacy for breast cancer, tamoxifen also has antifungal activity and inhibits the growth of var- ious tumours by a complex mechanism that requires CaM 2.3.1 Resonance and NOE assignment 2.3.1 Resonance and NOE assignment Resonance assignment of free CaM was based on the assign- ment of Drosophila CaM (BMRB entry 547) and it was veri- ﬁed using TOCSY and NOESY-HSQC experiments (Craven et al., 1996). For the idoxifene : CaM complex, the amide 1H-15N correlations were followed in the titration series of HSQC spectra using 3D TOCSY-HSQC and NOESY-HSQC experiments. The assignments of backbone and side-chain resonances of 13C, 15N-labelled CaM in the 2 : 1 idoxifene : CaM complex were conﬁrmed using previously described protocols (Craven et al., 1996; Osawa et al., 1998), including 3D 13C-edited NOESY-HSQC and 3D long-range 13C-13C correlation experiments for the assignment of the ε methyl resonances of methionine. The assignment of idoxifene in the absence of protein (Fig. S1) was carried out in D2O at pH 3.0, as the ligand is insoluble at pH 6.0. Idoxifene resonances in the complex were assigned using 2D TOCSY and a 2D 13C- 15N-double-half-ﬁltered NOESY experiment acquired with a mixing time of 100 ms, in line with previous solution struc- tures of CaM–small ligand complexes (Craven et al., 1996; Osawa et al., 1998). The identity of resonances involved in intermolecular NOEs in the complex was also discerned us- ing the 2D 13C-15N-double-half-ﬁltered NOESY experiment and, for protein resonances, conﬁrmed using a 3D ω1-13C- 15N-half-ﬁltered 13C-edited NOESY-HSQC spectrum with the same mixing time. For matching to the assignment data, tolerances of 0.03 and 0.3 ppm were used for 1H and 13C frequencies respectively. The chemical shift values of the peak centres were converted to X-PLOR restraints using in-house software. For the idoxifene : CaM complex, 180 ligand-protein NOEs were observed, of which 110 were un- ambiguously assigned. Stock solutions of idoxifene were prepared at a concen- tration of 60 mM in CD3OD. Idoxifene was added to the CaM solution up to a maximum ligand : protein molar ratio of 2.4 : 1. 1H and 2D 1H-15N HSQC spectra were recorded on the protein solutions and, for each successive addition of idoxifene, in steps of 0.2 equivalents of idoxifene to CaM. A control titration with CD3OD was carried out under the same conditions to correct for the small effects of CD3OD on the protein chemical shifts. The pH of the protein solutions was monitored throughout the titrations, and when necessary small additions of acid or base were made to maintain the pH at 6.0. 2.2 Fluorescence spectroscopy Binding of CaM to idoxifene was monitored using changes in the intrinsic ﬂuorescence of idoxifene at 293 K. Titra- tions were performed using a Cary Eclipse ﬂuorescence spectrophotometer, with excitation at 295 nm and observa- tion of emission at 450 nm. Idoxifene and CaM solutions were prepared in 45 mM KCl and 9 mM CaCl2, pH 6.0 with 10 % methanol, and CaM was added to idoxifene in aliquots of 0.1 molar equivalents. Titrations were performed with 10 and 0.54 µM idoxifene, using 90 µM and 4.5 µM CaM respectively. Concentrations were veriﬁed by quantitative 1D 1H NMR relative to TSP for idoxifene and by ab- sorbance at 276 nm for CaM, using the extinction coefﬁcient 3300 cm−1 M−1. The 0.54 µM idoxifene titration was ﬁtted for Kd using a 2 : 1 idoxifene : CaM stoichiometry and in- dependently ﬁtted for both Kd and stoichiometry using in- house software. 2.3 NMR spectroscopy NMR experiments were carried out at 310 K. The H2O reso- nance was suppressed by on-resonance low-power presatura- tion (typically applying a 10 Hz ﬁeld for 800 ms) during the relaxation delay, followed by a SCUBA sequence employing two composite 5 pulses separated by 30 ms delay (Brown et al., 1988). The data were acquired using standard het- eronuclear NMR experiments, processed using the program FELIX, and deconvoluted as described previously (Craven et al., 1996). Proton, carbon, and nitrogen frequencies were referenced relative to TSP using values of 0.251449530 and 0.101329118 for γC/γH and γN/γH respectively (Wishart et al., 1995). 2.1 Sample preparation Idoxifene was a gift from the CRC Institute of Cancer Re- search at Sutton, Surrey, UK. Unlabelled and uniformly Magn. Reson., 2, 629–642, 2021 https://doi.org/10.5194/mr-2-629-2021 631 L. Milanesi et al.: High-afﬁnity tamoxifen L. Milanesi et al.: High-afﬁnity tamoxifen 13C/15N-labelled recombinant mammalian CaM were pre- pared as described previously (Vogel et al., 1983). Puriﬁed, lyophilized, calcium-free protein was dissolved to a con- centration of 3 mM in 50 mM KCl, 5 mM NaN3, and 10 % D2O. 3-(trimethylsilyl) propionic-2,2,3,3,-d4 acid, sodium salt (TSP, 0.1 mM), was added as a reference compound. Appropriate quantities of CaCl2 solution (6 mol per mole of CaM) were added to yield calcium-saturated protein and checked by observing diagnostic amide chemical shift changes in the 1D 1H NMR spectrum. The ﬁnal sample vol- ume was 450 µL. The pH of the solution was adjusted to 6.0 (uncorrected value) by adding microlitre quantities of 0.1 mM HCl and NaOH. CaM samples were otherwise un- buffered because the apo-protein has sufﬁcient buffering ca- pacity at pH 6.0 and the extrinsic buffers available without non-exchangeable protons compete for calcium binding. The KCl concentration used here mimics conditions in which the calcium afﬁnity of CaM was determined (Linse et al., 1991) and was chosen to minimize the likelihood of idoxifene pre- cipitation at the end of the titration when excess ligand is present. This unbuffered system and salt concentration are in keeping with previous studies of CaM (50–100 mM KCl) (Finn et al., 1995; Craven et al., 1996; Trevitt et al., 2005). 2.3.2 Line-shape analysis Line shapes were calculated for a simple two-state exchange model using standard equations (McConnell, 1958). The transverse relaxation times were adjusted to match the ob- served line width, and the value of the off-rate was varied https://doi.org/10.5194/mr-2-629-2021 Magn. Reson., 2, 629–642, 2021 632 L. Milanesi et al.: High-afﬁnity tamoxifen L. Milanesi et al.: High-afﬁnity tamoxifen to optimize the agreement between the calculated and exper- imental data. A normalizing factor was applied to all data to correct for the constant intensity loss observed through- out the titration as a result of dilution. This was calculated by determining the mean intensity loss of a number of peaks for which no change in chemical shifts was seen on bind- ing idoxifene and hence were unaffected by the exchange processes. In interpreting the line-shape analysis in terms of absolute stoichiometry, it is imperative to be certain of the ligand and protein concentrations used. For the protein this was initially determined using UV absorbance and for the ligand using dry weight. The ﬁnal concentration in the 2 : 1 complexes was then checked by comparison of peaks in the 1D spectrum, which conﬁrmed that the concentrations were correct to within 10 %. a box of side 60 Å centred on the centre of mass of CaM. For NOE-restrained docking calculations, protein coordi- nates were taken from either the 2.4 Å resolution structure of CaM in a complex with a myosin light-chain kinase pep- tide (pdb – Protein Data Bank – entry 1cdl, Meador et al., 1992) or the tr2c domain of the 1.7 Å resolution X-ray struc- ture of mammalian CaM (pdb entry 1cll, Chattopadhyaya et al., 1992). Two sets of starting positions of the idoxifene molecules were investigated – molecules placed at random in a box and molecules manually docked into the hydrophobic pocket – to exclude any bias away from occupancy of the hy- drophobic pocket through restrictions in the sampling of the relative positions of molecules. For the former, a fresh idox- ifene starting conformation for each calculation was created by ﬁrst transforming the coordinates in an extended confor- mation by a random rigid body rotation. The centre of mass was then placed at random within a box of side 60 Å cen- tred on the centre of mass of the protein molecule. The idox- ifene molecule was treated as ﬂexible, subject to restraints of covalent geometry and van der Waals contacts. 2.3.2 Line-shape analysis The side chains involved in intermolecular NOEs were either ﬁxed with the remainder of the protein or allowed the same in- ternal ﬂexibility as the idoxifene molecules, and the two cal- culations were compared. For the manual docking of idox- ifene molecules into the hydrophobic pocket of the protein, 50 structures were generated in which different hydropho- bic parts of the idoxifene molecule were placed deep inside the hydrophobic pocket of the domain, and the molecules were subjected to 250 steps of conjugate gradient energy minimization to remove steric clashes. In each case part of the idoxifene molecule remained within the hydrophobic pocket. These structures were then used as starting coordi- nates for the structure calculation protocols described above. For both sets of starting positions, the ﬁnal distribution of structures was indistinguishable. When more than one idox- ifene molecule per domain of CaM was included in the calcu- lation, the core repulsion terms between idoxifene molecules was set to zero, whereas those within each idoxifene and be- tween each idoxifene and the protein were increased during the course of the calculation as described above. This meant that there was no energy penalty to atoms from more than one idoxifene molecule occupying the same space. An NOE restraint was satisﬁed by the proximity to the protein atoms of atoms from any individual idoxifene molecule 2.4 Structure calculations Restraints were classiﬁed as strong, medium, or weak and were assigned upper bounds of (i) 2.5, 3.5, and 5.0 Å, (ii) 3.0, 4.0, and 5.0 Å, or (iii) all as 5.0 Å in separate calcula- tions to accommodate the inherent uncertainty involved in converting cross-peak intensities to more precise distances when there is the possibility of conformational exchange of the ligand in its binding site. All NOE restraints were in- troduced using 1/r6 sum averaging to accommodate reori- entation of aromatic rings and isopropyl groups via bond rotation. Structures were calculated with X-PLOR 3.1 us- ing a molecular dynamics simulated annealing protocol for conventional protein structure determination (Brünger, 1992; Nilges, 1995). For the ﬁrst 3 ps, a high temperature (1500 K) was maintained, and the weight on the core repulsion poten- tial energy term was kept very low. This was followed by an 18 ps cooling stage in which the temperature was reduced in 50 K steps, and the weight on the core repulsion term was gradually increased. Finally, the structures were subjected to 250 steps of conjugate gradient energy minimization. As a reﬁnement stage, the temperature was increased to 1500 K and the above cycle repeated. During the high-temperature stage of the protocol, a square-well NOE potential was used, with harmonic sides. During the second part of the protocol, the X-PLOR soft-square potential was used, which smoothly changes the harmonic potential to a linear potential for large restraint violations. The energy constant for the harmonic po- tential was 5.0 kcal mol−1 Å−2. The slope of the asymptote was 0.5 kcal mol−1 Å−1. The switching region between the two regimes was approximately between 0.5 and 2 Å above the upper restraint bound. The parallhdg.pro parameter set of X-PLOR was used, with the X-PLOR quartic repel poten- tial to represent the repulsive part on the interatomic inter- actions. No attractive or electrostatic terms were used. The ﬁnal weight on the repulsive term was 4 kcal mol−1 Å−4. 3.1 Titration of CaM with idoxifene D64 and N137 occupy equivalent positions in the CaM structure, be- ing located in position 9 of Ca2+ binding loops II and IV, and contribute to the three residue β strands in each domain. The resonances of T28 and S101, the equivalent residues in loops I and III, show slow exchange behaviour, as do all of the other residues in the short β sheets of CaM (e.g. I27 and I100 in Fig. 2c). The anomalous behaviour of D64 and N137 may result from a sensitivity of these residues to the occupa- tion of the other globular domain of CaM by idoxifene. Table 1. 1H chemical shifts of idoxifene, free in solution and bound to CaM. Resonance Freea Boundb 1H δ δ 1δ 16/18 7.52 7.42 −0.10 15/19 6.86 6.84 −0.02 21/25 6.69 6.71 +0.02 22/24 6.47 6.55 +0.08 7/11 6.99 6.98 −0.01 8, 9, 10c 7.06 7.08 +0.02 12 2.23 2.31 +0.08 13 0.73 0.78 +0.05 26 3.96 4.10 +0.14 27 3.43 3.50 +0.07 28/31 3.39/3.06 3.30 +0.07 29/30 1.96 1.99 +0.03 a D2O, pH 3.0. b D2O, pH 6.0. c Unresolved resonances. a D2O, pH 3.0. b D2O, pH 6.0. c Unresolved resonances. edited NOESY-HSQC experiments (seven of the nine were assigned). Some resonances from residues L69–K77 were at- tenuated, particularly their Cα resonances, indicative of con- formational exchange in the ﬂexible tether region between domains in the complex. A summary of the backbone chemical shift changes ob- served on binding of idoxifene to CaM is shown in Fig. 3 as a weighted average (WA) of the changes in all ﬁve backbone resonances, obtained using the equation \|WA\| ∼= XδCaM:drug −δCaM /\|1max\|, (1) (1) where the summation extends over the backbone atoms, δCaM:drug and δCaM are the chemical shifts observed in the complex and in free CaM, and 1max is the largest chemical shift change observed for each type of nucleus. The chemical shift changes in each of the ﬁve backbone atoms are shown in Fig. S2. 3.1 Titration of CaM with idoxifene D64 and N137 occupy equivalent positions in the CaM structure, be- ing located in position 9 of Ca2+ binding loops II and IV, and contribute to the three residue β strands in each domain. The resonances of T28 and S101, the equivalent residues in loops I and III, show slow exchange behaviour, as do all of the other residues in the short β sheets of CaM (e.g. I27 and I100 in Fig. 2c). The anomalous behaviour of D64 and N137 may result from a sensitivity of these residues to the occupa- tion of the other globular domain of CaM by idoxifene. ﬁt to a Kd of 340 ± 30 nM using a stoichiometry of 2 : 1 idoxifene : CaM. When both Kd and stoichiometry were al- lowed to vary, the data best ﬁt to a Kd of 180 ± 50 nM and a stoichiometry of 1.7 : 1 idoxifene : CaM, but there is co- variance between these parameters in the range that includes a 2 : 1 stoichiometry and a Kd of 340 nM. A chi2 analysis of ﬁts indicated a clear minimum at Kd = 300 nM. The bind- ing of idoxifene to CaM was monitored independently us- ing 1D 1H NMR, and the perturbations of low ﬁeld amide 1H resonances during the addition of up to 2.4 equivalents of idoxifene are shown in Fig. 2c. Where the chemical shift changes induced by complex formation are much greater than 0.05 ppm, two sets of resonances are detected during the titration (e.g. I27, I100, and G134 in Fig. 2c). Thus, for these resonances, the dissociation rate of the complex is predominantly in the slow exchange regime on the NMR timescale. The resonances corresponding to free CaM disap- peared when two equivalents of ligand were added, in line with the stoichiometry of 2 : 1 reported by the ﬂuorescence measurements using 10 µM idoxifene. Some resonances ex- perienced a reduction of peak height at intermediate ligand concentrations and re-sharpened when two equivalents of idoxifene were added (e.g. D64 and N137 in Fig. 2c). The extent of line broadening allowed an estimate for the off-rate of 30 ± 10 s−1 to be obtained, which, in combination with the measured Kd value, indicates that the complex forms with a diffusion-controlled on-rate of ca. 1 × 108 M−1 s−1. 3.1 Titration of CaM with idoxifene The binding stoichiometry and afﬁnity between idoxifene and CaM were measured using changes in idoxifene ﬂuo- rescence at 450 nm upon addition of increasing amounts of Ca2+-CaM. Using 10 µM idoxifene, the response on CaM addition was linear up to a stoichiometry of 2 : 1 idoxifene : CaM (Fig. 2a). Using 540 nM idoxifene (Fig. 2b), the data For ab initio structure calculations, extended protein co- ordinates with random initial velocities were used as a seed, and two idoxifene molecules were placed at random within Magn. Reson., 2, 629–642, 2021 https://doi.org/10.5194/mr-2-629-2021 633 L. Milanesi et al.: High-afﬁnity tamoxifen ﬁt to a Kd of 340 ± 30 nM using a stoichiometry of 2 : 1 idoxifene : CaM. When both Kd and stoichiometry were al- lowed to vary, the data best ﬁt to a Kd of 180 ± 50 nM and a stoichiometry of 1.7 : 1 idoxifene : CaM, but there is co- variance between these parameters in the range that includes a 2 : 1 stoichiometry and a Kd of 340 nM. A chi2 analysis of ﬁts indicated a clear minimum at Kd = 300 nM. The bind- ing of idoxifene to CaM was monitored independently us- ing 1D 1H NMR, and the perturbations of low ﬁeld amide 1H resonances during the addition of up to 2.4 equivalents of idoxifene are shown in Fig. 2c. Where the chemical shift changes induced by complex formation are much greater than 0.05 ppm, two sets of resonances are detected during the titration (e.g. I27, I100, and G134 in Fig. 2c). Thus, for these resonances, the dissociation rate of the complex is predominantly in the slow exchange regime on the NMR timescale. The resonances corresponding to free CaM disap- peared when two equivalents of ligand were added, in line with the stoichiometry of 2 : 1 reported by the ﬂuorescence measurements using 10 µM idoxifene. Some resonances ex- perienced a reduction of peak height at intermediate ligand concentrations and re-sharpened when two equivalents of idoxifene were added (e.g. D64 and N137 in Fig. 2c). The extent of line broadening allowed an estimate for the off-rate of 30 ± 10 s−1 to be obtained, which, in combination with the measured Kd value, indicates that the complex forms with a diffusion-controlled on-rate of ca. 1 × 108 M−1 s−1. 3.3 Structure determination changes observed for methionine residues, in particular the β and ε resonances of M71 and M144 (Fig. 4c), which oc- cupy equivalent positions near the C terminus of each do- main, on the rim of their hydrophobic pockets. Also of note are the chemical shift changes in phenylalanine ring reso- nances. Only F68 and F141 undergo large shifts upon idox- ifene binding, indicating that the structure of the individual domains is not signiﬁcantly perturbed in the complex. The aromatic rings of F68 and F141 directly contact the ε methyl groups of M71 and M144, and the combined chemical shift perturbations of these residues indicate a local disturbance in this region upon idoxifene binding. Overall, it is clear that while side-chain shift changes are localized around the hy- drophobic pockets of both domains, backbone shift changes extend to the tether and the rear of the domains. changes observed for methionine residues, in particular the β and ε resonances of M71 and M144 (Fig. 4c), which oc- cupy equivalent positions near the C terminus of each do- main, on the rim of their hydrophobic pockets. Also of note are the chemical shift changes in phenylalanine ring reso- nances. Only F68 and F141 undergo large shifts upon idox- ifene binding, indicating that the structure of the individual domains is not signiﬁcantly perturbed in the complex. The aromatic rings of F68 and F141 directly contact the ε methyl groups of M71 and M144, and the combined chemical shift perturbations of these residues indicate a local disturbance in this region upon idoxifene binding. Overall, it is clear that while side-chain shift changes are localized around the hy- drophobic pockets of both domains, backbone shift changes extend to the tether and the rear of the domains. Intramolecular NOEs within CaM and within idoxifene and intermolecular NOEs between idoxifene and CaM were quantiﬁed using a 2D 13C-15N-double-half-ﬁltered NOESY spectrum of a 2 : 1 idoxifene : CaM solution (Fig. 5), and the identity of protein resonances was conﬁrmed using a 3D ω1- 13C-15N-half-ﬁltered 13C-edited NOESY-HSQC spectrum. Most of the intermolecular NOEs arising from the phenyl, p-iodo-phenyl, p-phenoxy, and ethyl groups of the idoxifene (Fig. 1) are to protein side-chain resonances in the vicinity of the two hydrophobic pockets exposed on calcium binding; none are observed to the opposite faces of the CaM domains. 3.2 Chemical shift changes Substantial chemical shift changes are seen in many con- tiguous stretches of the backbone such as E67–I85 and F141– T146 and are mapped onto the open structure of CaM in Fig. 4a. In contrast, some residues such as V55 display large shift changes, whereas surrounding residues are hardly per- turbed. Residues E67–I85 include the ﬂexible tether between the two domains, and the chemical shift changes here are closely comparable with those in the same region observed on formation of the CaM : M13 complex (Ikura et al., 1991), where CaM wraps its two domains around a helical peptide in a compact, globular structure. The NMR resonances of free idoxifene were assigned on the basis of characteristic chemical shifts of model compounds (2-pyrrolidinoethanol, iodo-benzene, methoxy- benzene), NOEs, and correlations observed in a 2D 1H TOCSY spectrum (Table 1 and Fig. S1). The backbone and side-chain resonances of 13C, 15N-labelled CaM in the 2 : 1 idoxifene : CaM complex were assigned using previously de- scribed protocols (Craven et al., 1996; Osawa et al., 1998). The acquisition of intra- and inter-residue carbonyl shifts was essential in order to distinguish residues with degener- ate Cα shifts, such as D50 and D122. More severe overlap present in the aromatic region of the spectra of the complex prevented the assignment of the ζ resonances of the pheny- lalanine residues. The assignment of the ε methyl resonances of methionine residues was achieved using a combination of 3D long-range 13C-13C correlation (LRCC) and 3D 13C- The positions of the side-chain chemical shift changes ob- served on binding of idoxifene to CaM are shown in Fig. 4b. The majority of protein side-chain resonances move by less than 0.05 ppm for 1H and 0.3 ppm for 13C. The larger side- chain shift changes are conﬁned to residues around the hy- drophobic pockets of both CaM domains, with the largest https://doi.org/10.5194/mr-2-629-2021 Magn. Reson., 2, 629–642, 2021 634 L. Milanesi et al.: High-afﬁnity tamoxifen Figure 2. Titration of idoxifene with calmodulin. (a) The changes in ﬂuorescence intensity of 10 µM idoxifene at 450 nm on addition of CaM up to 15 µM. Saturation is reached at a concentration ratio of CaM to idoxifene of 0.5, indicating the presence of two binding sites with sub- micromolar binding afﬁnity. (b) Repeat of (a) but with 0.54 µM idoxifene and increasing CaM concentrations from 0.02 µM up to 0.6 µM. 3.2 Chemical shift changes The data were ﬁtted to a binding isotherm with a 2 : 1 idoxifene : CaM binding stoichiometry. (c) A region of the 1D 1H spectra acquired during the titration of idoxifene into 3 mM CaM. Solid lines highlight the completion of the slow exchange event for the assigned resonances at 2 : 1 idoxifene : CaM. Dotted lines are drawn for resonances undergoing shift changes beyond the end point of the titration. G134, I100, and I27 are representative of the slow exchange regime observed for CaM resonances during the titration, while N137 is representative of an intermediate exchange character. L. Milanesi et al.: High-afﬁnity tamoxifen 634 Figure 2. Titration of idoxifene with calmodulin. (a) The changes in ﬂuorescence intensity of 10 µM idoxifene at 450 nm on addition of CaM up to 15 µM. Saturation is reached at a concentration ratio of CaM to idoxifene of 0.5, indicating the presence of two binding sites with sub- micromolar binding afﬁnity. (b) Repeat of (a) but with 0.54 µM idoxifene and increasing CaM concentrations from 0.02 µM up to 0.6 µM. The data were ﬁtted to a binding isotherm with a 2 : 1 idoxifene : CaM binding stoichiometry. (c) A region of the 1D 1H spectra acquired during the titration of idoxifene into 3 mM CaM. Solid lines highlight the completion of the slow exchange event for the assigned resonances at 2 : 1 idoxifene : CaM. Dotted lines are drawn for resonances undergoing shift changes beyond the end point of the titration. G134, I100, and I27 are representative of the slow exchange regime observed for CaM resonances during the titration, while N137 is representative of an intermediate exchange character. Figure 3. Chemical shift changes for backbone CaM resonances upon binding idoxifene. Normalized, weighted average chemical shift changes are plotted against the primary sequence of CaM. Secondary structure elements are indicated above the histogram as solid boxes for α helices, solid arrows for β strands, and thin lines for unstructured regions. The ﬂexible tether (E67–V85) experiences the largest chemical shift changes upon complex formation. Figure 3. Chemical shift changes for backbone CaM resonances upon binding idoxifene. Normalized, weighted average chemical shift changes are plotted against the primary sequence of CaM. Secondary structure elements are indicated above the histogram as solid boxes for α helices, solid arrows for β strands, and thin lines for unstructured regions. 3.2 Chemical shift changes The ﬂexible tether (E67–V85) experiences the largest chemical shift changes upon complex formation. 3.3 Structure determination In (a) residues are coloured accord- ing to the weighted average (WA) backbone shift changes observed in the idoxifene : CaM complex, with gradation from white (WA shift = 0.0 ppm) to red (WA shift > 0.7 ppm) and the broadest rib- bon indicating the maximum shift changes. The locations of M109 and M124 by the hydrophobic pocket of the C domain are indi- cated. Residues I85 and E67 are indicated, as they deﬁne the central portion of the ﬂexible tether joining the two domains. The position of V55, which undergoes a large shift change as in the CaM : M13 complex, is also indicated. In (b) residues are coloured according to the largest side-chain 1H shift changes observed upon idoxifene binding (shifts > 0.50 ppm), with colour gradation and ribbon width depicted as in (a). The locations of key residues such as three of the nine methionines in the vicinity of the hydrophobic pockets and F68 and F141 are indicated. (c) A 2D 13C CT-HSQC spectrum showing the ε methyl methionine signals of free CaM (grey) and idoxifene- bound CaM (black). b i t d ith l t d l l f i diff i t Figure 5. Intermolecular NOEs between idoxifene and CaM. A region of the 2D 13C-15N-double-half-ﬁltered NOESY spec- trum of the 2 : 1 idoxifene : CaM complex, processed to exclude the 13C/15N bound resonances in the ω2 dimension and to in- clude only the 13C bound resonances in the ω1 dimension. The ligand resonances corresponding to each strip are marked on the top edge as follows: p-iodo-phenyl ring (H16/H18), phenyl ring (H8/H9/H10), phenyl ring (H7/H11), p-iodo-phenyl ring (H15/19), and p-phenoxy ring (H21/25 and H22/24). Assignments of the resonances from tr2c are shown on the right. A red circle over the cross peak indicates examples of NOEs that are violated in the structure calculation corresponding to the data in Fig. S3. spin-diffusion effects are not dominant between nuclei within the ligand when it is bound in the complex. Hence, spin dif- fusion alone cannot account for the unusual similarity in in- tensities of the intermolecular NOEs involving the phenyl, p-iodo-phenyl, p-phenoxy, and ethyl groups. I iti ll t d d NOE t i d t t l l ti L. Milanesi et al.: High-afﬁnity tamoxifen L. Milanesi et al.: High-afﬁnity tamoxifen 635 Figure 5. Intermolecular NOEs between idoxifene and CaM. 3.3 Structure determination A region of the 2D 13C-15N-double-half-ﬁltered NOESY spec- trum of the 2 : 1 idoxifene : CaM complex, processed to exclude the 13C/15N bound resonances in the ω2 dimension and to in- clude only the 13C bound resonances in the ω1 dimension. The ligand resonances corresponding to each strip are marked on the top edge as follows: p-iodo-phenyl ring (H16/H18), phenyl ring (H8/H9/H10), phenyl ring (H7/H11), p-iodo-phenyl ring (H15/19), and p-phenoxy ring (H21/25 and H22/24). Assignments of the resonances from tr2c are shown on the right. A red circle over the cross peak indicates examples of NOEs that are violated in the structure calculation corresponding to the data in Fig. S3. Figure 4. Comparison of backbone and side-chain chemical shift changes in CaM resonances upon binding idoxifene. (a) and (b): heat map representations of CaM chemical shift changes upon idox- ifene binding, shown for clarity using the coordinates of the unli- ganded CaM X-ray structure, 1cll. The hydrophobic pocket of the N terminus at the top and the rear of the C-terminal hydropho- bic pocket is at the bottom. In (a) residues are coloured accord- ing to the weighted average (WA) backbone shift changes observed in the idoxifene : CaM complex, with gradation from white (WA shift = 0.0 ppm) to red (WA shift > 0.7 ppm) and the broadest rib- bon indicating the maximum shift changes. The locations of M109 and M124 by the hydrophobic pocket of the C domain are indi- cated. Residues I85 and E67 are indicated, as they deﬁne the central portion of the ﬂexible tether joining the two domains. The position of V55, which undergoes a large shift change as in the CaM : M13 complex, is also indicated. In (b) residues are coloured according to the largest side-chain 1H shift changes observed upon idoxifene binding (shifts > 0.50 ppm), with colour gradation and ribbon width depicted as in (a). The locations of key residues such as three of the nine methionines in the vicinity of the hydrophobic pockets and F68 and F141 are indicated. (c) A 2D 13C CT-HSQC spectrum showing the ε methyl methionine signals of free CaM (grey) and idoxifene- bound CaM (black). Figure 5. Intermolecular NOEs between idoxifene and CaM. Figure 5. Intermolecular NOEs between idoxifene and CaM. 3.3 Structure determination However, it is immediately striking that the phenyl, p-iodo- phenyl, p-phenoxy, and ethyl groups mostly have substantial NOEs to the same resonances on the protein despite those resonances belonging to nuclei that are distributed widely across each domain (Fig. S3). Such an NOE distribution can Magn. Reson., 2, 629–642, 2021 https://doi.org/10.5194/mr-2-629-2021 L. Milanesi et al.: High-afﬁnity tamoxifen Figure 4. Comparison of backbone and side-chain chemical shift changes in CaM resonances upon binding idoxifene. (a) and (b): heat map representations of CaM chemical shift changes upon idox- ifene binding, shown for clarity using the coordinates of the unli- ganded CaM X-ray structure, 1cll. The hydrophobic pocket of the N terminus at the top and the rear of the C-terminal hydropho- bic pocket is at the bottom. In (a) residues are coloured accord- ing to the weighted average (WA) backbone shift changes observed in the idoxifene : CaM complex, with gradation from white (WA shift = 0.0 ppm) to red (WA shift > 0.7 ppm) and the broadest rib- bon indicating the maximum shift changes. The locations of M109 and M124 by the hydrophobic pocket of the C domain are indi- cated. Residues I85 and E67 are indicated, as they deﬁne the central portion of the ﬂexible tether joining the two domains. The position of V55, which undergoes a large shift change as in the CaM : M13 complex, is also indicated. In (b) residues are coloured according to the largest side-chain 1H shift changes observed upon idoxifene binding (shifts > 0.50 ppm), with colour gradation and ribbon width depicted as in (a). The locations of key residues such as three of the nine methionines in the vicinity of the hydrophobic pockets and F68 and F141 are indicated. (c) A 2D 13C CT-HSQC spectrum showing the ε methyl methionine signals of free CaM (grey) and idoxifene- bound CaM (black). L. Milanesi et al.: High-afﬁnity tamoxifen 635 Figure 4. Comparison of backbone and side-chain chemical shift changes in CaM resonances upon binding idoxifene. (a) and (b): heat map representations of CaM chemical shift changes upon idox- ifene binding, shown for clarity using the coordinates of the unli- ganded CaM X-ray structure, 1cll. The hydrophobic pocket of the N terminus at the top and the rear of the C-terminal hydropho- bic pocket is at the bottom. 3.3 Structure determination When the intermolecular NOEs were all assigned upper bounds of 5.0 Å and the contacting side chains within the in- dividual domains of CaM were allowed conformational ﬂex- ibility, the lowest-energy structures converged, whereby the idoxifene molecules occupied one of two sites on CaM in the vicinity of the hydrophobic pocket on each domain (Fig. 6a– b), and the NOE violations in the lowest-energy structures dropped to an average of 5. However, while the docked idox- ifene molecules delineated two binding sites in CaM, the ori- entation of the individual idoxifene molecules was very vari- able between structures. Figure 6. Representative 2 : 1 idoxifene : CaM complex structures. Figure 6. Representative 2 : 1 idoxifene : CaM complex structures. Panels (a) and (b) are orthogonal views of the 2 : 1 idoxifene : CaM complex calculated using NOE-restrained docking of two molecules of idoxifene to CaM coordinates based on the CaM : M13 complex (pdb entry 1cdl). In this calculation, the upper bounds of all observed intermolecular NOEs were set to 5 Å. CaM do- mains tr1c and tr2c are coloured green and grey respectively and the idoxifene molecules are coloured in pairs (blue, cyan, yellow, or beige) derived from single calculations. View B is orientated to co- incide with parts C and D. Panels (c) and (d) show the positions of four molecules of idoxifene derived from a single NOE-restrained docking calculation to the tr2c domain of CaM (from pdb entry 1cll), illustrating one arrangement of four conformations that sat- isfy the NOE restraints. The four idoxifene molecules are shown in pairs for clarity only. The two darker-coloured idoxifene molecules have either the phenyl group (c) or the p-iodo-phenyl group (d) by the pocket in the hydrophobic surface of the domain. The lighter- coloured molecules also satisfy NOEs that are within reach of the ligand-binding site associated with the tr1c domain. The surface representation of tr2c is coloured as follows: carbon (grey), nitro- gen (blue), oxygen (red), and sulfur (yellow). In order to establish whether the variability of orienta- tion of the idoxifene ligands in their sites could be reduced, a gradation of upper-bound limits according to intensities of the intermolecular NOEs was re-introduced. 3.3 Structure determination CaM do- mains tr1c and tr2c are coloured green and grey respectively and the idoxifene molecules are coloured in pairs (blue, cyan, yellow, or beige) derived from single calculations. View B is orientated to co- incide with parts C and D. Panels (c) and (d) show the positions of four molecules of idoxifene derived from a single NOE-restrained docking calculation to the tr2c domain of CaM (from pdb entry 1cll), illustrating one arrangement of four conformations that sat- isfy the NOE restraints. The four idoxifene molecules are shown in pairs for clarity only. The two darker-coloured idoxifene molecules have either the phenyl group (c) or the p-iodo-phenyl group (d) by the pocket in the hydrophobic surface of the domain. The lighter- coloured molecules also satisfy NOEs that are within reach of the ligand-binding site associated with the tr1c domain. The surface representation of tr2c is coloured as follows: carbon (grey), nitro- gen (blue), oxygen (red), and sulfur (yellow). almost invariant across all deposited X-ray structures and the intra-protein NOE distribution within the domains of the 2 : 1 idoxifene : CaM complex reﬂected closely that of free CaM, focus was shifted to a restrained docking strategy. This al- lowed the NOE violation energies in the restrained dynam- ics to be isolated to the intermolecular NOEs rather than be spread widely across the CaM domains. In contrast to the invariance of individual domain struc- tures within CaM complexes, the relative position of the two domains varies considerably between deposited structures. NOE-restrained structure calculations where the individual domain structures were ﬁxed but the ﬂexible tether between the two domains was allowed conformational freedom led to closed structures of the idoxifene : CaM complex. How- ever, there was little convergence in the relative position of the two domains, and widespread intermolecular NOE vio- lations remained. Consequently, NOE-restrained docking to a closed structure that well represented the chemical shift changes in the protein backbone (cf. Figs. 3 and S2), that of the CaM : M13 (myosin light-chain kinase peptide) complex, was investigated. With the intermolecular NOEs calibrated directly according to their intensities, the docked structures distributed the idoxifene molecules widely across the hy- drophobic surfaces of the two domains of CaM, but 64 of the 180 NOEs remained violated in the lowest-energy struc- ture. 3.3 Structure determination A region of the 2D 13C-15N-double-half-ﬁltered NOESY spec- trum of the 2 : 1 idoxifene : CaM complex, processed to exclude the 13C/15N bound resonances in the ω2 dimension and to in- clude only the 13C bound resonances in the ω1 dimension. The ligand resonances corresponding to each strip are marked on the top edge as follows: p-iodo-phenyl ring (H16/H18), phenyl ring (H8/H9/H10), phenyl ring (H7/H11), p-iodo-phenyl ring (H15/19), and p-phenoxy ring (H21/25 and H22/24). Assignments of the resonances from tr2c are shown on the right. A red circle over the cross peak indicates examples of NOEs that are violated in the structure calculation corresponding to the data in Fig. S3. spin-diffusion effects are not dominant between nuclei within the ligand when it is bound in the complex. Hence, spin dif- fusion alone cannot account for the unusual similarity in in- tensities of the intermolecular NOEs involving the phenyl, p-iodo-phenyl, p-phenoxy, and ethyl groups. Initially, standard NOE-restrained structure calculations were performed including intra-protein, intra-ligand, and in- termolecular NOEs, classiﬁed as strong, medium, or weak according to intensity (assigned upper bounds of 2.5, 3.5, and 5.0 Å). The resulting structures did not converge to a well-deﬁned ensemble, and only high-energy structures with widespread NOE violations resulted. Since the conforma- tions of the two individual domains of mammalian CaM are be associated with elevated levels of spin diffusion at ex- tended mixing times, but the mixing time employed here (100 ms) is the same as or shorter than that used in the struc- ture determination of other CaM–ligand complexes (Craven et al., 1996, Osawa et al., 1998, 1999), where spin diffusion was not signiﬁcant, and there is no signiﬁcant difference in rotational correlation times between these complexes, since NOEs over extended distances are not observed. Moreover, https://doi.org/10.5194/mr-2-629-2021 Magn. Reson., 2, 629–642, 2021 L. Milanesi et al.: High-afﬁnity tamoxifen L. Milanesi et al.: High-afﬁnity tamoxifen 636 L. Milanesi et al.: High afﬁnity tamoxifen Figure 6. Representative 2 : 1 idoxifene : CaM complex structures. Panels (a) and (b) are orthogonal views of the 2 : 1 idoxifene : CaM complex calculated using NOE-restrained docking of two molecules of idoxifene to CaM coordinates based on the CaM : M13 complex (pdb entry 1cdl). In this calculation, the upper bounds of all observed intermolecular NOEs were set to 5 Å. 3.3 Structure determination Increased deviation from a uniform upper bound of 5.0 Å led to in- creased restraint violations and, in general, pulled the idox- ifene molecules towards the centre of mass of CaM but with- out any discernible decrease in the variability of the orien- tation of individual idoxifene molecules. An alternative ap- proach was thus investigated where the protein in the dock- ing protocol was simpliﬁed to one domain – the tr2c domain. This approach removes NOE violation energies that inadver- tently pull an idoxifene molecule towards the binding site of the other domain. With the intermolecular NOEs assigned upper bounds of 2.5, 3.5, and 5.0 Å, a minimum of 26 of 82 NOEs were violated. tein (Fig. 5), points to a complex where the ligand undergoes conformational exchange while bound to the protein. In order to simulate this in the NOE-restrained docking, the number of idoxifene molecules included was increased until all of the NOE-assigned upper bounds of 2.5, 3.5, and 5.0 Å were sat- isﬁed. No interaction potentials were included between idox- ifene molecules in order that multiple binding conformations could be satisﬁed simultaneously in the calculations, where ligands occupied the same space. With the introduction of four idoxifene molecules, all of the NOE restraints could be satisﬁed. A representative structure with no NOE violations is shown in Fig. 6c–d. The NOEs are satisﬁed by idoxifene placing either the p-iodo-phenyl (Fig. 6c) or phenyl group (Fig. 6d) by the pocket in the hydrophobic surface of the domain and orienting the p-phenoxy and ethyl groups alter- The high proportion of intermolecular NOE violations, coupled with the substantial number of NOEs between dis- tant parts of the ligand and the same resonances on the pro- https://doi.org/10.5194/mr-2-629-2021 Magn. Reson., 2, 629–642, 2021 L. Milanesi et al.: High-afﬁnity tamoxifen 637 and a ligand dissociation rate of ∼30 s−1. This dissocia- tion constant indicates that idoxifene has at least an order of magnitude stronger afﬁnity for CaM than that observed for most other antagonists, with the exception of calmida- zolium (Kd ∼1–10 nM), DPD (Kd ∼18 nM), and other bi- functional ligands (Reid et al., 1990; Harmat et al., 2000; Trevitt et al., 2005). 4.2 Domain closure The evidence points to CaM closing to a compact, globular structure on binding idoxifene. The observed distribution and magnitude of backbone chemical shift changes in the 2 : 1 idoxifene : CaM complex are closely similar to those on for- mation of the CaM : M13 complex, which forms a tightly closed, compact conformation (Ikura et al., 1991; Barbato et al., 1992). In CaM complexes in which the ligand binds to only one of the two domains and the complex does not close, the chemical shift changes in the vast majority of the amide protons in the central linker are very small, usually less than 0.1 ppm (Elshorst et al., 1999). No direct NOEs were observed between the two domains of CaM in the 2 : 1 idoxifene : CaM complex, but this was also the case for the W-7 : CaM complex, which forms a tightly closed confor- mation similar to that of the CaM : M13 complex (Osawa et al., 1998, 1999). Indeed, the direction and magnitude of the backbone proton chemical shift changes for residues in the ﬂexible linker (e.g. E82 and D80) in the W-7 : CaM com- plex are similar to those observed on formation of the 2 : 1 idoxifene : CaM complex (cf. Fig. S2 and Fig. 6 in Osawa et al., 1999). Moreover, the backbone chemical shift changes within the central tether in the idoxifene : CaM complex can- not be attributed solely to proximity to idoxifene – the in- termolecular NOEs and the largest side-chain chemical shift changes are not located in the tether (Fig. 4a–b). In addition, backbone chemical shift changes for T110–L112 are indica- tive of the formation of an α helical structure, while those of M144–T146 are indicative of a loss of α helical structure, as reported for the CaM : M13 complex (Ikura et al., 1991). Fi- nally, in the NOE-restrained docking calculations, when the residues in the ﬂexible tether region were allowed confor- mational freedom, the lowest-energy structures were always closed. 3.3 Structure determination The Kd value determined here is lower than the previously reported IC50 value for idoxifene, 1.5 µM (Hardcastle et al., 1996), which was derived indirectly from the inhibition of calmodulin-dependent cyclic AMP phos- phodiesterase and under experimental conditions different from here. On addition of more than two equivalents of idox- ifene, further small chemical shift changes are observed for some of the CaM resonances (e.g. K13 and N137 in Fig. 2) in the fast exchange regime on the NMR timescale. Such be- haviour has been observed also for other small ligands bind- ing to CaM, such as W-7, J-8, and TFP (Craven et al., 1996; Osawa et al., 1998), and is attributed to secondary, weaker binding phenomena. nately towards or away from the position that would be occu- pied by the tr1c domain (cf. Fig. 6c–d and b). The two other idoxifene molecules also satisfy NOEs that are within reach of the ligand-binding site associated with the tr1c domain (left-hand side of Fig. 6b). When this calculation is repeated with full-length CaM (in the closed form derived from the CaM : M13 complex), the NOE restraints were satisﬁed us- ing four idoxifene molecules per domain, and the positions of the idoxifene molecules in the full-length complex followed the pattern observed in the binding to tr2c, where either the p-iodo-phenyl group or phenyl group occupies the mouth of the hydrophobic pocket and the p-phenoxy and ethyl groups lie along the hydrophobic surface in one of two directions (Fig. 6). 4 Discussion There is now a considerable body of data on CaM–ligand interactions, including solution and solid-state studies on the interactions with both peptides and small molecules (Meador et al., 1992; Ikura et al., 1992; Vandonselaar et al., 1994; Craven et al., 1996; Osawa et al., 1998; Harmat et al., 2000; Horvath et al., 2005; Kovesi et al., 2008). CaM–peptide inter- actions are normally satisfactorily described by a single set of coordinates for the ligand, representing an average structure, and this is reﬂected in the good agreement between the struc- ture of these complexes determined using X-ray and NMR spectroscopy methods (Ikura et al., 1991; Meador et al., 1992; Ikura et al., 1992; Maximciuc et al., 2006). However, this approach is less appropriate in the case of many CaM– small ligand interactions. For example, two DPD : CaM com- plex structures show substantial differences in the orientation of the ligand (Fig. S4b) and extent of CaM domain closure, while computation of the same complex suggests that CaM adopts a more globular structure in solution relative to the conformations observed in the X-ray structures (Harmat et al., 2000; Kovesi et al., 2008). Other anomalies also often appear in CaM–small ligand complexes. For example, while X-ray structures of TFP : CaM complexes have been solved with one, two, and four TFPs bound to CaM, NMR measure- ments and computation show conclusively that the principal binding mode in solution involves two TFP molecules bound with indistinguishable afﬁnity, one to the equivalent site in each domain (Vandonselaar et al., 1994; Craven et al., 1996). Both the above scenarios illustrate that CaM–small ligand complexes normally require a description including a combi- nation of stoichiometry, relative afﬁnity, exchange dynamics, and structure. 4.3 Ligand distribution 6), two where the p-iodo-phenyl group occu- pies the mouth of the hydrophobic pocket and the p-phenoxy group lies in the direction of either helix 1 or helix 7 and two where the phenyl group occupies the mouth of the hydropho- bic pocket and the p-phenoxy group has one or another of the above orientations. More precise positioning of idoxifene molecules is not readily obtained from the NOE-restrained docking calculations for several reasons. Firstly, the relative position and orientation of the tr1c and tr2c domains may dif- fer slightly from those in the CaM : M13 complex, and this is not readily determinable without multiple NOEs between the domains. Secondly, the conversion of NOE cross-peak intensities to distances is not unambiguous, as an accurate measurement of the relative populations of the conformers is not independently available. Thirdly, although four idox- ifene molecules per domain satisfy the NOE restraints, this is a minimum number of conformers rather than a uniquely determined number. g g The lack of substantial chemical shift perturbations in the phenylalanine rings in the vicinity of the hydrophobic pock- ets and the low number of NOEs observed between pheny- lalanine residues and idoxifene indicate that the ligand does not occupy the hydrophobic pockets fully (Fig. 4b). Only F68 and F141 signiﬁcantly change chemical shift on idoxifene binding, and these changes reﬂect the loss of α helical struc- ture in the M71–A73 and M144–T146 regions that is typi- cally seen on peptide binding (Ikura et al., 1991). A likely driver for this loss of α helix is that the unwinding allows the methyl group of, for example, T146 to join the hydropho- bic surface that interacts with the ligand. This interpretation is supported by the observation of strong NOEs between the hydrophobic groups of idoxifene and T146γ and the chem- ical shift changes for F141 and M144, which contact each other in the tr2c domain (and F68 and M71 in the equiva- lent positions in the tr1c domain). Strong NOEs and large chemical shift changes were observed for other hydropho- bic residues located over the surface around the hydrophobic pockets, such as I85 and A128 (Figs. 3 and 4). Overall, the distribution of intermolecular NOEs in the 2 : 1 idoxifene : CaM complex is unusual (Fig. 5) in that each of the substituents around the central double bond of idox- ifene (Fig. 4.3 Ligand distribution between sites must be signiﬁcantly faster than the dissoci- ation rate. This interpretation is supported by the failure of standard structure determination methods to converge to a single conformation for the complex and by NOE-restrained docking calculations being unable to satisfy the NOEs when only one idoxifene molecule per CaM domain was present in the calculation. When the upper bounds of the NOE re- straints were relaxed, the calculated structures delineated the two binding sites in the proteins, which matched well with the chemical shift perturbations observed on idoxifene bind- ing. However, the orientation of the idoxifene molecules was not deﬁned by these calculations, and the introduction of less conservative upper bounds required multiple orientations of idoxifene to be satisﬁed. The distribution of NOEs between CaM and idoxifene reso- nances reﬂects closely the distribution of the largest chemi- cal shift changes in protein side-chain resonances observed upon idoxifene binding. The contributing side chains are lo- cated around the hydrophobic pockets of both CaM domains, and no large chemical shift changes are observed for side chains in the tether or the rear of the domains (Fig. 4b). The strongest intermolecular NOEs were to the ε methyl groups of methionine residues, and these resonances undergo the largest chemical shift changes (Fig. 4c). Substantial upﬁeld chemical shift changes in these methionine resonances have been reported for numerous peptide and ligand complexes of CaM and attributed to ring current effects from the aromatic groups of the bound ligand. It is well established that the high abundance of methionines is essential in the biological function of CaM, the side chains of which can provide the conformational variability that enables the binding of a wide range of ligands (Osawa et al., 1998; Elshorst et al., 1999). Hence, the relatively even distribution of intermolecular NOEs (Fig. 5) is only readily explicable in terms of multi- ple binding orientations for the two idoxifene molecules and establishes that the complex cannot be described by a single structure. In the NOE-restrained docking, a minimum of four orientations of idoxifene per CaM domain were required to satisfy the NOEs. The positions and orientations of the four idoxifene molecules still varied between calculations but can be described approximately by a combination of four con- formers (Fig. 4.1 Binding afﬁnity and stoichiometry The principal binding mode of idoxifene to CaM has a stoi- chiometry of 2 : 1 (according to both ﬂuorescence and NMR measurements) and a dissociation constant of ∼300 nM, re- sulting from a diffusion-controlled on-rate of ∼108 M−1 s−1 https://doi.org/10.5194/mr-2-629-2021 Magn. Reson., 2, 629–642, 2021 638 L. Milanesi et al.: High-afﬁnity tamoxifen 4.3 Ligand distribution 1) appears to be close to the same atoms of CaM despite the size and relative rigidity of the propeller struc- ture in this part of the ligand. Moreover, the atoms on CaM that are close to each of these substituents of idoxifene are widely distributed across the hydrophobic surfaces of the do- mains (Fig. S3). The unusual distribution of NOEs is not the result of spin diffusion and therefore must reﬂect some posi- tional heterogeneity within the complex. As free and bound CaM is in the slow exchange regime on the NMR timescale and only a single set of resonances is observed for the 2 : 1 idoxifene : CaM complex, idoxifene must exchange between these positions while bound to CaM; i.e. the exchange rate 4.6 Role of charge Electrostatic interactions have previously been proposed to be important in the binding of small ligands to CaM; for example, W-7, J-8, DPD, TFP, and tamoxifen (Edwards et al., 1992; Vandonselaar et al., 1994; Craven et al., 1996; Os- awa et al., 1998; Harmat et al., 2000) all contain a ﬂexible chain connected to a basic nitrogen (Figs. 1 and Fig.4SC). A similar picture emerges in the idoxifene : CaM complex. The pyrrolidine ring appears to occupy multiple areas of the pro- tein surface located around the pockets where it can contact the glutamic acid residues; intermolecular NOEs from the pyrrolidine ring to the glutamate side chains of both domains indicate their close proximity. The two X-ray structures of CaM complexed with DPD, which has a propeller struc- ture similar to that of idoxifene, also show multiple orien- tations of the analogous basic side chain (Fig. S4b). In some idoxifene : CaM conformers, the pyrrolidine ring is oriented toward the ﬂexible tether that connects the two domains, bringing the pyrrolidine nitrogen close to E84 and E87. A similar orientation of the basic chain is observed in the solu- tion structure of the W-7 : CaM complex (Fig. S4c) and also proposed in the computational model of the CaM : idoxifene complex (Edwards et al., 1992). L. Milanesi et al.: High-afﬁnity tamoxifen L. Milanesi et al.: High-afﬁnity tamoxifen 639 (Takeuchi et al., 2014). There are two main differences be- tween the behaviour of idoxifene and previously determined CaM-small ligand complexes: the requirement for a broad ensemble rather than a well-positioned bound ligand and the degree of occupancy of the hydrophobic pockets. The re- quirement for multiple orientations of idoxifene to describe the complex was, however, proposed previously on the ba- sis of molecular modelling (Edwards et al., 1992). Although there are some differences in the conformations and orien- tations of the bound idoxifene molecules, in both this and an earlier computational model, the aromatic groups of the ligand are not located deep in the hydrophobic pockets, in contrast to the behaviour of the W-7, J-8, and DPD : CaM complexes (Fig. S4). Furthermore, based on the results of the molecular modelling and subsequent SARs of idoxifene ana- logues, it was suggested that the binding of idoxifene to CaM would produce a compact globular protein structure similar to that observed in the peptide and TFP complexes (Hardcas- tle et al., 1996). ible to maximize contacts with the acidic residues located over the surfaces of the two domains. 4.5 Role of iodine In the molecular modelling study, the increased CaM antag- onism of idoxifene relative to tamoxifen was attributed to the presence of a hydrophobic group such as iodine (Edwards et al., 1992). However, in subsequent SAR studies it was found that substitution of the iodine with a more hydrophobic group such as a butyl chain did not improve CaM antagonism (Hardcastle et al., 1995). Similarly, J-8, an analogue of W-7 with iodine in place of chlorine, was also found to be the most potent CaM antagonist in a series of derivatives bearing different halogens in the naphthalene rings (MacNeil et al., 1988). These data and the observation that in the J-8 : CaM complex the iodine is placed inside the hydrophobic pockets of the CaM domains led to the conclusion that the afﬁnity for CaM correlates with the van der Waals radius of the halo- gen (Craven et al., 1996). However, in the idoxifene : CaM complex, the iodine is not positioned deeply inside the hy- drophobic pockets (Fig. 6c–d) but contacts the sulfur atoms and methyl groups of M109 and M144 and the oxygen of E127. Similar interactions are observed between the chlo- rine in the ligand Kar-2 and M109 and E114 in the X-ray structure of the Kar2 : CaM complex (Horvath et al., 2005). In addition, Kar-2 shows a mode of binding similar to that of idoxifene: both ligands bind to the hydrophobic surface of the domains but not to residues located deep in the hy- drophobic pockets. This mode of binding is also observed for CaM in a complex with a peptide derived from the myristoy- lated alanine-rich C kinase substrate (MARCKS) and with a peptide derived from the HIV-1 matrix protein p17 (Ya- mauchi et al., 2003; Izumi et al., 2008). The X-ray structure of the CaM : MARCKS complex shows that the terminal ly- sine side chains of the peptide are disordered. The presence of multiple conformers is suggested to keep the peptide ﬂex- 4.4 Differences with previous models The positionally dynamic mode of binding of idoxifene to CaM contrasts with the common view that small ligands bind to proteins in a single orientation when the binding afﬁnity is sub-micromolar. However, this paradigm has been chal- lenged by a number of studies (Chattopadhyaya et al., 1992; Carroll et al., 2011; Hughes et al., 2012) that show dynamic binding modes not unrelated to the behaviour of idoxifene when bound to CaM. Indeed, the retention and redistribu- tion of ps–µs dynamic modes within protein residues in com- plexes involving a range of drugs have been shown to favour high-afﬁnity binding in a multidrug transcription repressor https://doi.org/10.5194/mr-2-629-2021 Magn. Reson., 2, 629–642, 2021 L. Milanesi et al.: High-afﬁnity tamoxifen 5 Conclusion The observation that four idoxifene molecules are sufﬁcient to satisfy the NOE restraints in the closed form of full-length CaM argues strongly that the 2 : 1 complex is an ensem- ble where each idoxifene molecule is predominantly in the vicinity of one of the two hydrophobic patches, ﬂuctuating between a conformational distribution. The CaM molecule is predominantly in the form where the N- and C-terminal domains are in close proximity, meaning that the idoxifene molecules are able to contact both domains simultaneously. In addition, these results show that the substantial occupation of the hydrophobic pocket observed with TFP, J-8, W-7, and DPD does not appear to be an essential component of high- afﬁnity binding. This is further supported by the observation that there are other CaM complexes with high-afﬁnity ligands that do not bind into the hydrophobic pockets. It also explains the results of many SARs on idoxifene: synthetic modiﬁca- tion of the aromatic rings has not led to substantial improve- ment in CaM antagonism. The model presented here opens up opportunities to design substantially higher-afﬁnity antag- onists of CaM activity. In addition to extending the repertoire of CaM antagonism, the dynamic mode of binding adds to the growing number of similar binding modes reported re- cently for small-molecule ligands. https://doi.org/10.5194/mr-2-629-2021 Magn. Reson., 2, 629–642, 2021 L. Milanesi et al.: High-afﬁnity tamoxifen 640 Data availability. The underlying research data can be accessed from the University of Shefﬁeld ORDA database (https://doi.org/ 10.15131/shef.data.15113511, Milanesi et al., 2021) Data availability. The underlying research data can be accessed from the University of Shefﬁeld ORDA database (https://doi.org/ 10.15131/shef.data.15113511, Milanesi et al., 2021) Data availability. The underlying research data can be accessed from the University of Shefﬁeld ORDA database (https://doi.org/ 10.15131/shef.data.15113511, Milanesi et al., 2021) Byer, S. J., Eckert, J. M., Brossier, N. M., Clodfelder-Miller, B. J., Turk, A. N., Carroll, A. J., Kappes, J. C., Zinn, K. R., Prasain, J. 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https://openalex.org/W4232880684	https://journal.trunojoyo.ac.id/pgpaudtrunojoyo/article/download/5366/3636	Indonesian	null	Kegiatan Senam Otak Anak Usia Dini	Jurnal PG-PAUD (Jurnal Pendidikan dan Pembelajaran Anak Usia Dini)/Jurnal PG-PAUD Trunojoyo	2,019	cc-by	2,853	KEGIATAN SENAM OTAK ANAK USIA DINI Yolanda Dwi Putri Rivda Yetti Pendidikan Guru Pendidikan Anak Usia Dini Fakultas Ilmu Pendidikan Universitas Negeri Padang Email: Yolandadwiputri27@gmail.com Keywords: Brain Gym, Early Childhood, learning children Abstrak: Senam otak memiliki banyak manfaat terutama untuk menyeimbangi otak kiri dan kanan, melalui kegiatan senam otak dapat menumbuhkan semangat dan belajar anak. Penelitian ini menjelaskan kegiatan senam otak yang dilaksanakan di Taman Kanak-Kanak & PlayGroup Kreatif Primagama Padang. Metode yang digunakan adalah kualitatif dengan pendekatan deskritif. Teknik pengumpulan data yaitu observasi, wawancara dan dokumentasi, sedangkan teknik analisis datanya adalah teknik triangulasi data. Hasil penelitian di Taman Kanak-Kanak & PlayGroup Kreatif Primagama Padang bahwa kegiatan senam otak sudah terlaksana dengan baik, dilihat dari perencanaan semester, mingguan dan harian serta evaluasi yang dilakukan setiap kegiatan senam otak. Pelaksanaan kegiatan senam otak ini banyak memberikan manfaat kepada anak. Kata Kunci: Senam Otak, Anak Usia Dini, Belajar Anak emosional (Mulyasa, 2012). Pendidikan PAUD dibutuhkan anak dalam mengupayakan perkembangan. Susanto menjelaskan bahwa pendidikan anak usia dini merupakan suatu bentuk pembelajaran untuk memfokuskan dasar pertumbuhan yang tepat dan tahap perkembangan yang dilalui anak usia dini (Susanto, 2017). Suryana menjelaskan tujuan dari pendidikan anak usia dini membantu peletak dasar perkembangan sikap, perilaku, pengakuan, keterampilan, dan kreativitas yang diperlukan oleh anak (Suryana, Pendidikan Anak Usia Dini Stimulasi & Aspek Perkembangan Anak, 2016). Berbagai media pembelajaran dan kegiatan yang dapat memberikan rangsangan terhadap perkembangan anak. Senam otak merupakan salah satu kegiatan yang dapat menstimulasi Received March 2019, Accepted April 2019, Published April 2019 Abstract: Brain exercise has many benefits, especially to balance the child’s brain and left, though brain gym activities can foster the spirit of learning children. This study describe brain gym activities carried out in the primagama padang creative kindergarden & playgroup. This study uses qualitative methods with a descriptive approach. Data collection techniques are observation, interview and documentation, while the data analysis technique is data triangulation technique. The results of research in the primagama padang creative kindergarden & playgroup that brain gym activities have been carried out well, seen from semester planning, weekly and daily planning and evaluations carries out in every brain gymnastic activity. The implementation of brain gym activities provides many benefits to children. Keywords: Brain Gym, Early Childhood, learning children Jurnal PG-PAUD Trunojoyo : Jurnal Pendidikan dan Pembelajaran Anak Usia Dini, Volume 6, Nomor 1, April 2019, hal 12 - 17 13 13 terutama pada perkembangan kognitif, motorik, dan sosial emosional anak. Senam otak (Brain Gym) dipopulerkan oleh Paul E. Dennison, Dr Phill dengan istrinya Gail E. Dennison yang merupakan pelopor pendidikan di Amerika Serikat dalam penerapan senam otak. observasi tersebut peneliti tertarik untuk meneliti kegiatan senam otak di TK & PlayGroup Kreatif Primagama Padang. METODE Pendekatan kualitatif dengan metode deskritif yang digunakan pada penelitian ini. Subjek penelitian ini adalah kelas TK B yang berjumlah 17 anak. Instrumen penelitian ini menggunakan format observasi, format wawancara dan format dokumentasi dengan informan penelitian yaitu kepala sekolah dan tiga guru kelas sebagai bukti bahwa peneliti telah melakukan pengamatan tentang kegiatan senam otak di Taman Kanak-kanak & Play Group Kreatif Primagama Padang. Dennison & Dennison (Priambodo, 2017) senam otak dapat mengimbangi antara otak yang berkaitan dengan fisik dan psikis. Kegiatan senam otak memiliki peran penting dalam pendidikan anak. Beberapa alasan pentingnya melaksanakan senam otak antara lain, sulit belajar nerusaha terlalu keras, sehingga membuat stress pada anak, kinerja integrasi otak melambat, dan membuat bagian otak tertentu kurang berfungsi dengan baik, bagian belakang otak menerima informasi sulit diekspresikan, membuat kurang fokus dan stress yang mengakibatkan semangat belajar berkurang, dan kurang belajar dan berusaha membuat prestasi menurun dan perasaan tidak berhasil semakin bertambah, akibatnya sulit untuk keluar dari lingkungan negatif tersebut (Sukri & Sukri, 2013). Melalui observasi, wawancara dan dokumentasi pada teknik pengumpulan data penelitian. Untuk pengumpulan data, reduksi data, penyajian data, dan verifikasi data yang digunakann dalam teknik analisis data. Sedangkan teknik keabsahan data yaitu triangulasi data. Triangulasi adalah teknik penyajian data dengan cara data yang disesuaikan dengan informan yang sama menggunakan teknik yang berbeda (Sugiyono, 2017). Dalam menguji kebenaran data mengenai pelaksanaan kegiatan senam otak di Taman Kanak-kanak & PlayGroup Kreatif Primagama Padang, data yang sudah didapat dari lapangan disesuaikan dengan wawancara dan dokumentasi bersama kepala sekolah dan guru. Berdasarkan penjabaran di atas serta awal pengamatan yang peneliti lakukan di Taman Kanak-kanak & Playgroup kreatif primagama Padang, peneliti menemukan keunikan dari Taman Kanak-kanak yaitu memiliki rancangan kegiatan program- program sendiri yang membedakan dari Taman Kanak-kanak pada umumnya seperti, berenang, fiel trip berdasa rkan tema, dan pelaksanaan kegiatan senam otak yang sudah terancang dengan baik. Peneliti memfokuskan penelitian pada senam otak karena Taman Kanak-kanak ini sudah menerapkan kegiatan senam otak sejak tahun 2008. Pelaksanaan kegiatan senam otak ini sudah terjadwal dalam perencanaan yaitu dilaksnakan pada hari selasa dan kami yang tertera dalam program rancangan pembelajaran harian (RPPH). Berdasarkan PAUD Tahapan awal pada perkembangan manusia berada pada masa dini, dimana semua aspek berkembang sangaat baik. Maka di tahap ini dibutuhkan stimulasi yang tepat. Pertumbuhan dan perkembangan manusia sangat penting yaitu pada masa usia anak (Suryana, Pendidikan Anak Usia Dini, 2013). Usia dini merupakan masa awal dalam proses perrkembangan yang pesat untuk kehidupan berikutnya. Anak memiliki karakteristik sendiri yang membeda antara satu dengan yang lainnya (Yulsyofriend, 2013). Melalui pendidikan dapat menyonsong potensi yang ada pada diri anak. Mulyasa menjelaskan anak usia dini mempunyai karakter tersendiri seperti, fisik, psikis, sosial, moral spiritual maupun 12 Jurnal PG-PAUD Trunojoyo : Jurnal Pendidikan dan Pembelajaran Anak Usia Dini, Volume 6, Nomor 1, April 2019, hal 12 - 17 13 1. HASIL Dalam penelitian tentang kegiatan senam otak di Taman Kanak-kanak & PlayGroup Kreatif Primagama Padang dapat disimpulkan bahwa kegiatan tersebut sudah terlaksana dengan baik yang disesuaikan dengan kemampuan anak. kegiatan senam otak terdapat 26 gerakan, 14 Jurnal PG-PAUD Trunojoyo : Jurnal Pendidikan dan Pembelajaran Anak Usia Dini, Volume 6, Nomor 1, April 2019, hal 12 - 17 14 Pelaksanaan kegiatan brain gym pertama, tahapan-tahapan kegiatan senam otak di Taman Kanak-kanak & PlayGroup Kreatif Primagama Padang. Berdasarkan observasi, wawancara dan dokumentasi kegiatan senam otak diawali dengan minum air putih, kemudian anak diminta untuk mangambil barisan dan ambil jarak agar tidak berdempetan dengan teman yang lain pada saat melakukan kegiatan senam otak. kedua pemanasan ringan dengan gerakan- gerakan sederhana, seperti pernafasan perut, geleng-geleng kepala, dan berdiri satu kaki untuk mengimbangi tubuh. Sebelum melakukan kegiatan senam otak guru menjelaskan kepada anak tujuan dilakukan senam otak tersebut yang membedakan dari kegiatan senam lainnya serta manfaat dan pentingnya untuk melakukan kegiatan senam otak (Brain Gym) tersebut. namun guru memberikan pada anak sebanyak 15 gerakan, karena disesuaikan dengan kemampuan anak, Gerakan-gerakan tersebut mulai dari yang sederhana sampai tersulit. Adapun kegiatan senam otak dilaksanakan yaitu perencanan, pelaksanaan dengan tahapan-tahapan senam otak, gerakan-gerakan senam otak, manfaat kegiatan senam otak dan evaluasi. Berdasarkan observasi, wawancara dan dokumentasi bahwa perencanaan kegiatan senam otak di Taman Kanak- kanak & Playgroup Kreatif Primagama Padang bahwa sudah memiliki perencanaan yang tertera dalam kurikulum, program semester, mingguan dan harian. Dalam perencanaan tersebut sudah muncul indikator-indikator pencapaian senam otak (Brain Gym) oleh anak. Jurnal PG-PAUD Trunojoyo : Jurnal Pendidikan dan Pembelajaran Anak Usia Dini, Volume 6, Nomor 1, April 2019, hal 12 - 17 15 15 gerakan-gerakan lainnya sudah disesuaikan dengan kemampuan anak. Belikut pembahasan dari hasil penelitian di atas : Perencanaan, berdasarkan hasil temuan yang di dapatkan melalui observasi, wawancara dan dokumentasi yang dilakukan peneliti serta berdasrakan analisis data yang peneliti lakukan tentang perencanaan kegiatan senam otak di Taman Kanak-kanak & Playgroup Kreatif Primagama Padang dimulai dari program tahunan, proses perencanaan kegiatan semester, mingguan dan harian. Rancangan kegiatan harian terbagi menjadi Opening, Focus Theme, dan Closing. Pembuatan perencanaan kegiatan senam otak sesuai dengan kebutuhan anak. kegiatan yang dilaksanakan disesuaikan berdasrkan jadwal kegiatan yang dirancang. Proses pengambilan keputusan pada hasil akhir yang tersusunnya dokumen yang berisi tentang hal tersebut, kemudian dijadikan sebagai acuan dan pedoman dalam melaksanakan proses pembelajaran. Ketiga, manfaat kegiatan senam otak untuk anak. berdasarkan observasi, wawancara dan dokumentasi, manfaat senam otak terutama pada anak sangat banyak. Pada pembelajaran anak lebih tenang belajar, lebih semangat, meningkatkan konsetrasi dalam belajar dan meningkatkan daya ingat anak, hal ini dapat dilihat ketika selesai belajar dan melakukan tanya jawab tentang pembelajaran yang sudah dipelajari hari ini, jika anak bisa menjawab angat tangan. Dan hampir semua anak mengankat tangan. Kemudian evaluasi, berdasarkan hasil temuan yang di dapatkan dari hasil observasi, wawancara dan dokumentasi oleh peneliti serta berdasarkan analisis data yang peneliti lakukan tentang perencanaan kegiatan senam otak (Brain Gym) di Taman Kanak-kanak & PlayGroup Kreatif Primagama Padang yaitu kurikulum, proses perencanaan kegiatan untuk semester (Semester Plan) dan rencana pembelajaran mingguan (Weekly Plan/RPPM) dan rencana pelaksanan pembelajaran harian (Lesson Plan/RPPH). Rancangan kegiatan harian dibagi menjadi Opening, Fokus Theme dan Closing. Pembuatan perencanaan kegiatan senam otak (Brain Gym) sesuai dengan kebutuhan anak. Kegiatan yang dilaksanakan berdasarkan jadwal kegiatan yang sudah dirancang. Tersusunnya dokumen yang dijadikan sebagai acuan dan pedoman dalam melaksnakan proses pembelajaran merupakan hasil akhir dari proses pengambilan keputusan tersebut. Pelaksanaan kegiatan senam otak pertama, tahapan-tahapan kegiatan senam otak. berdasarakan hasil temuan di lapangan, wawancara dan dokumentasi yang dilakukan peneliti serta analisis data yang peneliti lakukan diperoleh hasil bahwa kegiatan senam otak telah sesuai dnegan perencanaan yang telah dirancang oleh guru. Adapun sebelum melakukan kegiatan senam otak terdapat tahapan-tahapan dilakukan seperti secukupnya minum air putih, yang bertujuan untuk menetralisirkan dan memberikan oksigen ke otak sehingga memudahkan dalam melaksanakan kegiatan senam otak. Selanjutnya pemanasan sederhana, hal ini bertujuan untuk menghindari cedera pada otot-otot. Pemanasan sederhana yang baik untuk anak, seperti pernafasan perut sebanyak delapan kali dilanjutkan gerakan-gerakan sederhana. Tabel 1. Uraian Kegiatan di TK Kelompok B Hari Selasa dan Kamis (Mengenal Literasi dan Brain Gym) Jurnal PG-PAUD Trunojoyo : Jurnal Pendidikan dan Pembelajaran Anak Usia Dini, Volume 6, Nomor 1, April 2019, hal 12 - 17 15 Tabel 1. Uraian Kegiatan di TK Kelompok B Kedua, gerakan-gerakan senam otak (Brain Gym) memiliki gerakan sederhana dapat merangsang kemampuan otak kanan dan kiri anak. Observasi yang dilakukan di TK & PlayGroup Kreatif Primagama dalam kegiatan senam otak terdapat gerakan-gerakan yang termasuk dalam dimensi lateral pertama, gerakan silang dimana gerakannya untuk menggerakan organ tubuh kiri dan kanan secara bersamaan. Kedua, gajah dimana gerakan yang menyerupai belalai gajah dengan menyilangkan tangan kanan dan kiri, satu tangan memegang hidung dan satunya lagi memegang telinga dan dilakukan secara bergantian antara kiri dan kanan. Selanjutnya gerakan-gerakan yang termasuk dalam dimensi pemfokusan pertama lambaian tangan dengan tangan keatas berada pada samping telinga kemudian tangan kedua di siku bawah yang berguna untuk meregangkan ketegangan di otot pundak, mengontrol koordinasi mata dan tangan. Selanjutnya dimensi pemusatan yaitu, minum air putih yang bertujuan mempermudah pengaliran energy dari otak ke seluruh tubuh. pompa betis, luncuran gravitasi, dan pasang kuda-kuda. Adapun A. Senin, Selasa, Rabu, Kamis 1. Greeting (Penyambutan Siswa) (15 Menit) 2. Iqro/mengenal Literasi (45 Menit) 3. Opening (30 Menit)  Morning Carpet  Cirle Time  Brain Gym 4. Focus Theme 1 (45 Menit) 5. Take a Break (30 Menit) 6. Snack Time (30 Menit) 7. Focus Theme 2 (45 Menit) 8. Review and Closing (15 Menit) B. Jumat 1. Greeting (Penyambutan Siswa) (15 Menit) 2. Iqro/Mengenal Literasi (45 Menit) 3. Opening (30 Menit)  Morning Carpet  Circle Time 4. Religious Activities (45 Menit) 5. Take a Break (15 Menit) 6. Snack Time (15 Menit) 7. Review and Closing (15 Menit) 8. Talents Class (45 Menit) Keterangan 1. Hari Senin dan Rabu (Iqro) 2. Hari Selasa dan Kamis (Mengenal Literasi dan Brain Gym) A. Senin, Selasa, Rabu, Kamis 1. Greeting (Penyambutan Siswa) (15 Menit) 2. Iqro/mengenal Literasi (45 Menit) 3. Opening (30 Menit)  Morning Carpet  Cirle Time  Brain Gym 4. Focus Theme 1 (45 Menit) 5. Take a Break (30 Menit) 6. Snack Time (30 Menit) 7. Focus Theme 2 (45 Menit) 8. Review and Closing (15 Menit) B. Jumat 1. Greeting (Penyambutan Siswa) (15 Menit) 2. Iqro/Mengenal Literasi (45 Menit) 3. Opening (30 Menit)  Morning Carpet  Circle Time 4. Religious Activities (45 Menit) 5. Take a Break (15 Menit) 6. Snack Time (15 Menit) 7. Review and Closing (15 Menit) 8. Talents Class (45 Menit) Keterangan 1. Hari Senin dan Rabu (Iqro) 2. 16 Jurnal PG-PAUD Trunojoyo : Jurnal Pendidikan dan Pembelajaran Anak Usia Dini, Volume 6, Nomor 1, April 2019, hal 12 - 17 16 Kedua, gerakan senam otak. Berdasarkan temuan di Taman Kanak- kanak & Plagroup Kreatif Primagama Padang bahwa gerakan senam otak yang dilakukan disesuaikan dengan kemampuan anak. Adapun gerakan-gerakannya sebanyak 15 gerakan yang diiringi musik. Terdapat tiga dimensi dalam gerakan senam otak yaitu dimensi lateral, dimensi pemfokusan dan dimensi pemusatan. Menurut Paul Dennison “senam otak dilakukan melalui tida dimensi, yakni dimensi lateral, dimensi pemusatan dan dimensi pemfokusan”. Gerakan-gerakan lebih berpusat pada tangan, seperti jari kanan gerakan gajah dimana tangan kiri memegang telinga dan tangan kanan memegang hidung, bayangankan tangan menjadi belalai gajah, ikuti 8 tidur yang terletak agak jauh yang bertujuan untuk meningkatkan penglihatan, pendengaran dan gerakan seluruh tubuh, lambaian tangan dimana letakkan kuda dibawah siku lewat belakang kepala. Leher di putar dimana bahu dinaikkan, tundukan kepala ke depan dan putar dari satu sisi ke sisi lainnya. Putaran kepala, gelengkan kepala, putaran tangan. Semua gerakan termasuk dalam tiga dimensi, namun selebihnya gerakan di fariasikan oleh guru sesuai dengan iringan musik. Dalam kegiatan senam otak diiringi musik sehingga anak lebih semangat dalam kegiatan senam otak. Disamping itu anak tidak berebutan dalam mengambil media pembelajaran, dan sudah bisa mempratekan sifat antri dengan baik tanpa harus mendahulukan satu dengan yang lain. Sejalan dengan pendapat Zulaini menjelaskan manfaat senam otak seperti meningkatkan konsentrasi, mengurangi stress, meningkatkan daya ingat, dapat berfikir lebih cepat, pelajar dapat menangkap pelajaran dengan baik (Zulaini, 2016). Hal ini senada dengan Dennison (Priambodo, 2017) menjelaskan bahwa ada beberapa manfaat senam otak yaitu memberikan stumulus pada kegiatan belajar dan efektif dalam mengalami hambatan belajar, dan memudahkan siswa untuk berfikir, merefleksikan suatu pekerjaan/tugas atau karya. Disamping itu anak tidak berebutan dalam mengambil media pembelajaran, dan sudah bisa mempratekan sifat antri dengan baik tanpa harus mendahulukan satu dengan yang lain. Sejalan dengan pendapat Zulaini menjelaskan manfaat senam otak seperti meningkatkan konsentrasi, mengurangi stress, meningkatkan daya ingat, dapat berfikir lebih cepat, pelajar dapat menangkap pelajaran dengan baik (Zulaini, 2016). Hal ini senada dengan Dennison (Priambodo, 2017) menjelaskan bahwa ada beberapa manfaat senam otak yaitu memberikan stumulus pada kegiatan belajar dan efektif dalam mengalami hambatan belajar, dan memudahkan siswa untuk berfikir, merefleksikan suatu pekerjaan/tugas atau karya. Evaluasi kegiatan senam otak di Taman Kanak-kanak & Playgroup Kreatif Primagama berdasarkan hasil temuan penelitian, peneliti melihat guru mengevaluasi kegiatan melalui observasi yaitu dengan melihat proses anak melaksanakan kegiatan senam otak. Evaluasi kegiatan disesuaikan dengan perencanaan yang sudah dikembangkan. Jurnal PG-PAUD Trunojoyo : Jurnal Pendidikan dan Pembelajaran Anak Usia Dini, Volume 6, Nomor 1, April 2019, hal 12 - 17 15 Hal ini sesuai dengan pendapat Sulis Diana dkk dalam melakukan kegiatan senam otak ada beberapa yang perlu dilakukan yaitu, minum air putih dan pernafasan perut sebanyak 2-8 kali (Diana & dkk, 2017). 2. PEMBAHASAN Hasil penelitian tentang kegiatan senam otak anak usia dini di Taman Kanak- kanak & Playgroup Kreatif Primagama Padang akan dibahas berdasarkan teori, sehinggaa akan terlihat keterkaitan antara teori dengan fakta yang ada dilapangan. 2. PEMBAHASAN Hasil penelitian tentang kegiatan senam otak anak usia dini di Taman Kanak- kanak & Playgroup Kreatif Primagama Padang akan dibahas berdasarkan teori, sehinggaa akan terlihat keterkaitan antara teori dengan fakta yang ada dilapangan. 16 Jurnal PG-PAUD Trunojoyo : Jurnal Pendidikan dan Pembelajaran Anak Usia Dini, Volume 6, Nomor 1, April 2019, hal 12 - 17 SIMPULAN Berdasarkan hasil penelitian tentang kegiatan senan otak anak usia dini di Taman Kanak-kanak & Playgroup Kreatif Primagama Padang dapat disimpulkan bahwa kegiatan senam otak dilaksanakan dengan baik. Kegiatan senam otak melalui perencanaan yang dirancang oleh guru sehingga terjadwal dengan baik. Senam otak dilakukan dalam bentuk gerakan-gerakan sederhana yang dapat merangsang perkembangan otak, meningkatkan konsetrasi dalam belajar. Evaluasi kegiatan senam otak yang dilakukan guru melalui observasi yaitu melihat proses anak dalam melakukan kegiatan senam otak. Ketiga,manfaat senam otak. berdasarkan temuan di Taman Kanak-kanak & Playgroup Kreatif Primagam Padang bahwa manfaat kegiatan senam otak sudah terlihat pada diri anak, dibuktikan dalam proses pembelajaran anak dapat menjawab dan mengulangi kembali pembelajaran yang dipelajari hampir semua anak dapat menjawab pertanyaan yang di ajukan oleh guru. Kusuma menjelaskan dengan sering melakukan senam otak dapat meningkatkan pertumbuhan sel-sel otak, menyimpan memori dan meningkatkan fokus dalam belajar. Dilihat dari perkembangan emosional, dimana dalam proses suasana belajar anak terlihat tenang dan senang. Jurnal PG-PAUD Trunojoyo : Jurnal Pendidikan dan Pembelajaran Anak Usia Dini, Volume 6, Nomor 1, April 2019, hal 12 - 17 17 17 17 DAFTAR PUSTAKA Diana, S., & dkk. (2017). Brain Gym Stimulasi Perkembangan Anak Paud I. Surakarta: CV Kekata Group. Mulyasa. (2012). Manajemen PAUD. Bandung: Remaja Rosdakarya. Priambodo, M. (2017). Brain Gym. Retrieved from http://www.brain%gym/78032541.pdf Sugiyono. (2017). Metode Penelitian Pendidikan Pendekatan Kuantitatif, Kualitatif dan R&D. Bandung: Alfabeta. Sukri, A., & Sukri, P. (2013). Meningkatkan Hasil Belajar Siswa Melalui Brain Gym. Jurnal Edukasi Matematika dan Sains. Suryana, D. (2013). Pendidikan Anak Usia Dini. Padang: UNP Press. Suryana, D. (2016). Pendidikan Anak Usia Dini Stimulasi & Aspek Perkembangan Anak. Jakarta: PT Raja Grafindo Persada. Susanto, A. (2017). Pendidikan Anak Usia Dini. Jakarta: PT Bumi Aksara. Yulsyofriend. (2013). Permainan Membaca dan Menulis Anak Usia Dini. Padang: Sukabina Press. Zulaini. (2016). Manfaat Senam Otak. Retrieved from http://www.6140-12223-1-PB.pdf
https://openalex.org/W4302283372	https://bmcpalliatcare.biomedcentral.com/counter/pdf/10.1186/s12904-022-01062-8	English	null	Gender differences in the intention to withhold life-sustaining treatments involving severe dementia for self and on behalf of parent or spouse	BMC palliative care	2,022	cc-by	9,692	Abstract Background Few studies have explored gender differences in the attitudes toward advanced care planning and the intention to withhold life-sustaining treatments (LSTs) involving severe dementia in Asian countries. We examined gender differences in the attitude toward the Patient Autonomy Act (PAA) in Taiwan and how the gender differences in these attitudes affect the intention to withhold LSTs for severe dementia. We also investigated self–other differences in the intention to withhold LSTs between genders. Methods Between March and October 2019, a structured questionnaire was distributed to hospitalized patients’ family members through face-to-face contact in an academic medical center. Exploratory factor analysis and independent and paired-sample t-tests were used to describe gender differences. Mediation analyses controlled for age, marital status, and education level were conducted to examine whether the attitude toward the PAA mediates the gender effect on the intention to withhold LSTs for severe dementia. Results Eighty respondents filled out the questionnaire. Exploratory factor analysis of the attitude toward the PAA revealed three key domains: regarding the PAA as (1) promoting a sense of abandonment, (2) supporting patient autonomy, and (3) contributing to the collective good. Relative to the men, the women had lower average scores for promoting a sense of abandonment (7.48 vs. 8.94, p = 0.030), higher scores for supporting patient autonomy (8.74 vs. 7.94, p = 0.006), and higher scores for contributing to the collective good (8.64 vs. 7.47, p = 0.001). Compared with the women, the men were less likely to withhold LSTs for severe dementia (15.84 vs. 18.88, p = 0.01). Mediation analysis revealed that the attitude toward the PAA fully mediated the gender differences in the intention to withhold LSTs for severe dementia. Both men and women were more likely to withhold LSTs for themselves than for their parents. Compared with the women, the men were more likely to withhold resuscitation for themselves than for their parents (p = 0.05). Women were more likely to agree to enteral tube feeding and a tracheotomy for their husbands than for themselves; men made consistent decisions for themselves and their wives in those LST scenarios. Correspondence: Duan-Rung Chen duan@ntu.edu.tw Full list of author information is available at the end of the article © The Author(s) 2022. Gender differences in the intention to withhold life-sustaining treatments involving severe dementia for self and on behalf of parent or spouse Duan-Rung Chen1, Jih-Shuin Jerng2, Daniel Fu-Chang Tsai3 and Yuchi Young4 Duan-Rung Chen1, Jih-Shuin Jerng2, Daniel Fu-Chang Tsai3 and Yuchi Young4 © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. 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://creativecommons.org/licenses/by/4.0/. BMC Palliative Care BMC Palliative Care Chen et al. BMC Palliative Care (2022) 21:171 https://doi.org/10.1186/s12904-022-01062-8 Open Access Introductionh The treatments people choose near the end of life may differ from those they receive [1]. End-of-life (EOL) deci sions are commonly arranged through advance directives (ADs), legal documents that outline treatment prefer ences, or by designating power of attorney to ensure that patients receive care consistent with their wishes when incapacitated. Studies highlight the benefits of ADs, with patients in the United States having ADs being less likely to die in the hospital [1–3] and more likely to receive care that is consistent with their preferences [2], and have surrogates who report more effective communication with physicians near the end of life [3]. However, many patients may have limited knowledge of future treat ments, and thus they may be reluctant to complete an AD [4]. The absence of an AD may lead to the application of non-beneficial therapies that can increase suffering for patients and their families.h The Patient Autonomy Act (PAA), which was passed in Taiwan in 2019 [5], is the first relevant legislation in Asia to protect a patient’s right to natural death; it allows individuals aged ≥ 20 years to make decisions through advanced care planning, and complete a written AD to decline medical treatments in specific clinical scenarios in case of incapacity [5]. With the PAA, capable adults can provide written EOL care instructions and com plete a durable power of attorney for health care. The person appointed in this document will make treatment decisions for the patent in case of incapacitation. Such requests have long been subject to legal protection in Western countries, such as that provided by the Patient Self-Determination Act passed by the US Congress in 1990 and the Mental Capacity Act passed in the United Kingdom in 2005 [6, 7]. Despite the rights afforded by the Patient Self-Determination Act, the rate of advanced care planning remains low [8]. A recent systematic review examining studies published between 2011 and 2016 indicated that only approximately 36.7% of US adults have ADs [9]. In the UK, the estimated propor tion of adults with ADs is about 4% in England and just 2% in Wales, while in Germany, approximately 10% of the general population [10]. In Taiwan, less than 0.2% of the adult population (35,545 out of roughly 19.39 million adults) had completed an AD by the end of 2021 [11]. Page 2 of 12 (2022) 21:171 Chen et al. BMC Palliative Care Conclusion Gender influences the attitude toward advanced care planning and consequently affects the intention to withhold LSTs, indicating that there may be a difference in how men and women perceive EOL decision-making for severe dementia in Taiwan. Further studies are warranted. Keywords Gender differences, Life-sustainment treatments, The patient autonomy act Cultural norms influence attitudes regarding patient autonomy in decisions about EOL care [15]. Individuals with positive attitudes toward patient autonomy exhibit greater willingness to sign an AD [16]; moreover, will ingness to sign an AD is positively associated with older age, higher educational attainment [17], white race [3], employment, knowledge of hospice and palliative care [18], comprehensions of the illness condition [18, 19], and life satisfaction [20]. Several studies also point to sex-based gender differences in the preferences for life-sustaining treatments. For example, a qualitative study reported gender differences in attitudes concern ing advance care planning, with men fearing harm from health treatments and hesitant to disclose EOL wishes to physicians. By contrast, women anticipated the ben efits of ADs and believed that having one could pre vent unwanted mechanical life support [21]. A study in the United States, which tracked 301 advanced cancer patients, also found that male patients are more likely to receive life-prolonging medical treatments [22]. A muti- hospital survey of 2,329 patients with advanced cancer in Taiwan found that compared with men, women were less likely to regard prolonging life as a medical goal of end-stage cancer care and were more inclined not to receive intubation and ventilator therapy [23]. Another study in the United States indicated that the decisions to withhold or withdraw potentially life-prolonging treat ments are more often made in women (28.0% vs. 22.8%, P = 0.003) [24]. Men with advanced cancers are more likely than women to receive aggressive, non-beneficial ICU care near death [25]. A recent systematic review of the literature reveals that women are more likely to with draw or withhold life-prolonging treatments than men [26]. Although empirical findings point to sex-based dif ferences in the preferences for life-sustaining treatments, few studies discuss the underlying socialized gender roles that may affect how men and women perceive EOL decision-making. Introductionh In Chinese society, sons typically make surrogate decisions, although female family mem bers are more likely to care for the patient [35, 36].h After the respondents read the description, they indi cated their preferences for the following five medical interventions for prolonging life: (1) receiving enteral tube feeding if severe dementia hinders eating; (2) receiv ing dialysis for kidney failure; (3) being intubated on a ventilator if severe dementia makes breathing impos sible; (4) undergoing a tracheotomy following one month of intubation and if survival appears unlikely without a ventilator; and (5) undergoing CPR if a cardiac arrest because of end-stage dementia [45]. This study assumes that gender is socially constructed roles, behaviors, and attributes that a society considers appropriate for men and women. In Taiwan, although the gender gap has narrowed considerably in terms of educa tional attainment [37], the gender difference in the labor market participation rate persists (51.5% for women and 67% for men, Directorate-General of Budget, Account ing and Statistics [DGBAS], 2021). Wives, daughters, and daughters-in-law represent 63% of the caretakers of dis abled or ill household members [38, 39]. A recent study revealed different disability trends for Taiwanese men and women aged ≥ 50 years, with women progressing 18% more rapidly than men toward more substantial disabil ity after its initial onset; older age resulted in a 1.2 times faster rate of change in disability for women than for men (p < 0.001) [40]. Women may experience higher fatigue levels than men at the end of life [41]. Women were also less likely to regard prolonging life as a medical goal of end-stage cancer care and were more inclined not to receive intubation and ventilator therapy [23]. Therefore, Taiwanese women may be expected to follow the gender role to be family-centered and probably internalize the sense of sacrifice to reduce family burden [42]. When fac ing the choices about life-sustainment treatments at the end of life, they may be more likely to withhold prolong ing life treatments than men. Scenarios involving severe dementia were selected because providing a realistic prognosis for patients with dementia is challenging [46]. Realistic forecasts are essen tial for patients and caregivers to prepare for subsequent difficult situations. Introductionh Life-sustaining treatments (LSTs) are widely used for patients approaching the end of life [12, 13], highlight ing the importance of understanding the barriers to AD completion [14]. Despite their benefits, ADs might not be available for most hospitalized patients [2]; family members serve as surrogate decision-makers in such cases. Growing evi dence suggests that the decisions of surrogate decision- makers often differ from those of patients [27], probably because of psychological distance [27] or social distance [28] between the two parties. Research has revealed that surrogate decisions regarding EOL care might not ben efit the patient [29], whose wishes may be compromised in such situations [30]. These decisions might differ from Chen et al. BMC Palliative Care (2022) 21:171 Page 3 of 12 (2022) 21:171 Chen et al. BMC Palliative Care those the surrogate might make for themselves [31]. Fam ily members as surrogates commonly regard their role as being the patient’s voice, an advocate for the patient, an advocate for other family members, or an advocate for themselves [32]. description of severe end-stage dementia—based on the Functional Assessment Staging Test [43, 44]—was as follows: “When you have severe dementia, you do not remember many things, forget many people, can not express your needs, or understand others, and have a deteriorating mental state. You need someone to take care of your daily tasks for an extended period; for exam ple, you need help getting dressed, eating, and taking a bath. Gradually, you become incontinent, your mobility diminishes, and you eventually need to be in a wheelchair or stay in bed for long periods.” Surrogate decisions may also be affected by socialized gender roles. A study reported that surrogate choices in the United States are most likely to be made by daughters (58.9%) [33]. In Japan, female surrogates are more likely to change their preference from cardiopulmonary resus citation (CPR) to do-not-resuscitate (DNR) orders than male surrogates [34]. Introductionh Several reasons are listed: (1) doctors and family members do not regard advanced dementia as a terminal disease; (2) excessive focus on cognitive and physical impairments can limit palliative care options; (3) patients with dementia often present distinct illness tra jectories over a long period between diagnosis and death, often without an acute deterioration [47]. Before the introduction of the PAA, most doctors in Taiwan favored active treatment of patients with dementia to ensure that the patient’s medical needs were met and to avoid legal repercussions [48]. Study sample Thi d This study was conducted at the National Taiwan Uni versity Hospital in Taipei, Taiwan, from March to Octo ber 2019. The data were collected through a structured questionnaire through face-to-face contact with the fam ily members of patients treated in this hospital’s intensive care unit (ICU) and general internal medicine and surgi cal wards. All the participants provided written informed consent before participating in this study. This study was performed by the Declaration of Helsinki and approved by the National Taiwan University Research Ethics Com mittee (No. 201811035RINB). This study aims to examine gender differences in the attitude toward the Patient Autonomy Act (PAA) in Tai wan and how the gender differences in these attitudes affect the intention to withhold LSTs for severe dementia. We also hypothesize that gender differences might exist in self–other differences in the intention to withhold LSTs involving severe dementia. Study design The inclusion criteria for the interview participants were as follows: (a) related by blood or law to an in- patient who had been hospitalized for 48 h or longer, (b) visited the patient more than twice during the patient’s hospitalization (or served as the primary caregiver during We conducted a quantitative survey to investigate whether men and women differ in their intention to withhold LSTs in cases involving severe dementia for themselves and on behalf of their family members. The Page 4 of 12 (2022) 21:171 Care (2022) 21:171 Chen et al. BMC Palliative Care Fig. 1 Scheme of a simple mediation model The mediation models in Hayes’ PROCESS Macro (2022) were used to examine the direct, indirect, and total effect of gender on the intention to withhold LSTs for severe dementia (also see Fig. 1). Bootstrapping (5000 resamples) was used to estimate the 95% confidence interval (CI) for the aforementioned effects. All the sta tistical analyses were performed using SPSS 19.0 (SPSS, Chicago, IL, USA). the hospitalization), and (c) aged ≥ 20 years and able to answer interview questions. Potential participants were excluded if they could not speak or read Chinese fluently or could not be present at the hospital for the face-to-face interview. We assumed that recruiting family members of hospitalized patients would be more likely to reflect on matters concerning EOL decisions. the hospitalization), and (c) aged ≥ 20 years and able to answer interview questions. Potential participants were excluded if they could not speak or read Chinese fluently or could not be present at the hospital for the face-to-face interview. We assumed that recruiting family members of hospitalized patients would be more likely to reflect on matters concerning EOL decisions. To determine the necessary number of respondents, we used the Gpower program [49]. GPower [50]was designed as a general stand-alone power analysis pro gram for statistical tests in social and behavioral research. For studies with two to five predictors, the required sam ple size ranges from 68 to 92, for an effect size of 0.15, an error probability of 0.05, and a power (1 − B error prob ability) of 0.8. Therefore, we adopted a sample size of 100. Analysis plan First, univariate and bivariate analyses were conducted to examine the gender differences in variables of inter est in this study. Second, independent and paired-sample t-tests (for self–other differences) were used to describe the difference between men and women. Third, explor atory factor analysis and reliability tests were used to examine the respondents’ attitudes toward the PAA. Lastly, we investigated whether men and women differ in their attitudes toward the PAA and whether the differ ences in the attitudes affect their intention to withhold LSTs for severe dementia by using mediation models. Study design For the mediation analysis, we specifically examined (1) whether men and women differ in the intention to withhold LSTs for severe dementia (the direct effect “c”) and (2) whether men and women differ in their attitudes toward the PAA (the effect “a”), and (3) whether these attitudes toward the PAA affect the intention to withhold LSTs for severe dementia (the effect “b”). The total effect of gender is the direct effect (“c”) plus the indirect effect (“ab”) (see Fig. 1). Explanatory variables Gender was coded as 1 for men and 0 for women.i Attitude toward the PAA was defined according to responses to nine items on how respondents regarded the PAA. Items were rated on a 5-point Likert scale from 1 (strongly disagree) to 5 (strongly agree). Exploratory factor analysis was conducted, and three factors were identified: Factor 1, promoting a sense of abandonment, comprised four items: With the PAA, (1) I am basically waiting to die; (2) I feel insecure about my medical treat ments in the future; (3) my quality of care will not be guaranteed; (4) healthcare providers will no longer care for any of my medical conditions. This factor had a Cron bach’s α value of 0.79, and the total score ranged from 4 to 20. Factor 2, supporting patient autonomy, included two items: The PAA can (1) reduce the pressure on fam ily members when making decisions and (2) ensure that my medical treatment decisions are respected. The sec ond factor had a Cronbach’s α value of 0.74, and the total scores ranged from 2 to 10. The third factor, contributing to the collective good, comprised three items: With the PAA, (1) futile medical care can be reduced; (2) health care providers are protected from lawsuits while provid ing the treatment that I prefer; and (3) inefficient medical resource usage can be reduced. Factor 3 had a Cronbach’s α value of 0.88, and the total scores ranged from 4 to 20. Gender differences were discovered across all three factors for attitude toward the PAA (Table 2). The men had higher average scores regarding the PAA promot ing a sense of abandonment than the women (8.94 vs. 7.48; p < 0.05). However, the women had higher scores for supporting patient autonomy (8.74 vs. 7.94; p < 0.01) and contributing to the collective good (8.64 vs. 7.47, p < 0.001) than the men. Measures Based on the previous studies [45, 51], we adopted a modified Chinese version of the Life Support Prevalence Questionnaire (LSPQ) to measure respondents’ prefer ences for life-sustaining treatments for severe dementia. Second, we developed a questionnaire and invited three experts to review the feasibility of its contents. The ques tionnaire consisted of two parts, which evaluated the following: (1) general attitude toward the PAA and (2) Page 5 of 12 Chen et al. BMC Palliative Care (2022) 21:171 (2022) 21:171 Chen et al. BMC Palliative Care hypothetical LST decisions involving severe dementia for themselves and on behalf of people close to them. hypothetical LST decisions involving severe dementia for themselves and on behalf of people close to them. college diploma, we coded 1 as college and above and 0 as high school and below. Age was treated as a continuous variable. Gender differences in the intention to withhold LSTs for severe dementia As indicated in Table 3, compared to the women, the men were less likely to withhold LSTs for severe demen tia (total scores, 15.84 vs. 18.88, p = 0.01). The men less likely to agree to withhold invasive LSTs even in case of severe dementia than were the women (enteral tube feed ing, men vs. women 2.78 vs. 3.36, p = 0.05; dialysis, 2.66 vs. 3.43, p = 0.02; intubation with a ventilator, 3.23 vs. 3.93, p = 0.02; tracheotomy, 3.50 vs. 4.12, p = 0.01). Yet, men and women showed no difference in the intention to withhold resuscitation in case of a cardiac arrest (3.65 vs. 4.07, p = 0.2). Results One hundred potential participants were approached during the study, and 82 filled out the questionnaire suc cessfully. Because of the COVID-19 pandemic at the time of the survey, access to the hospital for face-to-face contact for filling out the questionnaire was denied, hin dering the recruitment of more respondents. Of the 82 questionnaires collected, 2 had missing data and were excluded from the analysis, yielding an effective response rate of 80%. Table 1 presents the demographic character istics of the respondents and gender differences in these characteristics. Of the study respondents, 52.5% were women, 87.6% had a college education or higher, and 40% resided in Taipei City (where the hospital was located). Approximately 41.3% of the respondents’ family mem bers (the patients) were admitted to the ICU at the time of the interview. About one-third (32.5%) of the respon dents were the patients’ parents, 22.5% were the patients’ children, and 19% were the patients’ spouses. No gen der differences were observed in these demographic characteristics. Outcome variables Marital status could be single or never married, mar ried, divorced, or cohabitating. Because of the small number of divorced or cohabitating respondents, we coded 1 as married and 0 as not married. Intention to Withhold LSTs for severe dementia was defined according to respondent ratings for the following five medical interventions for prolonging life: (1) receiv ing enteral tube feeding if severe dementia hinders eating; (2) receiving dialysis for kidney failure; (3) being intu bated on a ventilator if severe dementia makes breath ing impossible; (4) undergoing a tracheotomy following one month of intubation and if survival appears unlikely without a ventilator; and (5) undergoing CPR if a car diac arrest because of end-stage dementia. Respondents rated each medical intervention on a 5-point Likert scale from 1 (strongly agree) to 5 (strongly disagree). Explor atory factor analysis was performed, and one factor with an explained variance of 54% was found. This factor has a Cronbach’s α value of 0.92. We summed the ratings of these five medical interventions, yielding scores ranging from 5 to 25: a higher score indicates a stronger intention to withhold LSTs for severe dementia. Control variables Education level was defined as the highest diploma respondents obtained. Because most respondents had a Chen et al. BMC Palliative Care (2022) 21:171 Chen et al. BMC Palliative Care Page 6 of 12 Table 1 Demographic Characteristics of the Family Members of Hospitalized Patients (N = 80) Variables All (N = 80) %/SD Men (N = 38, 47.5%) %/SD Women (N = 42, 53.5%) %/SD X2/Indepen dent two-sam ple t-test Age (mean) 45.10yrs 12.07 47.1yrs 11.6 43.1yrs 11.9 1.52 ns Education level High school and below 10 12.5 4 10.5 6 14.3 2.60(2) ns College 53 66.3 23 60.5 30 71.4 Graduate school 17 21.3 11 28.9 6 14.3 Marital status Single 22 27.5 9 23.7 13 31 3.55(3) ns Married 55 6.8 27 71.1 28 66.7 Divorced 1 1.3 0 0 1 2.4 Cohabitation 2 2.5 2 5.3 0 0 Respondent’s relationship with the patient Spouse 15 18.8 6 15.8 9 21.4 8.92(6) ns Parents 26 32.5 10 26.3 16 38.1 Children 18 22.5 12 31.6 6 14.3 Siblings 3 3.8 3 7.9 0 0 Friends 8 10 3 7.9 5 11.9 Self 3 3.8 2 5.3 1 2.4 Unspecified 7 8.8 2 5.3 5 11.9 Family monthly income USD 0 ~ 1,011 7 8.9 3 8.1 4 9.5 2.93(2) ns USD 1,012 ~ 3,371 39 49.4 22 59.5 17 40.5 USD 3,372 and above 33 41.8 12 32.4 21 50 Employment Unemployed 4 5 2 5.2 2 4.8 Retired 12 15 6 15.8 6 14,3 0.04(2) ns Yes 64 80 30 78.9 34 80.9 Place of residence Taipei City 32 40 16 42.1 16 38.1 0.148(2) ns Outside Taipei City 46 57.5 21 55.3 25 59.5 Foreign countries 2 2.5 1 2.6 1 2.4 Hospital ward ICU 33 41.25 24 63.2 23 54.8 0.58(1) ns Internal/Surgical ward 47 58.75 14 36.8 19 45.2 Rounding differences to 100% are possible Table 1 Demographic Characteristics of the Family Members of Hospitalized Patients (N = 80) In terms of making hypothetical LST decisions for parents, both men and women tend to agree to treat ments for their parents rather than themselves. Control variables The self–parent differences in the respondents’ decisions regarding enteral tube feeding was 0.67 (p = 0.05) for men and 0.54 (p = 0.05) for women, regarding dialysis was 0.60 (p = 0.001) for men and 0.50 (p = 0.01) for women, regarding intubation on a ventilator was 0.72 (p = 0.01) for men and 0.75 (p = 0.01) for women, and regard ing tracheotomy was 0.28 (nonsignificant) for men and 0.33 (p = 0.001) for women. No gender differences were observed in these self–other differences. The only signifi cant gender difference was noted for conducting CPR if a cardiac arrest because of end-stage dementia (self–parent intention to withhold LSTs for severe dementia intention to withhold LSTs for severe dementia Regarding the intention to withhold LSTs for people close to the respondents, if those people had severe dementia, 42 (52.5%), 31 (38.8%), 3 (4%), and 4 (5%) of the respondents referred to parents, spouses, children, and friends, respectively, as people for whom they would make LSTs decisions. Table 3 summarizes the self–other differences in the respondents’ intention to withhold LSTs for severe dementia. The mean values indicate the differences between the respondents’ intention to with hold LSTs for themselves and those on behalf of their parents or spouses. A greater mean difference indicates greater discordance. Page 7 of 12 Page 7 of 12 Chen et al. BMC Palliative Care (2022) 21:171 Chen et al. BMC Palliative Care Table 2 Gender Differences in Attitude Toward the Patient Autonomy Act (PAA) (Mean, SD) Attitude Items1 All Men Women Indepen dent two- sample t-test Mean (SD) Mean (SD) Mean (SD) T-value Cron bach’s a Factor 1: Sense of abandonment 0.79 With the PAA, I am basically waiting to die. 1.83 (0.81) 2.05 (0.73) 1.62 (0.83) -2.47 With the PAA, I feel insecure about my medical treatments in the future. 2.29 (0.99) 2.45 (1.03) 2.14 (0.95) -1.37 With the PAA, my quality of care will not be guaranteed. 2.19 (0.86) 2.42 (0.83) 1.98 (0.84) -2.38* With the PAA, healthcare providers will no longer care for my medical conditions. 1.88 (0.83) 2.03 (0.82) 1.74 (0.83) -1.56 Sum scores (5–20) 8.18 (2.73) 8.94 (2.46) 7.48 (2.81) -2.48* Factor 2: Sup port for patient autonomy 0.74 PAA can reduce the pressure on family members to make decisions. 4.06 (0.80) 3.89 (0.80) 4.21 (0.78) 1.81 PAA can make how I wish to be medi cally treated to be respected. 4.30 (0.70) 4.05 (0.70) 4.52 (0.63) 3.17 Sum scores (5–10) 8.36 (1.34) 7.94 (1.37) 8.74 (1.21) 2.74 Factor 3: Collective good 0.88 With the PAA, futile medical care can be reduced. 4.10 (0.88) 3.79 (0.91) 4.38 (0.76) 3.17 With the PAA, healthcare providers will be protected from lawsuits while treating me how I wish to be medically treated. 4.19 (0.66) 3.92 (0.59) 4.43 (0.63) 3.72* With the PAA, waste in medical resources can be reduced. intention to withhold LSTs for severe dementia 3.99 (089) 3.68 (0.96) 4.26 (0.73) 3.04 Sum scores (5–15) 8.08 (1.07) 7.47 (1.74) 8.64 (1.41) 3.32 1: The higher score, the stronger agreement with the statement * P < 0.05; P < 0.01, * P < 0.001 Table 2 Gender Differences in Attitude Toward the Patient Autonomy Act (PAA) (Mean, SD) difference = 0.50, p = 0.01 for men, and 0.08 [not signifi cant] for women; gender difference, p = 0.05). f Regarding the intention to withhold LSTs for spouses, the hypothetical decisions men would make for them selves were consistent with those they would make for their spouses. However, the hypothetical decisions women would make for their spouses differed signifi cantly from those they would make for themselves. The women were more likely to accept enteral tube feed ing for their husbands than for themselves (self–spouse difference = 0.60, p = 0.05) and were more in favor of a tracheotomy for their husbands than for themselves in cases where survival appears unlikely without a ventila tor (self–spouse difference = 0.48, p = 0.05). However, no significant gender differences in these self–spouse differ ences were observed. Testing for the mediation effect BMC Palliative Care Table 3 Gender Differences in the Intention to Withhold LSTs involving Severe Dementia for Self and Self-others Differences (Mean, SD) Self Self-parent differences2 Self-spouse differences2 Table 3 Gender Differences in the Intention to Withhold LSTs involving Severe Dementia for Self and Self-others Differences (Mean, SD) Self Self-parent differences2 Self-spouse differences2 LSTs to be withheld Men (N = 38) Women (N = 42) Indepen dent two- sample t-test1 Men (N = 18) Women (N = 24) Inde pen dent two- sam ple t-test Men (N = 15) Women (N = 14) In de pen dent two- sam ple t-test Enteral tube feeding as a means of ingesting food 2.78 (1.17) 3.36 (1.43) p = 0.05 0.67 (1.19)* 0.54 (1.06)* ns 0.50 (1.02) 0.60 (0.99)* ns Dialysis if have kidney failure 2.66 (1.19) 3.43 (1.36) p = 0.01 0.60 (0.70)* 0.50 (0.78) ns 0.07 (0.83) 0.33 (1.05) ns To be intubated and utilizing a ventilator to breathe 3.23 (2.10) 3.90 (1.30) p = 0.02 0.72 (1.02)* 0.75 (1.03)* ns 0.07 (0.83) 0.20 (1.01) ns Undergo a tracheotomy If being intubated for a month and survival is not possible without a ventilator 3.50 (1.08) 4.12 (1.19) p = 0.01 0.28 (0.75) 0.33 (0.64)* ns 0.07 (0.73) 0.48 (0.74)* ns Undergo cardiopulmonary resuscitation if a cardiac arrest 3.65 (1.07) 4.07 (1.18) ns 0.50 (0.79)** 0.08 (0.58) p = 0.05 -0.07 (0.62) -0.33 (0.72) ns Total scores 15.84 (4.91) 18.88 (5.60) p = 0.01 - - - - - - 1 Independent two-sample t-test was used to test gender differences 2 Pair-sample t-tests were used to test self-other differences by gender, and independent two-sample t-tests were used to test gender differences in self-other differences * p < 0.05; p < 0.01; * p < 0.001 Table 3 Gender Differences in the Intention to Withhold LSTs involving Severe Dementia for Self and Self-others Differences (Mean, SD) Table 3 Gender Differences in the Intention to Withhold LSTs involving Severe Dementia for Self and Self-others Differences (Mean, ) Table 3 Gender Differences in the Intention to Withhold LSTs involving Severe Dementia for Self and Self-others Differences (Mean, p p gf 2 Pair-sample t-tests were used to test self-other differences by gender, and independent two-sample t-tests were used to test gender differences in self-other differences * < 0 05 < 0 01 * < 0 001 Table 4 Mediators as Outcome Variables: The Gender Effect on the Attitude Toward Patient Autonomy Act (PAA) Sense of abandonment Support for patient autonomy Collective good Variables B SE 95% CI B SE 95% CI B SE 95% CI Gender (ref: Women) Men 1.55 0.63 (0.31, 2.80)* -0.75 0.3 (-1.34, -0.16)* -1.77 0.48 (-2.73, -0.81)*** Control variables Age 0.001 0.03 (-0.06, 0.06) -0.01 0.02 (-0.05, 0.02) 0.01 0.02 (-0.04, 0.05) Education -1.35 1.22 (-3.77, 1.07) 0.21 0.42 (-0.48, 1.32) 1.08 0.73 (-0.38, 2.54) Married -0.78 0.67 (-2.11, 0.56) 0.06 0.37 (-0.69, 0.80) 0.69 0.66 (-0.62, 1.99) Constant 9.12 1.58 (5.98, 12.26) 8.94 0.96 (7.02, 10.85) 11.48 1.4 (8.70, 14.26) Model R2 0.11 0.11 0.19 * p < 0.05; p < 0.01; * p < 0.001 ble 4 Mediators as Outcome Variables: The Gender Effect on the Attitude Toward Patient Autonomy Act (PAA) Sense of abandonment Support for patient autonomy Collective good abandonment reduced the intention to withhold LSTs for severe dementia (− 0.53, 95% CI = − 0.99, − 0.01); however, regarding the PAA as supporting patient autonomy (1.52, 95% CI = 0.37, 2.69) and as contributing to the collective good increased the intention to withhold LSTs for severe dementia (1.1, 95% CI=0.44, 1.76). Testing for the mediation effect were fully mediated by differences in attitude toward the PAA between genders. This study contributes to the liter ature by exploring the disparity in how men and women perceive the PAA, an innovative piece of legislation in Taiwan that addresses a topic that generally receives little attention in Asian countries. It may be easy to understand that people choose not to receive LSTs when the survival period is very short. According to a study by Tang et al. in Taiwan in 2014, among 2452 terminal cancer patients, when they knew the prognosis, they were less likely to choose life-pro longing medical treatment[52]. Yet, when the survival period may last several years, such as dementia, whether to withhold LSTs to shorten life will reflect more on people’s value concerning what is meant by the quality of life. For example, patients with severe dementia rely on Testing for the mediation effect We examined whether men and women differ in their attitudes toward the PAA and whether their attitudes affect their intention to withhold LSTs for severe demen tia (the mediation model). Table 4 presents the regres sion results (“a” in Fig. 1) for gender on the three factors derived from the attitude toward the PAA (serving as three mediators in testing for the mediation effect). The results revealed that, in comparison with the women’s scores, the men’s scores were 1.55 higher for regard ing the PAA as promoting a sense of abandonment (95% CI = 0.31, 2.80), 0.75 lower for supporting patient auton omy (95% CI = − 1.38, − 0.12), and 1.87 lower for con tributing to the collective good (95% CI = − 2.89, − 0.84), when age, education, and marital status were controlled. None of the control variables were significantly associ ated with attitude toward the PAA. Table 5 presents the regression results (“b,” “c,” and “ab” in Fig. 1). The direct effect of gender on the intention to withhold LSTs for severe dementia were fully mediated through the three factors of attitude toward the PAA, respectively. The indirect effect (“ab” in Fig. 1) of gender through the factor of regarding the PAA as promoting a sense of abandonment (Mediator 1) on the intention to withhold LSTs was − 0.77 (95% CI = − 1.96, − 0.034), and that through the factor of supporting patient autonomy (Mediator 2) is − 0.91 (95% CI = − 1.96, − 0.09), and that through the factor of contributing to the collective good (Mediator 3) was − 1.65 (95% CI = − 2.99, − 0.52). These findings suggest that in comparison with the women, the men were significantly less likely to withhold LSTs involving severe dementia (the total effect [“c” + “ab” in Fig. 1] of gender was − 3.37 [95% CI = − 5.90, − 0.85]).f As indicated in Table 5, the different aspects of atti tude toward the PAA had distinct effects on the inten tion to withhold LSTs for severe dementia (“b” in Fig. 1). Regarding the PAA as promoting a sense of Chen et al. BMC Palliative Care (2022) 21:171 Page 8 of 12 Page 8 of 12 Chen et al. Discussionh The results reveal that men tend to perceive the PAA and the signing of ADs as the abandonment of patients. In contrast, women tend to have more positive attitudes toward the PAA and view the PAA as supporting patient autonomy and contributing to the collective good. Men were generally less likely to withhold LSTs for severe dementia than women. However, the gender differences in the intention to withhold LSTs for severe dementia Page 9 of 12 Page 9 of 12 Chen et al. BMC Palliative Care (2022) 21:171 Chen et al. BMC Palliative Care Table 5 Mediation Models: The Direct, Indirect, and Total Effect of Gender (X) on the Intention to Withhold LSTs (Y) involving Severe Table 5 Mediation Models: The Direct, Indirect, and Total Effect of Gender (X) on the Intention to Withhold LSTs (Y) involving Severe Dementia Intention to Withhold LSTs (Y) Model 1 (Mediator 1) Model 2 (Mediator 2) Model 3 (Mediator 3) B SE 95% CI B SE 95% CI B SE 95% CI Gender (X) effect Direct effect of X on Y -2.6 1.36 (-5.30, 0.10) -2.46 1.38 (-5.21, 0.28) -1.73 1.42 (-4.56, 1.11) Indirect effect of through M on Y -0.77 0.51 (-1.96, -0.034)* -0.91 0.49 (-1.96, -0.09)* -1.65 0.63 (-2.99, -0.52)* Total Effect of X on Y -3.37 1.27 (-5.90, -0.85)* -3.37 1.27 (-5.90, -0.85)* -3.37 1.27 (-5.90, -0.85)* Mediators Sense of abandonment -0.53 0.25 (-0.99, -0.01)* - - - - - - Support for autonomy - - - 1.52 0.58 (0.367, 2.687)* - - - Collective good - - - - - - 1.1 0.33 (0.44, 1.76)*** Control variables Age 0.06 0.07 (-0.07, 0.19) 0.08 0.06 (-0.05, 0.20) 0.05 0.06 (-0.10, 0.18) Education 0.18 2.67 (-5.15, 5.51) 0.34 2.38 (-4.41, 5.09) -0.16 2.38 (-0.07, 0.18) Married 1.07 1.45 (-1.83, 3.97) 1.39 1.49 (-1.59, 4.37) 0.82 1.46 (-2.09, 3.72) Constant 19.16 4 (11.19, 27.13) 3.87 4.91 (-5.90, 13.64) 3.94 4.06 (-4.21, 12.09) Model R2 0.18 0.2 0.24 * p < 0.05; p < 0.01; * p < 0.001 Table 5 Mediation Models: The Direct, Indirect, and Total Effect of Gender (X) on the Intention to Withhold LSTs (Y) involving Severe These findings can be situated among the relevant lit erature on self–other differences [27, 54]. Discussionh The risk-as- feelings theory holds that subjective risk preferences are attenuated in surrogate decisions [55]; when people make decisions on behalf of others, they are less influ enced by their interpretation of the risks involved. Other biasing factors, such as social norms, can determine individuals’ choices on behalf of others. When sons or daughters act as surrogate decision-makers for their parents of advanced age, they may place more weight on social norms than on the risk of compromising their parents’ quality of life. We speculate that the hypothetical LST decisions that adult children make for their parents are affected by the principle of filial piety emphasized in Asian societies. As a result, the respondents in the pres ent study indicated that they would agree to more hypo thetical LSTs for their parents than for themselves in cases where the patient’s quality of life would be severely affected because of end-stage dementia. life-supporting devices and other people for daily nutri tion and hygiene, resulting in severely impaired quality of life and poor dignity. Ultimately, attitudes toward patient autonomy and associated ideas such as the quality of life may determine LST-related decisions in real situations. This study found that women were hesitant to agree to LSTs for severe dementia if these treatments were per ceived to be futile. Moreover, women felt empowered because they believed that ADs can prevent unwanted life support and that healthcare practitioners would honor their ADs, consistent with the results from Per kins et al. [21]. By contrast, men felt disempowered and feared harm from the healthcare system. A recent review of studies conducted in China indicated that unfamiliar ity with the concept of advance care planning and ADs is the main reason for people’s reluctance to complete an AD [53]. Furthermore, although both the women and men in this study generally agreed to more LSTs for their parents than for themselves, this tendency was stron ger among the men, who were more likely than women to agree to CPR for their parents even if their hearts stopped because of end-stage dementia. However, when the men were making hypothetical decisions on behalf of their spouses, the self–other differences disappeared; that is, the men made the same decisions for themselves and their spouses. Data Availability h d d The datasets used and/or analyzed during the current study are available from the corresponding author, Duan-Rung Chen, upon reasonable request (email: duan@ntu.edu.tw). Ethics approval and consent to participant All the participants provided written informed consent to participate in this study. This study was performed by the Declaration of Helsinki and approved by the National Taiwan University Research Ethics Committee (No. 201811035RINB). Authors’ contributions All authors met the criteria for authorship defined by the International Committee of Medical Journal Editors and approved the final version of this manuscript for submission. DRC designed the study, analyzed the data, and drafted the manuscript. JSJ, FCT, and YY revised the manuscript. All authors reviewed and approved this manuscript. The current study provides a new perspective on how socialized gender roles affect the intention to withhold LSTs involving severe dementia. However, some limita tions are noted. First, the sample is not representative because we adopted a single-center design with conve nience sampling. Second, the questions regarding atti tudes toward the PAA and the hypothetical scenarios involving severe dementia in the questionnaire were developed specifically for this study; a more extensive survey must be conducted to validate the questionnaire. Third, the answers to the questionnaire may be affected by the patients’ conditions on the respondents’ attitudes toward the PAA was not assessed. Last, the small sample size call for caution in the findings. Declarations h h d l The authors declared no potential conflicts of interest concerning this article’s research, authorship, and publication. The authors declared no potential conflicts of interest concerning this article’s research, authorship, and publication. Conclusion The men in this study tended to perceive the PAA as a sign that patients are being abandoned by the health system and exhibited less interest than the women in regarding the PAA as a collective good and a means of protecting patient autonomy. Gender differences in the intention to withhold LSTs were fully mediated by their attitude toward the PAA. Both men and women were more likely to withhold LSTs for themselves than for their family members. Men were also more likely to agree to CPR for their parents than themselves, even if a cardiac arrest because of end-stage dementia, com pared with what women would do for their parents. Received: 28 April 2022 / Accepted: 22 September 2022 Received: 28 April 2022 / Accepted: 22 September 2022 Role of the funding source. The funders had no role in the study design, data collection and analysis, publication decision, or manuscript preparation. Consent for publication The authors give the publisher permission to publish the work. Author details 1 1Institute of Health Behaviors and Community Sciences, National Taiwan University, Taipei, Taiwan 2Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan 3Department & Graduate Institute of Medical Education and Bioethics, National Taiwan University College of Medicine, Taipei, Taiwan 4Department of Health Policy, Management & Behavior, School of Public Health, New York State University,, Albany, USA Finally, women were more likely to agree to enteral tube feeding and a tracheotomy for their husbands than for themselves. In contrast, men made consistent decisions for themselves and their spouses for these LST scenarios. The study results indicate that social ized gender roles may play a critical role in EOL deci sions in Taiwanese society. Because people still rely on family members to make LST decisions in real-life situations, patient education and public support for patient autonomy should be strengthened, especially in Asian countries. Discussionh By contrast, the women’s hypotheti cal LST decisions exhibited self–spouse differences: they were more likely to agree to tube feeding and tracheot omy for their husbands than for themselves. Because of the small sample size, no significant gender differences in self–spouse differences were observed in the intention to withhold LSTs for severe dementia. f The integrated model of surrogate decision-making proposed by Tunney and Ziegler [29] may also help eluci date intergeneration and gender differences in surrogate decisions in cases where a patient’s quality of life is highly compromised because of severe dementia. According to that model, the surrogate decision-maker may consider what is in the recipient’s best interest (benevolent per spective), what they would do if they were the recipient (projected perspective), what they believe the recipient would choose (simulated perspective), and what serves their interests irrespective of the recipient’s wishes (ego centric perspective). Familiarity [29] and closeness [27] with the recipient affect people’s ability to engage in simulated perspective-taking. Most Taiwanese families are still strongly influenced by patriarchy and traditional Chen et al. BMC Palliative Care (2022) 21:171 Page 10 of 12 Chen et al. BMC Palliative Care CPR Cardiopulmonary Resuscitation. DGBAS Directorate-General of Budget, Accounting, and Statistics. DNR Do-Not-Resuscitate. EOL End-of-Life. ICU Intensive Care Unit. LSPQ Life Support Prevalence Questionnaire. LSTs Life-Sustaining Treatments. social hierarchies; surrogate decisions made by adult children on behalf of their parents or by women on their spouses are expected to be different from those made for themselves. Therefore, when people make deci sions on behalf of others, they tend to be less influenced by the interpretation of risks involved and more influ enced by other biasing factors, such as patriarchal prin ciples and socialized gender roles. Several studies using health insurance claims data in Taiwan found that men, as fathers and husbands, are more likely than women to receive aggressive EOL care even at the end stage of ter minal cancer [12, 13].h 1. Fried TR, Bradley EH, Towle VR, Allore H. Understanding the treatment prefer ences of seriously ill patients. N Engl J Med. 2002;346:1061–6. 2. Silveira MJ, Kim SY, Langa KM. Advance directives and outcomes of surrogate decision making before death. N Engl J Med. 2010;362:1211–8. 3. Teno JM, Gruneir A, Schwartz Z, Nanda A, Wetle T. Association between advance directives and quality of end-of-life care: A national study. J Am Geriatr Soc. 2007;55:189–94. 4 P ki HS C t lli d th th f l i f d di ti A Acknowledgements Acknowledgements We thank Chia-Yin Wu for her help in data collection. Acknowledgements We thank Chia-Yin Wu for her help in data collection. Funding This work was supported by the Ministry of Technology and Science (MOST) in Taiwan (Grant number MOST- 109-2410-H-002 -054 -SS2). References Sources of concern about the patient self-determination act. N Engl J Med. 1991;325:1666–71. 34. Nakamura K, Kinugasa Y, Sugihara S, Hirai M, Yanagihara K, Haruki N, et al. Sex differences in surrogate decision-maker preferences for life-sustaining treatments of Japanese patients with heart failure. ESC heart failure. 2018;5:1165–72. 8. Khosla N, Curl AL, Washington KT. Trends in engagement in advance care planning behaviors and the role of socioeconomic status. Am J Hospice Pal liat Medicine®. 2016;33:651–7. 35. Cheng YH, Wang JJ, Wu KH, Huang S, Kuo ML, Su CH. Do-not-resuscitate orders and related factors among family surrogates of patients in the emer gency department. Support Care Cancer. 2016;24:1999–2006. 9. Yadav KN, Gabler NB, Cooney E, Kent S, Kim J, Herbst N, et al. Approximately one in three US adults completes any type of advance directive for end-of- life care. Health Aff. 2017;36:1244–51. 36. Ni P, Zhou J, Wang ZX, Nie R, Phillips J, Mao J. Advance directive and end- of-life care preferences among nursing home residents in Wuhan, China: a cross-sectional study. J Am Med Dir Assoc. 2014;15:751–6. f 10. Kitzinger J, Kitzinger C. Increasing understanding and uptake of advance decisions in Wales. 2016. 37. Chen DR, Chang LY, Yang ML. Gender-specific responses to social determi nants associated with self-perceived health in Taiwan: A multilevel approach. Soc Sci Med. 2008;67:1630–40. 11. Ministry of Health and Welfare. 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The Journals of Gerontology Series B: Psychological Sciences and Social Sciences. 2001;56:P78–84. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Page 12 of 12 Page 12 of 12 Chen et al. BMC Palliative Care (2022) 21:171 Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Chen et al. BMC Palliative Care (2022) 21:171 References Advance care planning for older people in mainland China: An integrative literature review. Int J Older People Nurs. 2021;16:e12409. away they are on the family tree. PLoS ONE. 2012;7:e49479. 29. Tunney RJ, Ziegler FV. Toward a psychology of surrogate decision making. Perspect Psychol Sci. 2015;10:880–5. 30. Sulmasy DP, Snyder L. Substituted interests and best judgments: an inte grated model of surrogate decision making. JAMA. 2010;304:1946–7. 54. Batteux E, Ferguson E, Tunney RJ. 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https://openalex.org/W4308761683	https://zenodo.org/records/7312615/files/41.17n.pdf	English	null	Enzyme assisted extraction of polyphenols from Niger seed meal: a review	Zenodo (CERN European Organization for Nuclear Research)	2,022	cc-by	5,872	Email: omkarpokharkar4@gmail.com Omkar V. Pokharkar2 Omkar V. Pokharkar2 Enzyme assisted extraction of polyphenols from Niger seed meal: a review Vivek K. Mukherjee1,. ABSTRACT The changing global economy and the food surpluses around the world have now stimulated an interest in seeking a new alternative crop especially an Oilseed Crop. Niger seed (G. abyssinica) is drawing commercial and academic attention because of its nutritional value, biological activities and excellent antioxidant properties. Niger seed has diverse application and hence this seed is of great industrial importance. Presence of polyphenols in Niger seed has been reported previously. Phenolic compounds, naturally found in plants are of considerable interest and have enticed researchers around the globe in recent years due to their bioactive functions. These components are known as secondary plant metabolites and have antimicrobial, antiviral and anti-inflammatory properties along with their high antioxidant capacity. And thus, presence of such properties makes polyphenol a functional food. Enzyme-assisted extraction has attracted interest among researchers across industries like food, pharmaceutical and academia. This extraction method of polyphenols from plants is a suitable alternative to conventional solvent based extraction methods. The application of hydrolytic enzymes for extraction of natural functional compounds from plants is widely investigated in recent years for its advantages in easy operation, high efficiency, eco-friendly nature and preserving the bioactive properties of the extracts. This review summarizes the nutritional composition of Niger seed and importance of polyphenols which are known to be present in Niger seed. It also discusses the advantages of using eco-friendly extraction methods known as enzyme assisted extraction. Keywords: Niger seed meal, Enzyme-assisted extraction, Phenolic compounds, G. abyssinica Up to this point, countless plants have been examined and some of these have been cultivated and developed as new oil crops. Among these new sources of oil crops, Niger (Guizotia abyssinica Cass.) seed oil is of interest and may play an important role in human nutrition. As per M F Ramadan, Niger is an oilseed that has been cultivated for approximately 5000 years. South India and Ethiopia are the two major producers of Niger seeds. It belongs to the same botanical family as that of sunflower and safflower (Compositae) (Baagø, 1974). It is a dicotyledonous herb, moderately to well branched, and grows up to 2m in height (Getinet and Teklewold, 1995). Niger is cultivated in both temperate and tropical climates, being considered a temperate-region plant that has adapted to a semi- tropical environment. It prefers moderate temperatures for growth, from about 19◦C to 30◦C. 1 D.Y. Patil University, School Of Biotechnology and Bioinformatics, C.B.D Belapur, Navi Mumbai – 400614, India. 1 D.Y. Patil University, School Of Biotechnology and Bioinformatics, C.B.D Belapur, Navi Mumbai – 400614, India. 2Wageningen University and Research Centre, 6708 PB Wageningen, Netherlands. AN INTERNATIONAL QUARTERLY JOURNAL OF BIOLOGY & LIFE SCIENCES 4(1):121-129 AN INTERNATIONAL QUARTERLY JOURNAL OF BIOLOGY & LIFE SCIENCES 4(1):121-129 ISSN (online): 2320-4257 www.biolifejournals.com R E V I E W A R T I C L E INTRODUCTION Non-traditional oilseeds are being considered because their constituents have unique chemical properties and may augment the supply of edible oils (Cherry and Kramer, 1989). Even today, no oil from any one source has been observed to be suitable for all edible applications. Since, oils from different sources mostly differ in their composition. This urges the need to search for new sources of novel oils. How to Site This Article: Vivek K. Mukherjee, Omkar V. Pokharkar (2016). Enzyme assisted extraction of polyphenols from Niger seed meal: a review. Biolife, 3(3), pp 121-129. doi:10.17812/blj.2016.4117 Published online: 10 February 2016 Biolife \| 2016 \| Vol 4 \| Issue 1 Biolife \| 2016 \| Vol 4 \| Issue 1 © www.globalsciencepg.org 121\| Copyright@2016 Vivek K. Mukherjee et al temperatures for growth, from about 190C to 300C (Quinn and Myers, 2002). In literature, Niger seeds are reported to contain 483 calories, 2.8–7.8% moisture, 17–30% protein, 34–39% total carbohydrate, 9–13% fiber, 1.8–9.9 g ash, 50–587 mg/100 g calcium, 180–800 mg/100 g phosphorus, 0.43 mg/100 g thiamine, 0.22–0.55 mg/100 g riboflavin, and 3.66 mg/100 g niacin (Bhardwaj and Gupta, 1977; Kachapur et al., 1978; Rao, 1994). Table-1 also gives a comparative picture of composition of Niger oil seeds. Polyphenols have been discovered in plants since their early appearance. These compounds, also called secondary metabolites, are indeed crucial for many important functional aspects of plant life, including structural roles in different supportive or protective tissues, involvement in defence strategies, and signalling properties, particularly in the interactions between plants and their environment. Collectively, higher plants synthesise several thousand different known phenolic compounds, and the number of these which have been fully characterized is continually increasing. There is a huge demand for novel natural compounds that rocketed the growth and development of plant based compounds called as bioactives that either are beneficial for health or are toxic upon consumption. Increased release of such bioactives from plant cells either by cell disruption or by extraction through the cell wall can be enhanced using a particular enzyme or mixture of different enzymes. However, in biotechnology the use of enzymes is not commonly exploited to its greatest potential within the food industry. Here, we discuss the application of nature friendly enzyme-assisted extraction of bioactive compounds. Niger seed (Guizotia abyssinica Cass): Botanical details, production, composition and uses: Botanical details, production, composition and uses: Niger (Guizotia abyssinica Cass) is one of the main oilseed crops having commercial importance. It has been under cultivation for long period of time spanning approximately 5000 years. Niger is a dicotyledonous herb, which is moderately to well branched, and attains up to two meters of height (Getinet and Teklewold, 1995). Niger seed belongs to the Compositae family and there are six species of which G. abyssinica is the only cultivated species (Baagø, 1974). India and Ethiopia are the major producers of Niger oil seed, with 90 kilotons of production reported in India during the year 2011- 2012 (IOPEPC 2012). INTRODUCTION In various parts of the world there is an excess production of some conventional crops and also there is a quest for new crops, these also includes plants that produce beneficial oils. Till date, we know that the agricultural industry has produced raw materials that were employed mostly for the food industries. Yet, later on use of a greater amount of its products will be coordinated for chemical applications. Table-1. Nutrient composition of Niger oil seed (Ref Rao, 1993) Table-1. Nutrient composition of Niger oil seed (Ref Rao, 1993) Components Value Starch g/100g 6.1 Phosphorus mg/100g 801 Calcium mg/100g 587 Magnesium mg/100g 323 Iron mg/100g 30 Zinc mg/100g 6.6 Manganese mg/100g 8.5 Copper mg/100g 5.7 Chromium mg/100g 0.4 Tannins mg/100g 158 Nicotinic acid mg/100g 0.57 Riboflavin mg/100g 0.22 Niger seeds are used after frying or used as a condiment, are milled into flour, or pressed with honey into cakes. Apart from this they are used for livestock feed after oil extraction while the plants are used as green manure (a type of cover crop grown primarily to add nutrients and organic matter to the soil) (Riley and Belayneh, 1989, Bhagya and Sastry, 2003). In countries like USA, Europe, and Japan, Niger seed is used as bird feed (Dutta et al., 1994; Marini et al., 2003; Shahidi et al., 2003). Niger seed is also used in human food mainly as a spice in the frying of vegetables (Bhagya and Sastry, 2003). Niger seed (Guizotia abyssinica Cass): Oil- Content, composition and uses: varieties of niger seed contains 25% oleic and 55% linoleic acids (Nasirullah et al., 1982), and have lower linoleic acid percent when compared with seeds grown in Ethiopia (75%) (Nasirullah et al., 1982). The variation in fatty acid level in different varieties of Indian and Ethopian origin is reported in Table 2. Table-2. Variation in the fatty acid levels (% of fame) of Indian and Ethiopian Niger seed oil (Ref, Ramadan 2012) Table-2. Variation in the fatty acid levels (% of fame) of Indian and Ethiopian Niger seed oil (Ref, Ramadan 2012) Bhatnagar and Gopal Krishna (2013) have characterized Niger seed oil components and reported the following parameters: linoleic acid (69.4–73.2 %) was the major fatty acid and trilinolein (31.2–33.4 %) was the major triacylglycerol; color (40.0–95.0 Lovibond units), free fatty acids (3.6–12.3 g/100 g), peroxide value (3.2–7.8 mequiv O2/kg), iodine value (137.6–140.3 cg I2/g), saponification value (177.3–185.9 mg KOH/g) and unsaponifiable matter (1 3–4 3 g/100 g); total tocopherols (171 9– Fatty acids Ethiopian seed oil Indian seed oil Palmitic acid (C16:0) 8.0-9.7 9.4-16.9 Behenic acid (C22:0) 0.3 0.4-0.5 Palmitoleic acid (C16:1) 0.2 0.2 Stearic acid (C18:0) 5.6-8.1 6.5-6.8 Oleic acid (C18:2) 5.9-11.0 8.3-9.2 Linoleic acid (C18:2) 70.7-79.2 63.0-71.1 Linolenic acid (C18:3) 0.4 0.3 Arachidic acid (C20:0) 0.4 0.5 Table-2. Variation in the fatty acid levels (% of fame) of Indian and Ethiopian Niger seed oil (Ref, Ramadan 2012) Fatty acids Ethiopian seed oil Indian seed oil Palmitic acid (C16:0) 8.0-9.7 9.4-16.9 Behenic acid (C22:0) 0.3 0.4-0.5 Palmitoleic acid (C16:1) 0.2 0.2 Stearic acid (C18:0) 5.6-8.1 6.5-6.8 Oleic acid (C18:2) 5.9-11.0 8.3-9.2 Linoleic acid (C18:2) 70.7-79.2 63.0-71.1 Linolenic acid (C18:3) 0.4 0.3 Arachidic acid (C20:0) 0.4 0.5 Table-3. Amino acid Composition of Niger oil seeds (Ref Rao, 1993) Table-3. Oil- Content, composition and uses: , p The oil content in Niger seed is in the range of 30–50% (Getinet and Teklewold, 1995; Dange and Jonsson, 1997) and is having pale yellow colour with a nutty taste. It is very similar to sunflower and safflower oils with respect to linoleic acid content (Francis and Campbell, 2003). Recently, Bhatnagar and Gopal Krishna (2013) reported 31.8–41.3 g/100 g oil content of Niger seeds obtained after extraction with solvents of different polarities. In India, 20,000 tons of oil production was reported in 2009-10. In India, Niger is grown mainly in the states of Maharashtra, Orissa, Madhya Pradesh, Karnataka and Andhra Pradesh. In India, Ootacamund, N-5, RCR-18 and IGP-76 are the different varieties of Niger seed that are grown chiefly (Bhagya and Sastry, 2003). The crop is known to grow mainly on poorly drained, heavy clay soils (Alemaw and Wold, 1995; Francis and Campbell, 2003) in both temperate and tropical climates. However, it prefers moderate The fatty acid composition of Niger seed oil is typical for seed oils belonging to the Compositae family (e.g., safflower and sunflower) with linoleic acid being the dominant fatty acid. Getinet and Teklewold, (1995) reported fatty acid composition of the seed to be 7–8% palmitic and stearic acids, 5– 25% oleic acid, and 55–80% linoleic acid. The Indian 122 \|© 2016 Global Science Publishing Group, USA Biolife \| 2016 \| Vol 4 \| Issue 1 Copyright@2016 Vivek K. Mukherjee et al compared to Indian variety) and an unacceptable dark cake colour (Seegeler, 1983). The Niger meal contains approximately 30% protein and 23% crude fiber after oil extraction and the oil, protein, and crude fiber contents are dependent on the hull thickness with thick-hulled having less oil and protein and more crude fiber (Getinet and Teklewold, 1995). The meal was reported to contain more crude fiber than most oilseed meals and is free from any toxic substance. Earlier, Bhagya and Sastry, (2003) reported an integrated method of processing to dehull the niger seed with a dehulling efficiency of 96–98%. Further, the oil extracted from dehulled seeds was of good quality and the cake was high in protein and low in fiber. The amino acid composition of Niger protein was deficient in tryptophan (Table-3). Oil- Content, composition and uses: Amino acid Composition of Niger oil seeds (Ref Rao, 1993) Constituents Values Lysine 4.0 Histidine 2.1 Arginine 3.3 Aspartic acid 9.6 Threonine 5.3 Serine 5.5 Glutamic acid 27.0 Proline - Glycine 4.8 Alanine 3.7 Valine 4.0 Methionine 1.2 Isoleucine 3.7 Leucine 6.5 Tyrosine 2.7 Phenylalanine 2.8 Tryptophan 1.0 Chemical score 33 First limiting amino acid Methionine Bhatnagar and Gopal Krishna (2013) have characterized Niger seed oil components and reported the following parameters: linoleic acid (69.4–73.2 %) was the major fatty acid and trilinolein (31.2–33.4 %) was the major triacylglycerol; color (40.0–95.0 Lovibond units), free fatty acids (3.6–12.3 g/100 g), peroxide value (3.2–7.8 mequiv O2/kg), iodine value (137.6–140.3 cg I2/g), saponification value (177.3–185.9 mg KOH/g) and unsaponifiable matter (1.3–4.3 g/100 g); total tocopherols (171.9– 345.8 ppm with α-tocopherol 154–276 ppm as major component); total phenolics (247.1–2,647.7 ppm; vanillic acid with 176–1,709 ppm as major component); total sterols (1,249.6–6,309.3 ppm) and total carotenoids (18.9–181.0 ppm). The lipoprotein contained 4% moisture, 12% ash, 46% protein, 20% fat, 7% crude fiber, and 11% soluble carbohydrate (Eklund, 1971a; 1971b; 1974). The antioxidant potential of defatted niger seed and its extracts was reported by Shahidi et al. (2003). The meal remaining after oil extraction was tested as a source of antioxidants. Three solvent systems, A [80:20 (vol/vol) ethanol/water], B [80:20 (vol/vol) acetone/water], and C (water) were evaluated as extraction media. Extract A, which exhibited superior antioxidant activity over B and C, was studied further by using column chromatography and HPLC. Out of four fractions obtained (I–IV) were obtained, Fraction IV was also highly effective in scavenging the 2, 2- diphenyl-1-picrylhydrazyl radical but was less active when used in a bulk oil model system. UV spectroscopy suggested that the major active component present was a chlorogenic acid-related compound (Table 4). Niger seed or its oil also used in perfumes as a carrier of the scents and fragrances. Niger seed oil is used as a substitute for olive oil and as a pharmaceutical substitute for sesame oil. It also finds usage in the manufacture of soap and as a lubricant or lighting fuel. The oil is also documented to find use though to a limited extent in paints (being slow- drying), for which the Ethiopian seed is superior to the Indian seed as it has higher linoleic acid content. Meal- Content, composition and uses: Meal- Content, composition and uses: The utilization of Niger seed proteins in human food is very limited due to the presence of a high fiber content (more in Ethiopian varieties when 123 \|© 2016 Global Science Publishing Group, USA Biolife \| 2016 \| Vol 4 \| Issue 1 Copyright@2016 Vivek K. Mukherjee et al able-4. Phenolic acids in Niger seed crude extract (mg/g extract) (Shahidi et al., 2003) Phenolic Acids Chlorogenic Caffeic p-Coumaric Ferulic Sinapic Free 2.6 0.6 Trace - 0.1 Esterified - 40.7 0.2 0.2 Trace Glycosylated - 2.1 - - - Total 2.6 43.4 0.2 0.2 0.1 Table-4. Phenolic acids in Niger seed crude extract (mg/g extract) (Shahidi et al., 2003) phenylpropane units. The sensorial food properties such as flavor and color are influenced by polyphenols, and they also contribute to the aroma and taste of numerous food products of plant origin (Lule and Xia, 2005). The contribution of polyphenols towards aroma is mainly due to the presence of volatile phenols. Polyphenols apart from above- mentioned diversity are also present in plant tissues mainly as glycosides and/or associated with various organic acids and/or as complex polymerized molecules with high molecular weights, such as tannins. Furthermore, HPLC analysis established that chlorogenic acid was dominant in the free phenolics fraction (2.6 mg/g). It was concluded in the study that that defatted niger seed and its extracts can provide a natural source of antioxidants (Shahidi et al., 2003). The use of niger seed was advisable in applications where the functionality of seed components, such as their fat- and moisture binding ability and textural effects, are important in addition to their antioxidant activity. yp Occurrence, structure and types Polyphenols are secondary metabolites found in plants, and characterized by the presence of more than one phenol unit or building block per molecule. They play important roles in defense against plant pathogens and animal herbivore aggression and as response to various abiotic stress conditions, such as rainfall and ultraviolet radiation. Fruits, vegetables, and beverages like berries, tea, beer, grapes/wine, olive oil, chocolate/cocoa, coffee, walnuts, peanuts, borojo, pomegranates, popcorn, yerba mate, and other fruits and vegetables (Vinson et al., 2001; 2002) are the main sources of phenolic compounds in the human diet (Kapur and Kapoor, 2001; Dimitrios, 2006) (Table-5). Polyphenols: The term "polyphenol" includes more than 8,000 compounds with great structural diversity (although each has at least one aromatic ring with one or more hydroxyl groups). They can be divided into 10 different classes depending on their basic chemical structure. Table 1 shows the main families of phenolic compounds, most of which are found in nature associated with mono- or polysaccharides (glycosides) or functional derivatives such as esters or methyl esters. Moreover, the main sources where phenolic compounds are found have been classified. Anti-microbial activities of polyphenol: Proanthocyanidins also showed antiviral effects against influenza A virus and type-1 herpes simplex virus (HSV) by preventing the entry of the virus into the host cell, which is the first critical step in primary HSV-1 infection (Geschera et al., 2011). Lignans obtained in hexane extract from Aristolochia taliscana roots, a plant used in traditional Mexican medicine, contains neolignans, among which Licarin A was found to be the most active against four mono- resistant variants and 12 clinical isolates of Mycobacterium tuberculosis strains with MICs ranging from 3.12 to 12.5 mg/ml (Leon-Dı´az et al., 2010). Friedman et al., (2006) reported inhibition of food- borne pathogenic bacteria, such as Bacillus cereus by tea catechins, such as (-)-gallocatechin-3-gallate, (-)-epigallocatechin- 3-gallate, (-)-catechin-3-gallate, and (-) - epicatechin-3-gallate, at nanomolar levels. It was reported that many of these compounds were more active than antibiotics, such as tetracycline or vancomycin, at comparable concentrations. The study indicated the tested tea catechins could exert a positive effect against gastrointestinal diseases. The pre-treatment of a human cultured cell line (HL cells), with flavonols at polyphenol concentrations ranging from 0.5 to 50 mM decreased the infectivity of Chlamydia pneumoniae by 50% as compared to untreated controls (Alvesalo et al., 2006). Proanthocyanidins derived from berries inhibit the growth of several pathogenic bacteria, such as uropathogenic Escherchia coli, cariogenic Steptococcus mutans, and oxacillin-resistant Staphylococcus aureus (Cote et al., 2010). The proliferation inhibition activities on Escherichia coli, Staphylococcus aureus and Bacillus subtilis were also measured for evaluating the antimicrobial activity of polyphenols from tobacco leaf. The diameters of inhibition zones were 20.23 ± 0.42, 17.66 ± 0.86 and 12.89 ± 0.29 mm, respectively (Wang et al., 2008). Enzyme assisted extraction of bioactives from plant sources: 125 \|© 2016 Global Science Publishing Group, USA Biolife \| 2016 \| Vol 4 \| Issue Table-5. Plant, fruit and vegetable-based biological compounds classified as polyphenols and separated according to differences in chemical structure (Myburgh, 2014) Groups Classes Examples and Derivatives Excellent sources Phenolics Cinnamic Caffeic acid Coffee beans Benzoic Gallic acid Tea leaves Flavonoids Flavonols Quercetin ; Kaempferol Yellow onions, deep green vegetables. Quercetin glucosides Cherry tomatoes Flavones Intensely flavored vegetables and herbs(celery,parsley) Isoflavones Soy products Anthocyanins Dark blue, red and deep red fruit and vegetables; red wine Flavonones Citrus fruit and pith of peel, Concentrated tomato( paste) Flavonols Naringin; Hesperetin; Eriodictyol; Naringenin Catechins and derivatives Tannins, grapes, seeds, cocoa. Biological effects of polyphenols: Polyphenols have been studied in the past two decades for their potential involvement in the prevention of many chronic diseases, like cardiovascular disease, cancer, osteoporosis, diabetes mellitus, and neurodegenerative diseases. Many of the effects that dietary polyphenols exhibit are likely through impacts on transcriptional networks or signalling cascades that modulate gene expression, promoting anti-inflammatory mediators, and NO production (Christy C. Tangney et al.,2013). The protective activity of polyphenols have been attributed to their role as an anti-oxidant, free radical scavenger, and metal chelator, or to due to inhibition of telomerase, cycloxygenase, or lipoxygenase, interaction with signal transduction pathways and cell receptors, acting as anti-microbial agents, anti- allergic and effect on inflammation and endothelial dysfunction by in part affecting the recruitment or “homing” of inflammatory cells from the circulation and down-regulating the production of adhesion molecules by the endothelium, thereby hindering cellular migration into the subendothelial space and reducing atherosclerotic plaque formation (Christy C. Tangney et al.,2013) (Table-5). Polyphenols may be classified into different groups as a function of the number of phenol rings that they contain and of the structural elements that bind these rings to one another. In general polyphenols are divided into flavonoids and non- flavonoids. Flavonoids share a common carbon skeleton of diphenyl propanes, two benzene rings (ring A and B) joined by a linear three-carbon chain. The central three-carbon chain forms a closed pyran ring (ring C) with A benzene ring. They are subdivided into many subclasses: flavonols, flavones, flavanones, anthocyanidins, flavanols, and also isoflavones (Daglia, 2012). The main groups of nonflavonoids include phenolic acids, which are subdivided into derivatives of benzoic acid, like gallic acid and protocatechuic acid, along with derivatives of cinnamic acid, that consist chiefly of coumaric, caffeic, and ferulic acid. Stilbenes, constitutes the second group whose main representative is resveratrol, that exists in both cis and Trans isomeric forms, while the third group comprises of lignans which are produced by oxidative dimerization of two Biolife \| 2016 \| Vol 4 \| Issue 1 124 \|© 2016 Global Science Publishing Group, USA Vivek K. Mukherjee et al Copyright@2016 Copyright@2016 Anti-microbial activities of polyphenol: Epicatechin Gallocatechin Epigallocatechin Epigallocatechin- gallate Proanthocyanidin Stilbenes Resveratrol Trans and cis resveratrol Wine, red grapes Piceid Trans and cis piceid Red grape juice Lignans For example enterolignans Seeds, legumes, wholegrains Allicin Bulbs, certain fruits Table-5. Plant, fruit and vegetable-based biological compounds classified as polyphenols and separated according to differences in chemical structure (Myburgh, 2014) Copyright@2016 input, low product quality, environment risk and toxicological effects [5 from enzyme assisted]. As an alternative technology, enzyme-assisted extraction has enticed considerable interest among researchers across industries like food, pharmaceutical and academia. The application of hydrolytic enzymes for extraction of natural functional compounds from plants is widely investigated in recent years for its advantages in easy operation, high efficiency, eco- friendly nature and preserving the bioactive properties of the extracts. A variety of enzymes like cellulases, pectinases and hemicellulases, which attack cell wall components, are used to disrupt the structural integrity of the plant cell wall, thereby enhancing the extraction of bioactives like polyphenols from plants (Table-7). These enzymes increase extraction yields of bioactives by increasing the cell wall permeability. Various microorganisms like bacteria, fungi, animal organs or vegetable/fruit extracts are the sources of these enzymes and it is imperative to understand their catalytic property and mode of action, optimal operational conditions and which enzyme or enzyme combination for extraction applications. input, low product quality, environment risk and toxicological effects [5 from enzyme assisted]. As an alternative technology, enzyme-assisted extraction has enticed considerable interest among researchers across industries like food, pharmaceutical and academia. The application of hydrolytic enzymes for extraction of natural functional compounds from plants is widely investigated in recent years for its advantages in easy operation, high efficiency, eco- friendly nature and preserving the bioactive properties of the extracts. A variety of enzymes like cellulases, pectinases and hemicellulases, which attack cell wall components, are used to disrupt the structural integrity of the plant cell wall, thereby enhancing the extraction of bioactives like polyphenols from plants (Table-7). These enzymes increase extraction yields of bioactives by increasing the cell wall permeability. Various microorganisms like bacteria, fungi, animal organs or vegetable/fruit extracts are the sources of these enzymes and it is imperative to understand their catalytic property and mode of action, optimal operational conditions and which enzyme or enzyme combination for extraction applications. α-L-rhamonisidase treatment have been shown to increase flavonoid release from plant material while minimizing the use of solvents and heat (Kaur et al., 2010). In the processing of pectic polysaccharide from mangosteen for enhancing extraction of an antioxidant, utilization of pectinase has been reported (Gan et al., 2010). The enzyme at 0.1% w/w increased extraction from 1.7 to 7.4 g/kg of raw material dry weight. Dobozi et al. Vivek K. Mukherjee et al Vivek K. Mukherjee et al Copyright@2016 Phenols are classified as cell-wall phenols (which are bound to polysaccharides) and non-cell- wall phenols (associated with vacuoles and the cell nucleus) in higher plants (Pinelo et al., 2006 EA 2). The retention of phenols in the cell wall depends on physical traits, compositional and structural characteristics of cell wall polysaccharides as the latter contains hydrogen groups as well as aromatic with polyphenols (Le Bourvellec et al., 2004 EA 3). As cross-linking between cell wall polysaccharides act as the main barrier for the release of intracellular substances, degradation of cell-wall polysaccharides is one of the key steps in releasing phenols from cell wall. Conventional methods such as cold pressing, super-critical fluid and solvent extraction which are used to extract bioactives from plants sources suffers 126 \|© 2016 Global Science Publishing Group, USA Biolife \| 2016 \| Vol 4 \| Issue 1 and glycosidic oxygen atoms that have the ability to form hydrogen bonds and hydrophobic interactions from several drawbacks, including low selectivity, low extraction efficiency, safety hazards, high energy Table-6. Chemical structure of Polyphenol classes and microorganisms sensitive to them (Daglia, 2012). O OH Flavon-3-ol O OH O Flavonol Antibacterial V.cholerae - S.mutans- C.jejuni C.perfringes - E.coli- B.Cereus H.pylori - S.aureus - L.acidophilus A.naeslundii - P.oralis - P.gingivalis P.melaninogenica - F.nucleatum C.pneumonia Antiviral Adenovirus-Enterovirus-Fluvirus Antifungal Candida albicans Microsporum gypseum Trichophyton mentagrophytes Trichophyton rubrum O OH OH OH O H OH O OH OH OH O H OH Condensed Tannin Antibacterial S.mutans, E.coli, S. aureus Antiviral Influenza A virus, type-1Herpes Simplex virus O O H O H Phenolic acids Antibacterial S.aureus,L.monocytogenes E.coli, P.aeruginosa O OH Flavon-3-ol OH Flavon-3-ol O OH O Flavonol OH Flavonol O OH OH OH O H OH O OH OH OH O H OH Condensed Tannin OH Condensed Tannin OH Condensed Tannin S.aureus,L.monocytogenes E.coli, P.aeruginosa and glycosidic oxygen atoms that have the ability to form hydrogen bonds and hydrophobic interactions from several drawbacks, including low selectivity, low extraction efficiency, safety hazards, high energy Biolife \| 2016 \| Vol 4 \| Issue 1 126 \|© 2016 Global Science Publishing Group, USA 126 \|© 2016 Global Science Publishing Group, USA 126 \|© 2016 Global Science Publishing Group, USA Vivek K. Mukherjee et al Copyright@2016 Copyright@2016 Vivek K. Mukherjee et al Glucose oxidase offered a better performance in enhancement of the extraction yields of FMPS, antioxidant and activate alcohol dehydrogenase activities. Miron et al., (2014) reported enzyme- assisted extraction (EAE) and pressurized liquid extraction (PLE) for extraction of phenolic compounds from lemon balm (Melissa officinalis) by using cellulase, endo-β-1,4 xylanase and pectinase. The results showed that EAE enhanced the total phenolic content and the antioxidant capacity compared to a non-enzymatic control. Among the bioactive phenolic compounds identified in lemon balm, rosmarinic acid was the main component, although other important compounds were also identified, such as caffeic acid derivatives (salvianolic acids, lithospermic acid) and rosmarinic acid derivatives (rosmarinic acid hexoside, sagerinic acid, sulfated rosmarinic acid). The t study confirmed that EAE can be considered alternative method for the extraction of natural compounds with biological activity from natural sources. [5]. Bhardwaj, S.P., & Gupta, R.K. (1977). Tilangi, a potential rich yielding oil seed crop. IndianFarming. 27: 18–19. [6]. Cerda. A., et al (2012). The enhancement of antioxidant compounds extracted from Thymus vulgaris using enzymes and the effect of solvent. Food Chem. 139: 138-143. [7]. Choudhari, S.M. and Ananthanarayan, L. (2007) Enzyme aided extraction of lycopene from tomato tissues. Food Chem. 102, 77–81. [8]. Christy C. Tangney & Heather E. Rasmussen. (2013). Polyphenols, Inflammation, and Cardiovascular Disease. Curr Atheroscler Rep 15:324. [9]. Cote. J., Caillet. S., Doyon. G., Sylvain JF, Lacroix M: Bioactive compounds in cranberries and their biological properties. Crit Rev Food Sci Nutr 2010, 50:666-679. [10]. Daglia, M. (2012). Polyphenols as antimicrobial agents. Curr Opin Biotechnol. 23:174–181. [11]. Dange, K., & Jonsson, A. (1997). Oil content & fatty acid composition of seeds of Guizotia Cass (compositae). J. Sci. Food Agric. 73: 274–278. Niger seed has huge diverse applications and it can be used as an alternative to traditional Oilseed crop. It has been extensively studied for its antioxidant properties. Polyphenols which are known to be present in Niger seed exhibit wide range of biological activity such as anti-microbial, anti- inflammatory, prebiotics and antioxidant activity. Polyphenols may play an important role in the prevention and treatment of highly widespread diseases such as cancer and cardiovascular diseases. Use of enzymes to extract bioactives from plants is a suitable alternative for solvent based extraction. Application of enzymes is still not widely known, but there is a huge scope for the eco-friendly extraction methods for production of variety of bioactives. [12]. Copyright@2016 Dehghan-Shoar, Z. et al. (2011) Lycopene extraction from extruded products containing tomato skin. Int. J. Food Sci. Technol. 46, 365– 371. [13]. Dimitrios, B. (2006). Sources of natural phenolic antioxidants. Trends Food Sci.Technol. 17:505– 512. [14]. Dobozi. M.S., Halasz. A., E. Kozma-Kovacs., & Szakacs. G., (1988) Enhancement of mustard oil yield by cellulolytic pretreatment: Appl. Microbiol Biotechnol 29:39-43. [15]. Dominguez. H., et al (1995). Enzyme-assisted hexane extraction of soyabean oil. Food Chem. 54 223-231. [16]. Dutta, P.C., Helmersson, S., Kebedu, E., Getinet, A., & Appliqvist, L. (1994). Variation in lipid composition of niger seed (Guizotia abyssinica Cass) samples collected from different regions in Ethiopia. J. Am. Oil Chem. Soc. 71: 839–843. Conflict of Interests Authors declare that there is no conflict of interests regarding the publication of this paper. [17]. Dutta, P.C., Helmersson, S., Kebedu, E., Getinet, A., and Appliqvist, L. (1994).Variation in lipid composition of niger seed (Guizotia abyssinica Cass) samples collected from different regions in Ethiopia. J. Am. Oil Chem. Soc. 71: 839–843. Copyright@2016 (1988) reported that treatment of mustard seeds with cellulolytic enzymes results in an increase (20–30%) in the yield of oil. Santamaria et al. (2000) showed that enzyme preparations of cellulase, hemicellulase and pectinase when used for the treatment of chilli and an increase in the yield of carotenoids (11%) and capsaicinoids (7%). Chowdhary and Satyanarayana (2007) reported significant increase (206%) in lycopene extraction from tomato tissues using cellulases and pectinases versus control experiments. Similarly, a 2.5-fold increase in yield of lycopene from tomato skin was reported by using pancreatin digestion. Deng et al. (2014) reported extraction of Fructus Mori polysaccharides (FMPS) from Fructus Mori using four kinds of enzymes. Table-7. List of bioactive compounds of industrial importance obtained by enzyme-assisted extraction from plants (Puri et al., 2012). Product type Product Source Enzyme used Maximum yield % Oils and carotenoids Oil Grape seed Cellulase, protease, xylanase and pectinase 17.5 Carotenoids Marigold flower Viscozyme, Pectinex, neutrase, corolase and HT- proteolytic 97 Volatile oil Mandarin peel Xylan-degrading enzymes 15 Carotene Carrot pomace Pectinex Ultra SP-L 0.0064 Lycopene Tomato Pancreatin 2.5-fold Tomato Cellulase and pectinase 206 Capsaicin Chilli Cellulase, hemicellulase and pectinase N.D. Anthocyanin Grape skin Pectinex BE3-L N.D. Glycosides Sugar Grapefruit peel waste Cellulase and pectinase 0.6377 Oligosaccharide Rice bran Cellulase 39.9 Starch Cassava Pectinase enzyme 45.6 Pectin Pumpkin Xylanase, cellulose, b- glucosidase 14.0 Others Polyphenols Grape pomace Pectinolytic 98.0 Flavonoid (naringin) Kinnow peel Recombinant rhamnosidase N.D. Phenolics Citrus peel Celluzyme MX 65.0 Proteins Lentils and white beans Glucoamylases 50.3 Catechins Tea beverage Pepsin 80.0 f bioactive compounds of industrial importance obtained by enzyme-assisted extraction uri et al., 2012). 127 \|© 2016 Global Science Publishing Group, USA Biolife \| 2016 \| Vol 4 \| Issue 1 Biolife \| 2016 \| Vol 4 \| Issue 1 127 \|© 2016 Global Science Publishing Group, USA 127 \|© 2016 Global Science Publishing Group, USA Copyright@2016 Copyright@2016 nigerseed (Guizotia abyssinica Cass.) soluble in hot aqueous ethanol. Acta Physiol. Scand. 90: 602–608. [34]. Le Bourvellec, C., Guyot, S., & Renard, C. M. G. C. (2004). Non covalent interaction between procyanidins and apple cell wall material. Part 1: Effect of some environmental parameters. Biochimica and Biophysica Acta. 1672, 192– 202. [21]. Eydelnant, I., & Tufenkji, N. (2008). Cranberry derived proanthocyanidins reduce bacterial adhesion to selected biomaterials. Langmuir. 24, 10273-10281. [35]. 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https://openalex.org/W2586070161	https://europepmc.org/articles/pmc5332610?pdf=render	English	null	Stability of vertical magnetic chains	Proceedings - Royal Society. Mathematical, physical and engineering sciences	2,017	cc-by	23,959	JS, 0000-0002-3417-3239; EF, 0000-0001-5329-3394 JS, 0000-0002-3417-3239; EF, 0000-0001-5329-3394 A linear stability analysis is performed for a pair of coaxial vertical chains made from permanently magnetized balls under the inﬂuence of gravity. While one chain rises from the ground, the other hangs from above, with the remaining ends separated by a gap of prescribed length. Various boundary conditions are considered, as are situations in which the magnetic dipole moments in the two chains are parallel or antiparallel. The case of a single chain attached to the ground is also discussed. The stability of the system is examined with respect to three quantities: the number of balls in each chain, the length of the gap between the chains, and a single dimensionless parameter which embodies the competition between magnetic and gravitational forces. Asymptotic scaling laws involving these parameters are provided. The Hessian matrix is computed in exact form, allowing the critical parameter values at which the system loses stability and the respective eigenmodes to be determined up to machine precision. A comparison with simple experiments for a single chain attached to the ground shows good agreement. Subject Areas: mechanics Keywords: constrained mechanics, many body, non-local, athermal, buckling, bifurcation Author for correspondence: Eliot Fried e-mail: eliot.fried@oist.jp Author for correspondence: Eliot Fried e-mail: eliot.fried@oist.jp Johannes Schönke and Eliot Fried Research Cite this article: Schönke J, Fried E. 2017 Stability of vertical magnetic chains. Proc.R. Soc.A 473: 20160703. http://dx.doi.org/10.1098/rspa.2016.0703 Received: 15 September 2016 Accepted: 10 January 2017 Subject Areas: mechanics Keywords: constrained mechanics, many body, non-local, athermal, buckling, bifurcation Author for correspondence: Eliot Fried e-mail: eliot.fried@oist.jp Okinawa Institute of Science and Technology Graduate University, Onna, Okinawa 904-0495, Japan Okinawa Institute of Science and Technology Graduate University, Onna, Okinawa 904-0495, Japan Research JS, 0000-0002-3417-3239; EF, 0000-0001-5329-3394 Stability of vertical magnetic chains Johannes Schönke and Eliot Fried rspa.royalsocietypublishing.org 1. Introduction Permanently magnetized spherical balls provide fascinating avenues for studying many fundamental phenomena in physics. Being millimetre to centimetre- sized, they can be assembled by hand to form very complex structures. A simple but important question concerns the mechanical stability of chains formed from these magnetic balls: What is the maximum length of a chain of balls that can be balanced vertically without buckling by ﬁxing the position and dipole orientation of its lowest element? For instance, although this is easily achieved with a chain of ﬁve commercially available toy balls of diameter 5 mm, mass 0.5 g and magnetic ﬂux 2017 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. (a) (b) (c) Figure 1. Stability of chains made from magnetic balls of diameter 5 mm, mass 0.5 g and magnetic flux density 1.19 T. A stack of no more than nine balls is stable (a,b) but a stack of 10 balls buckles under its own weight (c). 2 rspa.royalsocietypublishing.org Proc. R.Soc. A 473: 20160703 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (a) 2 Figure 1. Stability of chains made from magnetic balls of diameter 5 mm, mass 0.5 g and magnetic flux density 1.19 T. A stack of no more than nine balls is stable (a,b) but a stack of 10 balls buckles under its own weight (c). density 1.19 T, it is impossible with a chain of 10 such balls, as shown in ﬁgure 1! How does the maximum length of a chain of balls that can be balanced vertically without buckling depend on the diameter, mass and magnetic ﬂux density of the constituent balls? Deﬁnitive answers to this and other related questions are provided in this paper. It is a rare opportunity in modern physics to encounter a macroscopic, globally interacting many-body system which can be manipulated directly in our hands, watching and feeling its response, while at the same time having access to an accurate yet simple theory that encompasses its behaviour. 1. Introduction In this paper, we present a rigorous mathematical framework to study the stability of many-body constrained mechanics including global interactions. Although we apply this framework only to chains of magnetic balls, it can readily be used for more complex assemblies such as sheets, as we discuss in §8. With reference to the observation, due to Vandewalle & Dorbolo [1], that for many-body athermal systems ‘. . . the interplay between small-scale physics and global properties is still a mystery’, we contribute here to this barely explored ﬁeld. For athermal dipolar systems, phenomena that remain either completely or only partially understood include pattern formation in self-assembled structures (for example, in granular dipolar media under vibrations or external ﬁelds), the importance of metastable states, defects, frustration memory, structural stability and analogies to classical elastic materials. Our work can be viewed as a step towards increasing the understanding of such phenomena. In one of the earliest studies of the collective behaviour of spherical dipoles, Weis & Levesque [2] found that such particles exhibit a strong tendency to form chains. In subsequent works, Clarke & Patey [3] and Messina et al. [4] found, depending on the number of particles, that the ground state is given by a sequence of straight chains, rings and tubes. In this regard, it is important to recognize that insofar as the dipole orientations are concerned, a tube formed by bending a sheet of spherical dipoles into a cylinder is equivalent to a tube formed by stacking appropriately sized rings. Since chains provide a fundamental building block for many structures made from magnetized balls, it is natural to seek information concerning their effective elastic properties. Magnetic interactions make it energetically costly to bend such chains. In colloidal science, effects associated with dipolar particles have been explored in considerable detail (as evidenced by the review of Teixeira et al. [5]) and studies of the elastic behaviour of chains made from such particles have been conducted by Biswal & Gast [6], Cerda et al. [7] and Coq et al. [8]. The elastic properties of chains comprised by bacteria that contain magnetic nanoparticles have been studied by Kiani et al. [9]. Chains of magnetic toy balls provide alternative macroscopic systems for investigation. Vandewalle & Dorbolo [1] showed that mechanically stable defects can result from removing the central branch of a triple junction of chains made from these balls. rspa.royalsocietypublishing.org Proc. R.Soc. A 473: 20160703 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . In this paper, we study the stability of vertical chains of magnetic balls under the inﬂuence of gravity. Instead of considering a single chain, we consider a system of two coaxial chains, one being attached to the ground and the other hanging from above with a gap of prescribed length. There are actually four different pathways along which this double-chain system can become unstable. Aside from the buckling of the lower chain due to gravity, the upper chain can rupture between its two uppermost balls as a consequence of the combined inﬂuences of gravity and the magnetic pull of the lower chain. If the lower chain is not ﬁxed at its base, it can lose contact with the ground due to the magnetic pull of the upper chain. Finally, if the chains have opposing dipole orientations, then they can both buckle due to mutual repulsion. Two classes of boundary conditions are considered. For the clamped case, the position and dipole orientation of the ball next to the boundary is ﬁxed. For the free case, the ball closest to the boundary is free to move tangentially without detaching from the boundary, and the dipole orientation is free. In our model, the particular problem of a single chain ﬁxed at its base is recovered by allowing the gap to become inﬁnite. We employ a linearized stability analysis to obtain explicit stability conditions in terms of eigenvalues and associated eigenmodes. Our methods allow us to evaluate these results to machine precision. We also provide new insights to the analogy between magnetic chains and classical elastic rods. After introducing the model and the linear stability analysis in §2 and §3, we discuss four examples: the single clamped chain (§4), two equal clamped chains (§5), two equal free chains (§6) and two clamped chains with opposing dipole orientations (§7). The conclusions appear in §8. 1. Introduction Questions about the elastic properties of chains made from such balls were ﬁrst addressed by Vella et al. [10] and Hall et al. [11], who introduced and demonstrated the utility of the notion of an effective magnetic bending stiffness. The ﬁrst analysis of the buckling of a vertical magnetic chain under its own weight in a gravitational ﬁeld was performed by Vella et al. [10]. Moreover, Hall et al. [11] used a discrete-to-continuum asymptotic analysis to derive an expression for the magnetic energy of a chain with local radius of curvature much larger than the diameter of the constituent balls. This result was used to determine the spectrum for the vibrations of a circular ring of spherical magnets, from which it was demonstrated that the small-amplitude oscillations of a ring of magnetic balls are effectively identical to those of an equivalent elastic ring made from an inextensible ﬁlament. In a related work, Boisson et al. [12] considered the dynamics of a horizontal chain of diametrically magnetized cylinders. Restricting attention to situations where neighbouring cylinders can only roll over one another without sliding, these authors determined the eigenfrequencies and associated eigenmodes arising from solving the relevant linearized equations of motion and found a close correspondence between the behaviour of their magnetic system and an elastic beam. 3 2. Constrained systems of magnetic balls Moreover, we deﬁne the dimensionless counterpart E of the energy U by E := α 6 i<j (ni · nj)x2 i,j −3(ni · xi,j)(nj · xi,j) x5 i,j + e2 · i wi, (2.4) (2.4) where the dimensionless parameter α := B2 4μ0ρgd > 0 (2.5) (2.5) incorporates the competition between magnetic and gravitational forces. d b ll i d j f h corporates the competition between magnetic and gravitational forces. For adjacent balls i and j to remain in contact, xi,j must satisfy the constraint x2 i,j = 1. (2.6) (2.6) Additionally, since the magnitude of the dipole moment of each ball i is constant, the associated orientation ni must satisfy the constraint Additionally, since the magnitude of the dipole moment of each ball i is constant, the associated orientation ni must satisfy the constraint \|ni\|2 = 1. (2.7) (2.7) To incorporate these constraints, we work with an augmented (dimensionless) potential energy of the form To incorporate these constraints, we work with an augmented (dimensionless) potential energy of the form F := E + xi,j=1 λi,j(x2 i,j −1) + i νi(\|ni\|2 −1), (2.8) (2.8) and λi,j and νi are Lagrange multipliers associated, respectively, with the constraints (2.6) and (2.7). and λi,j and νi are Lagrange multipliers associated, respectively, with the constraints (2.6) and (2.7). Bearing in mind that xi,j and ni can be varied independently, we observe the system is in equilibrium only if the condition ∂F ∂xi,j = ∂F ∂wi −∂F ∂wj = 0 (2.9) (2.9) holds for each pair i and j of balls that are adjacent and in contact and the condition holds for each pair i and j of balls that are adjacent and in contact and the condition ∂F ∂ni = 0 (2.10) (2.10) holds for each ball i. 2. Constrained systems of magnetic balls We consider a ﬁnite system of identical magnetic balls—each of which is of diameter d and has spatially uniform mass density ρ and spatially uniform magnetic ﬂux density B—in a spatially uniform gravitational ﬁeld. On denoting the centre position of ball i by pi, the vector ri,j directed from the centre position of ball j to the centre position of ball i is given by ri,j := pi −pj, (2.1) (2.1) with ri,j := \|ri,j\| being the associated distance. Since each ball i has a spatially uniform magnetic ﬂux density, its outer magnetic ﬁeld is that of a point dipole and is fully speciﬁed by the magnetic dipole moment mi. The total potential energy U of the system incorporates the dipole–dipole interactions between each pair of balls and the inﬂuence of the external gravitational ﬁeld. On introducing the permeability of free space μ0 and the mass M = πρd3/6 of a generic ball, this energy is given by U = μ0 4π i<j (mi · mj)r2 i,j −3(mi · ri,j)(mj · ri,j) r5 i,j + Mg e2 · i pi, (2.2) (2.2) where e2 denotes the unit vector directed upward antiparallel to the gravitational acceleration, which is of magnitude g > 0. 4 4 rspa.royalsocietypublishing.org Proc. R.Soc. A 473: 20160703 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . It is convenient to work in a dimensionless setting where spatial distances are measured relative to d, magnetic dipole moments are measured relative to m := πBd3/6μ0, and energies are measured relative to Mgd. Speciﬁcally, we deﬁne dimensionless counterparts wi, xi,j, xi,j, and ni, of pi, ri,j, ri,j, and mi by wi := pi d , xi,j := ri,j d , xi,j := \|xi,j\| and ni := mi m , (2.3) (2.3) respectively. Moreover, we deﬁne the dimensionless counterpart E of the energy U by respectively. 5 rspa.royalsocietypublishing.org Proc. R.Soc. A 473: 20160703 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Figure 2. Two coaxial vertical magnetic chains with an equal number of balls. We will eventually relax these assumptions to allow for (i) rupture of the upper chain at the junction between the two balls closest to its apex, (ii) lift off of the lower chain, and (iii) the dipole orientations of the upper chain to be directed downward and opposite to those in the lower chain. Note that reversing the orientation of all magnetic dipole moments leaves the energy unchanged and results in an equivalent state. We refer to this property as the polarity symmetry. System of two coaxial vertical magnetic chains separated by a finite gap We consider a system of two identical coaxial vertical chains each of them having N magnetic balls of diameter d separated by a distance Γ , as depicted in ﬁgure 2. It is easily conﬁrmed that this conﬁguration is an equilibrium conﬁguration. The (dimensionless) gap is denoted by γ := Γ d . (3.1) γ := Γ d . (3.1) Until further notice, we assume that (i) the balls at the base of the lower chain and the apex of the upper chain must remain in contact with the adjacent surfaces, (ii) the balls comprising each chain remain in contact, and (iii) the dipole orientation of each ball is directed upward. 5 rspa.royalsocietypublishing.org Proc. R.Soc. A 473: 20160703 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d 1 2 2N N + 1 N G Figure 2. Two coaxial vertical magnetic chains with an equal number of balls. 5 (b) Symmetries: planar version of the problem It is evident that the system exhibits a rotational symmetry around the central vertical axis. Beyond that symmetry, an important question concerns the possible shapes of the ﬁrst unstable mode arising from the stability analysis. Is that mode two-dimensional, so that the chains lie in a plane? Or is it three-dimensional, a helix for example? If we introduce cylindrical polar coordinates with central axis chosen to coincide with the axis shared by the undistorted chains and express the position wi of each ball i = 1, . . . , 2N with respect to those coordinates, we ﬁnd that, for any perturbations of the system, the gravitational potential energy is independent of the azimuthal angle. By contrast, the magnetic interactions energetically favour shapes in which all particle positions have the same azimuthal angle because such shapes have lower total curvatures. The ﬁrst unstable mode must therefore lie in a plane that passes through the central axis of the coordinate system. In combination with the aforementioned rotational symmetry, it therefore sufﬁces to restrict attention to perturbations involving only planar modes. (a) Lagrange multipliers The 2(N −1) Lagrange multipliers λi,i+1, i = 1, . . . , N −1, N + 1, . . . , 2N −1, can be interpreted as the (dimensionless) reactive forces needed to ensure that immediately adjacent pairs of balls in the lower and upper chains remain in contact. These multipliers must satisfy the linear system ∂F ∂wi − ∂F ∂wi+1 · xi,i+1 = 0, i = 1, . . . , N −1, N + 1, . . . , 2N −1, (3.2) (3.2) which has a tridiagonal banded coefﬁcient matrix. The solution to that system can be expr which has a tridiagonal banded coefﬁcient matrix. The solution to that system can be expressed as λi,i+1 = ⎧ ⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎨ ⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎩ −α 4 N−1 k=i ⎛ ⎝zk+1 · xk,k+1 + k l=2 [zl · (xl−1,l −xl,l+1)] ⎞ ⎠ + 1 2(N −i)e2 · x1,2, i = 1, 2, . . . , N −1, α 4 3N+1−i k=N+2 zk+1 · xk,k+1 + 2N−1 l=k [zl · (xl−1,l −xl,l+1)] + 1 2(N −i)e2 · x2N−1,2N, i = N + 1, . . . , 2N −1, (3.3) (3.3) where we have used the convention that b i=a · · · = 0 if b < a and we have introduced 6 rspa.royalsocietypublishing.org Proc. R.Soc. A 473: 20160703 zi := j̸=i 1 x5 i,j (ni · xi,j)nj + (nj · xi,j)ni + ni · nj −5 x2 i,j (ni · xi,j)(nj · xi,j) xi,j . (3.4) (3.4) The remaining 2N Lagrange multipliers νi, i = 1, . . . , 2N, can be interpreted as the reactive couples needed to ensure that the magnetic moment of each ball is of constant magnitude. These multipliers must satisfy the linear system ni · ∂F ∂ni = 0, i = 1, . . . , 2N, (3.5) (3.5) which can be solved to yield Proc. R.Soc. A 473: 20160703 νi = α 12 j,j̸=i 3(ni · xi,j)(nj · xi,j) −(ni · nj)r2 i,j r5 i,j . (3.6) (3.6) (c) Structure of the Hessian Consistent with the discussion in the previous section, we consider modes that lie in a plane characterized by a positively oriented orthonormal basis {e1, e2}, where e2 introduced in (2.2) is antiparallel to the gravitational acceleration. The constraints (2.6) requiring that balls in contact remain in contact dictate that the equilibrium position wi of each ball i has at most a single translational degree of freedom in the direction of the horizontal basis element e1 and thus admits a representation of the form wi = tie1 + ie2, i = 1, . . . , N, tie1 + (i + γ )e2, i = N + 1, . . . , 2N, (3.7) (3.7) where ti, i = 1, . . . , N −1, N + 1, . . . , 2N −1, must obey where ti, i = 1, . . . , N −1, N + 1, . . . , 2N −1, must obey where ti, i = 1, . . . , N −1, N + 1, . . . , 2N −1, must obey \|ti\| ≪1. (3.8) \|ti\| ≪1. (3.8) \|ti\| ≪1. (3.8) In view of (3.8), the difference xi+1,i = (ti+1 −ti)e1 + e2 (3.9) (3.9) describes an inﬁnitesimal rotation of the axis between two immediately adjacent balls i and i + 1 for each i = 1, . . . , N −1, N + 1, . . . , 2N −1. describes an inﬁnitesimal rotation of the axis between two immediately adjacent balls i and i + 1 for each i = 1, . . . , N −1, N + 1, . . . , 2N −1. The remaining constraints (2.7) dictate that the equilibrium magnetic moment ni of each ball i has at most a single degree of freedom in the direction of the horizontal basis element e1, describing rotation of the moment in the plane, and thus admits a representation of the form scribing rotation of the moment in the plane, and thus admits a representation of the form describing rotation of the moment in the plane, and thus admits a representation of the form 7 7 rspa.royalsocietypublishing.org Proc. R.Soc. A 473: 20160703 ni = sie1 + e2, (3.10) \|si\| ≪1. (3.11) ni = sie1 + e2, (3.10) (3.10) ni = sie1 + e2, (3.1 ni = sie1 + e2, rspa.royalsocietypublishing.org Proc. R.Soc. (c) Structure of the Hessian A 473: 20160703 where si, i = 1, . . . , 2N, must obey \|si\| ≪1. (3.11) \|si\| ≪1. (3.11) In the view of (3.11), ni as given by (3.10) describes an inﬁnitesimal rotation of the magnetic moment of ball i. In the view of (3.11), ni as given by (3.10) describes an inﬁnitesimal rotation of the magnetic moment of ball i. In terms of the column vectors In terms of the column vectors t = ⎛ ⎜⎜⎝ t1 ... t2N ⎞ ⎟⎟⎠ and s = ⎛ ⎜⎜⎝ s1 ... s2N ⎞ ⎟⎟⎠, (3.12) (3.12) the base conﬁguration in which both chains are straight and coaxial is captured by setting t = s = 0. For perturbations of the form (3.7) and (3.10), evaluating the Hessian of (2.8) at the base conﬁguration yields a symmetric 4N × 4N matrix the base conﬁguration in which both chains are straight and coaxial is captured by setting t = s = 0. For perturbations of the form (3.7) and (3.10), evaluating the Hessian of (2.8) at the base conﬁguration yields a symmetric 4N × 4N matrix H = Htt Hts Hst Hss , (3.13) (3.13) where the (i, j)th entries of the blocks Htt, Hts = Hst and Htt are given by ∂2F ∂ti∂tj t=s=0 , ∂2F ∂ti∂sj t=s=0 = ∂2F ∂si∂tj t=s=0 and ∂2F ∂si∂sj t=s=0 , (3.14) respectively. The Hessian can, alternatively, be expressed as the sum respectively. The Hessian can, alternatively, be expressed as the sum H = C + αA (3.15) H = C + αA (3.15) ely gravitational component C and a purely magnetic component αA. of a purely gravitational component C and a purely magnetic component αA. 8 rspa.royalsocietypublishing.org Proc. R.Soc. A 473: 20160703 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Figure3. Schematicdiagramdepictingtheremoval(×)ofrowsandcolumnsfromHstemmingfromtheimpositionofcertain boundary conditions. Figure3. Schematicdiagramdepictingtheremoval(×)ofrowsandcolumnsfromHstemmingfromtheimpositionofcertain boundary conditions. The 2N × 2N matrix A that determines the remaining term in the additive decomposition (3.15) of the Hessian H is generally a full 2N × 2N matrix of the form The 2N × 2N matrix A that determines the remaining term in the additive decomposition (3.15) of the Hessian H is generally a full 2N × 2N matrix of the form A = A0 + 2N−1 n=1 2 l=0 Alnγ l (γ + n)5 , (3.22) (3.22) with A0 and Aln being 6N −2 matrices consisting of rational entries that depend only on N for each combination of l = 0, 1, 2 and n = 1, . . . 2N −1. However, a symbolic manipulation programme can be used to obtain and evaluate these entries up to machine precision. In the subsequent analysis, which rests on an eigenvalue decomposition of H, we use Mathematica for all symbolic calculations. Without providing a proof, we note that A is positive-semideﬁnite. (d) Boundary conditions So far, the system can explore all possible degrees of freedom in the sense that each ball i = 1, . . . , 2N can move horizontally and its dipole moment can rotate. A collective rigid translation of the entire system in the direction e1 is therefore possible. This translational symmetry is reﬂected in the degeneracy of H. However, ﬁxing the horizontal position of one ball alleviates this difﬁculty. This can be achieved by ﬁxing the horizontal positions of either ball i = 1 or ball i = 2N and, thus, can arise as a natural consequence of imposing certain boundary conditions. If, for instance, the horizontal positions and dipole moments of balls i = 1 and i = 2N are ﬁxed, so that t1 = s1 = t2N = s2N = 0, then the corresponding Hessian can be obtained by deleting the rows and columns for t1, t2N, s1, and s2N from the general expression (3.13), as depicted in ﬁgure 3. Alternative boundary conditions are discussed subsequently. (c) Structure of the Hessian Consistent with its gravitational nature, C is independent of the magnetic moments of the balls and thus has block structure p y g p p y g p Consistent with its gravitational nature, C is independent of the magnetic moments of the balls and thus has block structure C = Ctt 0 0 0 , (3.16) (3.16) where Ctt is a block diagonal 2N × 2N matrix of the form where Ctt is a block diagonal 2N × 2N matrix of the form Ctt = CL tt 0 0 CU tt , (3.17) (3.17) with CL tt and CU tt being symmetric and tridiagonal N × N matrices that stem from the action of gravity on the balls that, respectively, comprise the lower and upper chains. Whereas CL tt has diagonal with CL tt and CU tt being symmetric and tridiagonal N × N matrices that stem from the action of gravity on the balls that, respectively, comprise the lower and upper chains. Whereas CL tt has diagonal {−N + 1, (−2N + 2i + 1)N−1 i=1 } (3.18) (3.18) and neighbouring diagonals {(N −i)N−1 i=1 }, (3.19) {(N −i)N−1 i=1 }, {(2N −2i −1)N−1 i=1 , N −1} (3.20) {(2N −2i −1)N−1 i=1 , N −1} and neighbouring diagonals and neighbouring diagonals {(i −N)N−1 i=1 }. (3.21) (3.21) With (3.18)–(3.21), a direct calculation shows that CL tt and CU tt are, respectively, negative- and positive-semideﬁnite. This is consistent with the observation that only the lower chain can undergo a buckling instability due to gravity. 8 rspa.royalsocietypublishing.org Proc. R.Soc. A 473: 20 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . t1 t2 tN tN+1 sN+1 s2N–1 s2N t2N–1 t2N s1 s2 sN t1 t2 tN tN+1 sN+1 s2N–1 s2N t2N–1 t2N s1 s2 sN 8 (e) Criteria for stability The stability of a given conﬁguration of the system is determined by the spectrum ηi, i = 1, . . . , Q of eigenvalues of the Hessian H evaluated at that conﬁguration. The number Q of eigenvalues of H depends on the chosen boundary conditions. If ηi > 0 for all i = 1, . . . , Q, then the conﬁguration is stable. If ηi < 0 for one or more i = 1, . . . , Q, then the conﬁguration is unstable. If the smallest eigenvalue ηi of H vanishes for some value of a system parameter (such as α deﬁned in (2.5)), then the conﬁguration loses stability and the corresponding value of the parameter in question is referred to ‘critical’. This is an example of a bifurcation point. The system is said to undergo a bifurcation, and therefore change its stability properties, each time an eigenvalue ηi of H becomes zero. In the view of the representation det H = η1η2 · · · ηQ, the bifurcation points correspond to the zeros of det H. 9 rspa.royalsocietypublishing.org Proc. R.Soc. A 473: 20160703 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Stability of a chain clamped at its base We ﬁrst explore the stability of the lower chain in isolation, with the stipulation that it is clamped at its base. This situation can be achieved formally by taking the limit γ →∞and neglecting the upper chain entirely. Under these circumstances, the stability of the lower chain involves only two parameters, namely the number N of balls it contains and the measure α of the competition between magnetic and gravitational forces. We assume that the lowermost ball i = 1 of the lower chain is clamped in the sense that it can neither translate nor rotate. The Hessian of the base conﬁguration then reduces to the 2(N −1) × 2(N −1) matrix ¯H that arises on removing the rows and columns for ti and si, i = 1, N + 1, . . . , 2N, from (3.13). Moreover, on noting that the sum on the right-hand side of (3.22) vanishes in the limit γ →∞, the decomposition (3.15) specializes to an expression of the form ¯H = ¯C + α ¯A0, (4.1) (4.1) where, with reference to (3.16) and (3.17), only the upper left block ¯CL tt of ¯C, which is of rank N −1, is non-vanishing. Since the lowermost ball is clamped, the degeneracy discussed in §3d is removed and it follows that ¯CL tt is negative-deﬁnite and A0 is positive-deﬁnite. It is crucial that the ball at the base of the lower chain is clamped. Otherwise, the system is unconditionally unstable. It might seem natural to consider particular values of α—or, equivalently, particular combinations of the parameters entering the deﬁnition (2.5) of α—and then determine the associated critical number Nc of balls that can be placed on top of one another before the chain becomes unstable. However, since N is integer valued, Nc must be discontinuous. It is therefore advantageous to ﬁx N and determine the associated critical value αc of α below which the chain is unstable. Hence, αc must be a bifurcation point of the Hessian ¯H, namely a point at which det ¯H = 0. To determine all bifurcation points of ¯H, we invoke the representation (4.1) and the invertibility of ¯A0 to express the determinant of ¯H in the form where, with reference to (3.16) and (3.17), only the upper left block ¯CL tt of ¯C, which is of rank N −1, is non-vanishing. 4. Stability of a chain clamped at its base Since the lowermost ball is clamped, the degeneracy discussed in §3d is removed and it follows that ¯CL tt is negative-deﬁnite and A0 is positive-deﬁnite. It is crucial that the ball at the base of the lower chain is clamped. Otherwise, the system is unconditionally unstable. It might seem natural to consider particular values of α—or, equivalently, particular combinations of the parameters entering the deﬁnition (2.5) of α—and then determine the associated critical number Nc of balls that can be placed on top of one another before the chain becomes unstable. However, since N is integer valued, Nc must be discontinuous. It is therefore advantageous to ﬁx N and determine the associated critical value αc of α below which the chain is unstable. Hence, αc must be a bifurcation point of the Hessian ¯H, namely a point at which det ¯H = 0. To determine all bifurcation points of ¯H, we invoke the representation (4.1) and the invertibility of ¯A0 to express the determinant of ¯H in the form det ¯H = det ¯A0 det(αI2(N−1) + ¯A−1 0 ¯C) = αN−1PN−1(α), (4.2) (4.2) where IK denotes the K × K identity matrix and where P(α) := det ¯A0 det[αIN−1 + ( ¯A−1 0 )tt ¯Ctt] (4.3) (4.3) is a polynomial of order N −1 in α. Since α > 0 and ¯A0 is positive deﬁnite, we infer from (4.2) that (4.4) det ¯H = 0 ⇐⇒P(α) = 0, (4.4) and, thus, that ¯H has at most N −1 distinct bifurcation points corresponding to the roots of P(α) = 0 or, by (4.3), the eigenvalues of the (N −1) × (N −1) matrix −( ¯A−1 0 )tt ¯Ctt. In appendix A, we establish the connection between the determinants in (4.2) and (4.3) and also show that the roots of P(α) = 0 must always be real and positive. If the roots of P(α) are labelled in ascending order, so that α1 ≤· · · ≤αN−1, then the critical value αc of α below which the chain is unstable must be given by αc = αN−1. (4.5) (4.5) αc = αN−1. Up to N = 50, the eigendecomposition shows that only a single eigenmode exhibits an exchange of stability at each bifurcation point. Moreover, when regarded as functions of α, two distinct eigenvalues ηi and ηj, i ̸= j, do not cross each other for the named range of N. 4. Stability of a chain clamped at its base In combination h- a 0 v- 2 v- 1 –1 0 1 2 0 1 2 3 4 5 6 ac h- 1 h- 2 a a = 4 a • Figure4. Eigenvalues ¯η1 and ¯η2 forthesingleclampedchainwithtwoballsasfunctionsofα andcorresponding(eigen)modes ¯v1 and ¯v2 for α →0, α = 4 and α →∞. For α < 4, the base configuration is unstable against perturbations in the direction of mode ¯v1. h- a –1 0 1 2 0 1 2 3 4 5 6 ac h- 1 h- 2 0 v- 2 v- 1 a a = 4 a • 10 rspa.royalsocietypublishing.org Proc. R.Soc. A 473: 20160703 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 Figure4. Eigenvalues ¯η1 and ¯η2 forthesingleclampedchainwithtwoballsasfunctionsofα andcorresponding(eigen)modes ¯v1 and ¯v2 for α →0, α = 4 and α →∞. For α < 4, the base configuration is unstable against perturbations in the direction of mode ¯v1. with (4.2) and (4.4), we can therefore conclude that ¯H has one family of N −1 conditionally stable (eigen)modes, where each mode loses stability at an α value associated with one unique root of P(α) = 0. Furthermore, there is a family of N −1 unconditionally stable modes which never destabilize for α > 0. These two families of modes exist because the gravitational contribution to the energy (2.8) is independent of the orientations of the dipole moments. To see this connection, note that the factoring of αN−1 in det ¯H is only possible because of the N −1 rank deﬁciency in ¯C (see appendix A). This rank deﬁciency arises simply because the gravitational energy is independent of the orientations of the dipole moments. (a) Stability threshold for a chain consisting of two balls Despite its simplicity, the case of N = 2 balls affords valuable insight. In this case, ¯H = ¯C + α ¯A0 = −1 0 0 0 + α 6 6 −3 −3 2 . (4.6) (4.6) ing −( ¯A−1 0 )tt ¯Ctt, we ﬁnd that P(α) = α −4 and, thus, that the critical value αc of α is given αN=2 c = 4. αN=2 c = 4. (4.7) (4.7) Moreover, we ﬁnd that ¯H possesses a conditionally stable eigenvalue Moreover, we ﬁnd that ¯H possesses a conditionally stable eigenvalue ¯η1 = 4α −3 −q 6 (4.8) (4.8) with q := 13α2 −12α + 9, satisfying ¯η1 < 0 for α < 4 and ¯η1 > 0 for α > 4, along with an unconditionally stable eigenvalue with q := 13α2 −12α + 9, satisfying ¯η1 < 0 for α < 4 and ¯η1 > 0 for α > 4, along with an unconditionally stable eigenvalue ¯η2 = 4α −3 + q 6 (4.9) (4.9) satisfying ¯η2 > 0. Plots of these eigenvalues and depictions of the corresponding modes, which obey satisfying ¯η2 > 0. Plots of these eigenvalues and depictions of the corresponding modes, which obey ¯v1 ∝ 3 −2α + q 3α and ¯v2 ∝ 3 −2α −q 3α (4.10) (4.10) appear in ﬁgure 4. appear in ﬁgure 4. Since our focus is on the stability of the base conﬁguration, ¯v1 and ¯v2 should not be mistaken for new equilibrium shapes. Rather, they describe the directions in which an inﬁnitesimal perturbation can be applied to the base conﬁguration. The sign of the corresponding eigenvalue 1 2 3 4 5 6 7 8 9 10 20 30 40 50 2 3 4 5 N ac= 750 log ac N = 8 acµ N3 Figure 5. Critical value αc of the dimensionless parameter α defined in (2.5) for the stability of the single clamped chain as a function of the number of balls N (red curve). Curves in black show the consecutive values of α corresponding to bifurcation points of the Hessian ¯H (4.1) at which a mode higher than the first mode loses stability. For a given N, we have N −1 conditionally stable modes and therefore N −1 bifurcation points. (a) Stability threshold for a chain consisting of two balls The dashed black lines demonstrate that, for α = 750 (which is the experimental value for the balls which appear in figures 1, 14 and 15), a chain consisting of no more than N = 8 balls is stable. The thick black line segment shows the asymptotic slope of αc as N →∞. 1 2 3 4 5 6 7 8 9 10 20 30 40 50 2 3 4 5 N ac= 750 log ac N = 8 acµ N3 11 Figure 5. Critical value αc of the dimensionless parameter α defined in (2.5) for the stability of the single clamped chain as a function of the number of balls N (red curve). Curves in black show the consecutive values of α corresponding to bifurcation points of the Hessian ¯H (4.1) at which a mode higher than the first mode loses stability. For a given N, we have N −1 conditionally stable modes and therefore N −1 bifurcation points. The dashed black lines demonstrate that, for α = 750 (which is the experimental value for the balls which appear in figures 1, 14 and 15), a chain consisting of no more than N = 8 balls is stable. The thick black line segment shows the asymptotic slope of αc as N →∞. then dictates whether or not the base conﬁguration is stable with respect to any inﬁnitesimal perturbation in the direction of the associated mode. (b) General stability threshold: asymptotic scaling for long chains The magnetic forces must be increasingly strong relative to gravity to keep a chain clamped at its base from bending as the number N of balls in the chain increases. Consistent with this observation, the critical value αc of α at which the base conﬁguration loses stability increases monotonically with N. What is perhaps surprising is how rapidly this increase occurs. In particular, for a chain of three balls αN=3 c = 4(293 + √ 50009)/105 ≈19.7, which represents a nearly 400% increase over the value αN=2 c = 4 for a chain of two balls. Figure 5 contains a plot of αc as a function of N together with various other non-vanishing values of α corresponding to bifurcations involving higher modes. Experiments conducted with balls of diameter 5 mm, mass 0.5 g and magnetic ﬂux density 1.19 T demonstrate that a chain of N = 8 such balls can be held straight without difﬁculty and that, with sufﬁcient care, it is also possible to hold a chain of N = 9 such balls straight. However, these experiments also demonstrate that it is impossible to hold a chain of N = 10 such balls straight. Photographs for chains with 9 and 10 balls are shown in ﬁgure 1. For the given experimental parameters, deﬁnition (2.5) of α yields α ≈750, which is slightly less restrictive than the theoretical prediction αN=9 c ≈790 that can be read off from ﬁgure 5 but well above the next lowest prediction αN=8 c ≈544. This small disparity between theory and experiment is likely due to the absence of frictional effects in our model. Inspection of ﬁgure 5 shows that, for sufﬁciently large values of N, αc scales with N3, as do the curves corresponding to bifurcations involving higher modes. Consistent with the discussion of Vella et al. [10], this scaling suggests that, in the continuum limit, a straight chain of magnetic balls behaves like a classical heavy elastica. Hall et al. [11] showed that the contributions to the 1 rspa.royalsocietypublishing.org Proc. R.Soc. A 473: 201607 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (b) General stability threshold: asymptotic scaling for long chains ac/N3 0.5 2 4 6 8 10 20 30 40 50 0.6 0.7 0.8 0.9 1.0 k¢ª 1.1187 k ª 1.2738 1.1 1.2 1.3 N Figure 6. Critical value αc (red curve) as in figure 5. The plot is scaled by N3. The solid blue line shows the critical asymptote α∞ c (4.12) for N →∞with the prefactor k from (4.13) obtained under the assumption of only local magnetic interactions. The dashed blue line is an asymptote analogue to (4.12) with the prefactor k′ obtained from Vella etal. [10]. ac/N3 0.5 2 4 6 8 10 20 30 40 50 0.6 0.7 0.8 0.9 1.0 k¢ª 1.1187 k ª 1.2738 1.1 1.2 1.3 N 12 rspa.royalsocietypublishing.org Proc. R.Soc. A 473: 20160703 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 Figure 6. Critical value αc (red curve) as in figure 5. The plot is scaled by N3. The solid blue line shows the critical asymptote α∞ c (4.12) for N →∞with the prefactor k from (4.13) obtained under the assumption of only local magnetic interactions. The dashed blue line is an asymptote analogue to (4.12) with the prefactor k′ obtained from Vella etal. [10]. energy of a chain of magnetic balls due to local and non-local magnetic interactions give rise to terms that also scale with N3 as N →∞. With this in mind, the local contribution to the energy calculated by Hall et al. [11] can be used to yield an effective bending stiffness Kloc = ζ(3)κ, κ := πB2d4 288μ0 = Mgd2α 12 , (4.11) (4.11) where ζ(3) = ∞ i=1 i−3 ≈1.202. Then, following Vella et al. [10] and using (4.11) in conjunction with Wang’s [13] reﬁnement of Greenhill’s [14] expression for the maximum height to which a heavy elastic standing column that is cantilevered at its base can be constructed without becoming unstable, we are led to the approximation where ζ(3) = ∞ i=1 i−3 ≈1.202. Then, following Vella et al. (b) General stability threshold: asymptotic scaling for long chains [10] and using (4.11) in conjunction with Wang’s [13] reﬁnement of Greenhill’s [14] expression for the maximum height to which a heavy elastic standing column that is cantilevered at its base can be constructed without becoming unstable, we are led to the approximation α∞ c = kN3, (4.12) (4.12) with the prefactor k being given by with the prefactor k being given by k = 16 3ζ(3)z2 1 ≈1.2738, (4.13) (4.13) where z1 ≈1.86635 is the ﬁrst zero of the Bessel function of the ﬁrst kind of order −1/3. The approximation (4.12)–(4.13) should become increasingly accurate in the continuum limit, namely as N →∞. The plots in ﬁgure 6 show that αc approaches α∞ c asymptotically from below and, thus, that α∞ c provides a reasonable approximation to αc for chains consisting of as few as N ≈30 balls. We therefore refer to α∞ c as the ‘critical asymptote’. In contrast to the generally valid local contribution (4.11) to the effective bending stiffness, the non-local contribution Knonloc depends on the current shape of the chain. For a closed ring, this contribution leads to Kring nonloc = κ/6, as shown by Vella et al. [10] and Hall et al. [11]. Even though the resulting total effective bending stiffness, Kring = Kloc + Kring nonloc = (ζ(3) + 1/6)κ, is exact only for the closed ring (see the ﬁnal sentence of Vella et al. [10, §2(d)]), Vella et al. [10] use it for the vertical clamped chain as well. With this assumption, Vella et al. [10, eqn. (3.2)] obtain an estimate for the critical value Nc of N as N →∞. In combination with the relation α = B2 4μ0ρgd = 1 8G (4.14) (4.14) h- 1= 0 h- 2ª 4.79 h- 3ª 28.51 h- 8ª 490.33 Figure 7. Thefirstthreemodes ¯v1, ¯v2 and ¯v3 andthefirstunconditionallystablemode ¯v8 forthesingleclampedchainofeight ballsatthecriticalαN=8 c ≈544wherethefirstmodedestabilizes.Thecorrespondingeigenvalueisindicatedbeloweachmode. h- 8ª 490.33 13 rspa.royalsocietypublishing.org Proc. R.Soc. A 473: 20160703 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 Figure 7. Thefirstthreemodes ¯v1, ¯v2 and ¯v3 andthefirstunconditionallystablemode ¯v8 forthesingleclampedchainofeight ballsatthecriticalαN=8 c ≈544wherethefirstmodedestabilizes.Thecorrespondingeigenvalueisindicatedbeloweachmode. between α and the magneto-gravitational number G of Vella et al. [10, eqn. (b) General stability threshold: asymptotic scaling for long chains (3.3)], that estimate leads to a relation of the form (4.12) but with a value k′ ≈1.1187 of the prefactor k slightly lower than that appearing in (4.13). Figure 6 shows that the corresponding approximation to αc does not provide a valid asymptote because it crosses αc between N = 11 and N = 12. Using the relation, Nc ≈0.4613G−1/3, between Nc and G corresponding to (4.12) and (4.13) also leads to an improvement on the ﬁt appearing in Fig. 5 of Vella et al. [10]. Since non- local interactions are neglected from the calculation leading to (4.12), this improvement raises a question about the extent to which the effective bending stiffness is sensitive to those interactions. Since the asymptotic analysis of Hall et al. [11] breaks down near the ends of a chain, a different strategy is needed to determine the non-local contribution to the effective bending stiffness. We expect several effects to be operative. One of these stems from the non-local interactions away from the boundaries and would yield a non-local term corresponding to that arising in the work of Hall et al. [11, eqn. (3.51)]. If, however, this were the only non-local effect, we would certainly obtain Kstraight chain nonloc < Kring nonloc = κ/6, because otherwise, as mentioned above, the relation αc = kN3 would intersect our theoretical estimate and, thus, would no longer provide an asymptote for large N. Another non-local effect stems from the endpoints of the chain, which in the continuum limit act as oppositely charged monopoles. Since bending a straight chain brings these attractive poles closer together and therefore decreases the energy, this effect lowers the non-local bending stiffness. The boundary conditions (such as the clamping of the chain at its base) would also exert an inﬂuence. The problem of determining the exact form of the prefactor entering the critical asymptote is an interesting problem but is beyond the scope of this paper. The results reported here nevertheless support the ﬁndings of Hall et al. [11] and Vella et al. [10] showing that, for inﬁnitesimal strains, it is reasonable to describe a chain of magnetic balls as an elastic rod. 5. Two clamped chains separated by a finite gap 14 We now return to the original problem involving two chains separated by a gap γ that is both positive and ﬁnite, with the stipulation that the lower chain is clamped at its base and the upper chain is clamped at its apex. For sufﬁcient small values of γ , we intuitively expect that magnetic interactions with the upper chain should stabilize the lower chain. Additionally, however, the magnetic force exerted on the upper chain by the lower chain can lead to rupture of the upper chain. We explore this possibility before considering the problem of stability in the absence of rupture. rspa.royalsocietypublishing.org Proc. R.Soc. A 473: 20160703 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (c) Sample modes for a chain consisting of eight balls For a chain of N = 8 balls with α = αN=8 c ≈544, several different modes are depicted in ﬁgure 7. Being at the critical α value, the ﬁrst mode destabilizes at this point and the corresponding eigenvalue vanishes. The shapes of the ﬁrst three modes are reminiscent of bending modes for a classical heavy elastic rod, which is reasonable in view of the discussion in the previous paragraph. Figure 7 also shows the eighth mode (out of 14 modes in total), which is the ﬁrst unconditionally stable mode. The nature of this mode differs fundamentally from the lower bending modes. The second ball (counting upward from the base) performs a strong sliding motion and the uppermost ball moves essentially longitudinally. (i) Rupture due solely to gravity We ﬁrst consider the upper chain in isolation, which can be achieved formally by taking the limit γ →∞and neglecting the lower chain entirely. In this context, the threshold for rupture is reached when the magnitude of the attractive magnetic force between the ball at the apex of the upper chain and its other N −1 balls is balanced by the gravitational force acting on those remaining balls. In dimensionless form, this criterion yields a relation between N and α: α N−1 i=1 1 i4 = N −1. (5.1) (5.1) Since the per cent error between the partial sum N−1 i=1 i−4 and its inﬁnite counterpart ζ(4) = ∞ i=1 i−4 = π4/90 decreases monotonically with N and is below 1% for N ≥4, (5.1) yields, to good approximation, an expression Since the per cent error between the partial sum N−1 i=1 i−4 and its inﬁnite counterpart ζ(4) = ∞ i=1 i−4 = π4/90 decreases monotonically with N and is below 1% for N ≥4, (5.1) yields, to good approximation, an expression Nr = 1 + π4α 90 (5.2) (5.2) for the critical number of balls at which rupture occurs. Thus, Nr grows linearly with α. In combination, the deﬁnition (2.5) of α and (5.2) give rise to an expression for the critical length for the critical number of balls at which rupture occurs. Thus, Nr grows linearly with α. In combination, the deﬁnition (2.5) of α and (5.2) give rise to an expression for the critical length ℓr := Nrd = d + π4B2 360μ0ρg (5.3) (5.3) of a chain on the threshold of rupture. Granted that α ≫1, (5.2) can be approximated by Nr = π4α/90 and (5.3) leads us to infer that the critical length ℓr at which a vertically hanging chain clamped at its apex ruptures is independent of the size of the balls from which it is made. of a chain on the threshold of rupture. Granted that α ≫1, (5.2) can be approximated by Nr = π4α/90 and (5.3) leads us to infer that the critical length ℓr at which a vertically hanging chain clamped at its apex ruptures is independent of the size of the balls from which it is made. al rupture (a) Rupture of the upper chain Although we formulated the problem under the assumption that any pair of balls which are in contact in the base conﬁguration remain in contact, it is important to recognize that the magnetic interactions that maintain the integrity of the upper chain must compete with the downward forces exerted by gravity and by magnetic interactions with the balls of the lower chain. Since the upper chain is clamped at its apex, we expect that any rupture must occur at the junction between the clamped ball i = 2N at its apex and its immediate neighbour, ball i = 2N −1. (ii) General rupture If the magnetic force exerted by the lower chain on the upper chain is taken into consideration, then the balance (5.1) between magnetic and gravitational forces generalizes to If the magnetic force exerted by the lower chain on the upper chain is taken into consideration, then the balance (5.1) between magnetic and gravitational forces generalizes to α N−1 i=1 1 i4 − N k=1 1 (i + k + γ −1)4 = N −1. (5.4) (5.4) Numerical methods can be used to extract from (5.4) a relation for the onset of rupture in which one of the three parameters N, α and γ is given in terms of the other two. Numerical methods can be used to extract from (5.4) a relation for the onset of rupture in which one of the three parameters N, α and γ is given in terms of the other two. A relatively simple case arises in the limit α →∞corresponding to situations where gravity is dominated by magnetic interactions. Consistent with intuition, it is evident from (5.4) that rupture induced by the attractive magnetic interactions between the chains can still occur in the absence of gravity. Increasing N incrementally, we ﬁnd that the critical gap γr corresponding to the onset of rupture varies between γ N=2 r ≈0.0157 and γ N→∞ r ≈0.0296, which represents a relatively small range of gaps. Also consistent with intuition is the observation that, in the limit α →∞, the lower and upper chains become indistinguishable. Thus, rupture should occur not only in the upper chain but also mirror-symmetrically (with respect to the line through the midpoint of the gap and orthogonal to the chains) in the lower chain as well. 15 rspa.royalsocietypublishing.org Proc. R.Soc. A 473: 20160703 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (i) Stability threshold for chains consisting of two balls In contrast to what occurs for a single isolated chain consisting of N = 2 balls, it is impractical to provide expressions for the eigenvalues and associated modes of ˜H, at least for arbitrary choices of α > 0 and 0 < γ < ∞. With some effort, however, αN=2 c (γ ) can be expressed in full generality (as shown in appendix B). For instance, for γ = 1 (which corresponds to a gap length equal to the diameter of a single ball), we obtain a critical value αN=2 c (1) ≈1.784 of α that is less than one- half of the corresponding value αc = 4 for a single isolated chain clamped at its base. In general, the function αN=2 c (γ ) increases monotonically from αN=2 c (γ →0) ≈0.3166 to the asymptotic value αc = 4, with a steep initial rise; in particular, at γ = 5 we ﬁnd that αN=2 c (5) ≈3.8622. (b) Stability of the base configuration For clamped chains separated by a gap γ , the Hessian ˜H of the base conﬁguration is now the 4(N −1) × 4(N −1) matrix that results on removing the rows and columns of (3.15) as described in §3d and illustrated in ﬁgure 3. Proceeding as in the derivation of ¯H, we ﬁnd that det ˜H =: α2(N−1)P−(α; γ )P+(α; γ ), (5.5) det ˜H =: α2(N−1)P−(α; γ )P+(α; γ ), (5.5) where P±(α; γ ) is a polynomial of order N −1 in α for each ﬁnite choice of γ . Whereas the roots α+ i (γ ), i = 1, . . . , N −1, of P+ are positive, the roots α− i (γ ), i = 1, . . . , N −1, of P−are negative. Thus, for each 0 < γ < ∞, there are at most N −1 bifurcation points α+ i (γ ), i = 1, . . . , N −1, associated with the solutions to P+(α; γ ) = 0. Therefore, there are N −1 conditionally stable modes but 3(N −1) unconditionally stable modes. If, as in the treatment of the problem involving a single chain, the roots of P+(α; γ ) are arranged in ascending order, so that α+ 1 ≤· · · ≤α+ N−1, then the critical value αc of α below which the lower chain is unstable must be α+ N−1. (iii) Sample modes for chains of eight Balls The ﬁrst three modes for a chain of N = 8 balls with α = 75 at the critical gap γc ≈2.907 are depicted in ﬁgure 9. The ﬁrst mode is unstable for this combination of parameter values and the corresponding eigenvalue vanishes. 6. Two equal free chains We now consider the consequence of relaxing the boundary conditions to allow the ball at the base of the lower chain to both rotate and slide while the ball at the apex of the upper chain is allowed to rotate but cannot slide. Under these conditions, the lower chain will always topple without the presence of the upper chain. That presence is not, however, sufﬁcient to guarantee that the lower chain will not topple. Moreover, the magnetic force exerted by the upper chain may cause the lower chain to lose contact with the base. We explore this possibility before considering the possibility of stability in the absence of such a lift off. (ii) General stability threshold . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Figure8. Criticalgapγc (black)belowwhichtwoequalclampedchains(eachcontainingN balls)arestable.Severalcurvesfor different values of α are plotted. Orange lines show the γr below which the upper chain will rupture (§5a). Cyan areas indicate the allowed ranges for N and γ to have stable chains that do not rupture for a given α. The blue line exemplarily shows the asymptotic behaviour of γc as N →∞. (ii) General stability threshold For each combination of N and α, it is generally possible to avoid rupture only for a certain range of the gap γ . If, in particular, conditions are such that the lower chain is stable in isolation, then γ can be arbitrarily large. If, alternatively, the lower chain is not stable in isolation, then γ must be small enough for the upper chain to stabilize the lower one but not so small that the upper chain ruptures. Figure 8 contains a plot of the critical value γc of the gap at which the base conﬁguration loses stability versus the number N of balls in each chain for various values of α. In particular, for α = 750, the plot shows that the base conﬁguration is stable for N = 20 balls if γ is less than γ N=20 c ≈4.7. c From the stability analysis, we obtain the scaling for the asymptotic behaviour of γc for N →∞ γc = ˜k α N 1/3 −1, (5.6) γc = ˜k α N 1/3 −1, (5.6) with ˜k being a constant of order unity. with ˜k being a constant of order unity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 10 1 2 3 4 5 6 7 8 910 20 30 40 50 0.1 0.01 N γc a = 7.5 a = 7.5 a = 75 a = 750 γc= k˜(a/N)1/3– 1 a = 750 a = 75 a • Figure8. Criticalgapγc (black)belowwhichtwoequalclampedchains(eachcontainingN balls)arestable.Severalcurvesfor different values of α are plotted. Orange lines show the γr below which the upper chain will rupture (§5a). Cyan areas indicate the allowed ranges for N and γ to have stable chains that do not rupture for a given α. The blue line exemplarily shows the asymptotic behaviour of γc as N →∞. 30 10 1 2 3 4 5 6 7 8 910 20 30 40 50 0.1 0.01 N γc a = 7.5 a = 7.5 a = 75 a = 750 γc= k˜(a/N)1/3– 1 a = 750 a = 75 a • 16 16 rspa.royalsocietypublishing.org Proc. R.Soc. A 473: 20160703 . . . . . . (b) General stability threshold If the ball at the base of the lower chain is free to slide and rotate and the ball at the apex of the upper chain is free to rotate, the Hessian ˆH of the base conﬁguration is the (4N −1) × (4N −1) matrix that results on deleting the row and column corresponding to t2N from (3.15). Figure 10 contains a plot of the critical value γc of the gap γ at which the base conﬁguration becomes unstable versus the number N of balls in each chain for various values of α. Owing to the possibility of lift off, the range of feasible gaps diminishes monotonically with increasing N. Since the lower chain is never stable without the upper chain, the curves for γc extend to N = 2 for all values of α. For α = 750, the range of γc shrinks from [γ N=2 l , γ N=2 c ] = [4.42, 7.47] to [γ N=50 l , γ N=50 c ] = [1.44, 1.53]. The curves appearing in ﬁgure 10 that depict the critical value γ r of γ associated with the rupture of the upper chain at the junction between its two uppermost balls are identical to those contained in ﬁgure 8. The results show that the values of N and α can combine to decide whether the upper chain ruptures or the lower chain lifts off. If, for example, α = 7.5 and N = 5, then rupture occurs at a value of γ higher than that required to induce lift off. The scaling of γc for N →∞differs from that appearing in (5.6) only to the extent that the f ˆk k diff l I l h l i l f d i p γ g q The scaling of γc for N →∞differs from that appearing in (5.6) only to the extent that the prefactor ˆk takes a different value. Importantly, that value is also of order unity. (a) Lift off of the lower chain The threshold for lift off of the lower chain should occur when the magnitude of the attractive magnetic force between the upper chain and the lower chain is balanced by the gravitational force acting on the lower chain. In dimensionless form, this criterion yields a condition analogous to conditions (5.1) and (5.4) that arise in our discussion of rupture: α N i=1 N k=1 1 (i + k + γ −1)4 = N. (6.1) (6.1) It is intuitively evident that for given values of N and α, the critical gap γl for the lift off of the lower chain must be less than the critical value γc associated with deviations from the coaxial, rectilinear shape of the base conﬁguration. It is intuitively evident that for given values of N and α, the critical gap γl for the lift off of the lower chain must be less than the critical value γc associated with deviations from the coaxial, rectilinear shape of the base conﬁguration. h˜1= 0 h˜2ª 0.389 h˜3ª 0.738 Figure 9. The first three modes ˜v1, ˜v2 and ˜v3 for two clamped chains, each containing eight balls with α = 75, separated by the critical gap γc ≈2.907 where the first mode destabilizes. The corresponding eigenvalue is indicated below each mode. 17 Figure 9. The first three modes ˜v1, ˜v2 and ˜v3 for two clamped chains, each containing eight balls with α = 75, separated by the critical gap γc ≈2.907 where the first mode destabilizes. The corresponding eigenvalue is indicated below each mode. (c) Sample modes for chains of eight balls The ﬁrst three modes for a chain of N = 8 balls with α = 75 at the critical gap γc ≈1.330 are depicted in ﬁgure 11. The ﬁrst mode is unstable for this combination of parameters and the corresponding eigenvalue vanishes. It is interesting to observe that for the ﬁrst mode (leftmost panel in ﬁgure 11), the upper chain is very nearly straight and, thus, remains very close to its base conﬁguration. For that ﬁrst mode, the lowest ball in the upper chain has the largest horizontal 2 3 4 5 6 7 8 910 20 30 40 50 N γc 0.01 0.1 1 10 a = 750 a = 75 a = 7.5 a = 7.5 a = 75 a = 750 a • γc= kˆ(a/N)1/3– 1 Figure10. Criticalgapγc (black)belowwhichtwofreechains,eachcontainingN balls,arestableagainstbucklingofthelower chain.Severalcurvesfordifferentvaluesofα areplotted.Orangelinesshowthevalueγr ofγ belowwhichtheupperchainwill rupture (§5a). Magenta lines show the value γl of γ below which the lower chain will exhibit lift off (§6a). Cyan areas indicate the allowed ranges for N and γ to have stable chains that neither rupture nor lift off for a given α. The blue line exemplarily shows the asymptotic behaviour of γc as N →∞. 2 3 4 5 6 7 8 910 20 30 40 50 N γc 0.01 0.1 1 10 a = 750 a = 75 a = 7.5 a = 7.5 a = 75 a = 750 a • γc= kˆ(a/N)1/3– 1 18 rspa.royalsocietypublishing.org Proc. R.Soc. A 473: 20160703 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 Figure10. Criticalgapγc (black)belowwhichtwofreechains,eachcontainingN balls,arestableagainstbucklingofthelower chain.Severalcurvesfordifferentvaluesofα areplotted.Orangelinesshowthevalueγr ofγ belowwhichtheupperchainwill rupture (§5a). Magenta lines show the value γl of γ below which the lower chain will exhibit lift off (§6a). Cyan areas indicate the allowed ranges for N and γ to have stable chains that neither rupture nor lift off for a given α. The blue line exemplarily shows the asymptotic behaviour of γc as N →∞. hˆ1= 0 hˆ2ª 0.121 hˆ3ª 0.714 Figure 11. The first three modes ˆv1, ˆv2 and ˆv3 for two free chains, each containing eight balls with α = 75, separated by the critical gap γc ≈1.330 where the first mode destabilizes. (c) Sample modes for chains of eight balls The corresponding eigenvalue is indicated below each mode. Figure 11. The first three modes ˆv1, ˆv2 and ˆv3 for two free chains, each containing eight balls with α = 75, separated by the critical gap γc ≈1.330 where the first mode destabilizes. The corresponding eigenvalue is indicated below each mode. deﬂection (to the right) of all balls in the upper chain, which is only about 0.5% of the diameter of a single ball. 7. Chains with opposing dipole orientations We now brieﬂy consider a class of base conﬁgurations in which the dipole moments are arranged so that the chains interact repulsively. Speciﬁcally, we suppose that the dipole moments of 19 rspa.royalsocietypublishing.org Proc. R.Soc. A 473: 20160703 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 2 4 6 8 10 20 40 60 3 4 5 6 7 8 910 20 30 40 50 N γc γc µ N a = 75 a = 750 a = 7500 Figure12. Criticalgapγc (black)abovewhichtwochains(eachcontainingNballs)withopposingdipoleorientationsarestable. Severalcurvesfordifferentvaluesofα areincludedintheplot.Whiledashedlinesrefertosituationswheretheballattheapex of the upper chain can rotate, the solid lines pertain to situations where that ball is clamped. The red line depicts the limit α →∞for the clamped upper chain and the thick black line segment shows the respective asymptotic slope for N ≫1. 2 2 4 6 8 10 20 40 60 3 4 5 6 7 8 910 20 30 40 50 N γc γc µ N a = 75 a = 750 a = 7500 19 19 rspa.royalsocietypublishing.org Proc. R.Soc. A 473: 20160703 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Figure12. Criticalgapγc (black)abovewhichtwochains(eachcontainingNballs)withopposingdipoleorientationsarestable. Severalcurvesfordifferentvaluesofα areincludedintheplot.Whiledashedlinesrefertosituationswheretheballattheapex of the upper chain can rotate, the solid lines pertain to situations where that ball is clamped. The red line depicts the limit α →∞for the clamped upper chain and the thick black line segment shows the respective asymptotic slope for N ≫1. the balls contained in the upper chain point downward while the balls in the lower chain point upward. For sufﬁciently large values of the parameter α and the gap γ , the effective bending stiffness of the chains is enough to ensure that the base conﬁguration is stable. However, the base conﬁguration is unstable below a certain critical value γc of γ depending on N and α. 7. Chains with opposing dipole orientations As in the study of a single clamped chain presented in §4, it is crucial that the ball at the base of the lower chain be clamped. Otherwise the system is unconditionally unstable. Importantly, however, no analogous restriction exists for the upper chain and we will consider situations in which the ball at the apex of that chain is either clamped or ﬁxed horizontally while being allowed to rotate. p p y g Before presenting results, we summarize the most salient differences in the formulation that ensue if the dipole moments are arranged as explained above. First, (3.10) is replaced by ni = sie1 + e2, i = 1, . . . , N, sie1 −e2 i = N + 1, . . . , 2N. (7.1) (7.1) Second, although the Hessian still admits a decomposition of the form (3.15), the magnetic contribution ˇA is indeﬁnite instead of positive-semideﬁnite. This is in keeping with the intuitive expectation that repulsive magnetic interactions can also destabilize the system independent of gravity. Moreover, ˇA takes the form ˇA = ˇA0 + 2N−1 n=1 4 l=0 ˇAlnγ i (γ + n)7 (7.2) (7.2) and thus depends on γ at different orders. Here, ˇA0 and ˇAln are 10N −4 coefﬁcient matrices with rational entries depending only on N for each combination of l = 0, 1, 2, 3, 4 and n = 1, . . . , 2N −1. and thus depends on γ at different orders. Here, ˇA0 and ˇAln are 10N −4 coefﬁcient matrices with rational entries depending only on N for each combination of l = 0, 1, 2, 3, 4 and n = 1, . . . , 2N −1. (a) General stability threshold Figure 12 contains a plot of the critical value γc of the gap at which the base conﬁguration loses stability versus the number N of balls in each chain for various values of α. If both chains are clamped, then γc is bounded below as α →∞. In this limit, the base conﬁguration is symmetric (again with respect to the line through the midpoint of the gap and orthogonal to the chains). Applying the additional limit of N ≫1, we ﬁnd a linear dependence hˇ1= 0 hˇ2ª 0.682 hˇ3ª 18.011 hˇ4ª 19.151 hˇ1= 0 hˇ2ª 0.383 hˇ3ª 9.290 hˇ4ª 18.423 γ = γcª 6.789 (upper chain clamped) γ = γcª 8.370 (upper chain not clamped) (b) (a) Figure13. Thefirstfourmodes ˇv1, ˇv2, ˇv3 and ˇv4 fortwochains,eachcontainingsixballswithα = 750,separatedbythecritical gap γc ≈6.789 (a) and γc ≈8.370 (b) where the first mode becomes unstable. In (a), both chains are clamped. In (b), only the lower chain is clamped and the upper is fixed and allowed to rotate at its apex. The corresponding eigenvalue is indicated below each mode. γ = γcª 8.370 (upper chain not clamped) (b) 20 rspa.royalsocietypublishing.org Proc. R.Soc. A 473: 20160703 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 γ = γcª 6.789 (upper chain clamped) (a) Figure13. Thefirstfourmodes ˇv1, ˇv2, ˇv3 and ˇv4 fortwochains,eachcontainingsixballswithα = 750,separatedbythecritical gap γc ≈6.789 (a) and γc ≈8.370 (b) where the first mode becomes unstable. In (a), both chains are clamped. In (b), only the lower chain is clamped and the upper is fixed and allowed to rotate at its apex. The corresponding eigenvalue is indicated below each mode. of γc on N with a prefactor of order unity (γc ≈1.172N). It can therefore be reasoned that the base conﬁguration is stable only if the chains are separated by a gap no less than their length. The collection of stable combinations of γ and N must lie above the curve provided by γc(N) as α →∞. That such a lower bound exists is intuitively clear, because increasing α not only increases the effective bending stiffness of the chains but also increases the repulsive forces between the chains. (a) General stability threshold Whereas the ﬁrst of these effects is stabilizing, the second is destabilizing. If the upper chain is not clamped, the threshold for stability decreases relative to the clamped case and the stability is lost at a larger value of the gap. The difference is more pronounced for small values of N and large values of α. Furthermore, γc(N) has a global minimum for given α (dashed lines in ﬁgure 12). For decreasing N, however, γc is again increasing because with fewer balls the upper chain can overcome gravity more easily and rotate away from vertical. (b) Sample modes for chains of six balls Figure 13 contains the ﬁrst four modes of the system with N = 6 for α = 750 at the critical gap γc, for situations in which both chains are clamped and in which the lower chain is clamped while the upper chain is ﬁxed horizontally with the ball at its apex being allowed to rotate. 21 rspa.royalsocietypublishing.org Proc. R.Soc. A 473: 20160703 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . While we treated only problems involving chains with equal numbers of balls, our method is immediately applicable to chains with different numbers of balls. Moreover, with a minor extension, our approach can be used to treat chains consisting of balls of different sizes. A potentially limiting feature of our model arises from neglecting the frictional forces that act between the balls and which might, at least under certain circumstances, act to stabilize chains against shape changes. A ﬁrst step towards incorporating these potentially important effects would be to allow for frictionless rolling contact between the balls while neglecting sliding contact. Such a generalization would couple the positional and orientational degrees of freedom while reducing by half the total number of degrees of freedom. Another possible approach would involve including static friction. However, this would make it necessary to consider discontinuous bifurcations, which fall outside the scope of our current approach. Many other possible equilibrium conﬁgurations involving assemblies of magnetic balls could be studied for their stability properties. One simple generalization of the conﬁguration studied in this paper arises on rotating the vertical chains by 90◦, so that their ends are attached to vertical walls and form a shape reminiscent of the catenary curve of a classical heavy chain supported at the end points (ﬁgure 14). Under such circumstances, the equilibrium conﬁguration is generally curved and its description would almost certainly need to be determined numerically, preventing the possibility of calculating the energy and, hence, the Hessian in exact form. Another fascinating problem involves cylindrical tubes made from sheets of magnetic balls packed and oriented in various ways. In analogy to the single vertical chain, there should be critical parameter values at which such cylinders buckle under the inﬂuence of gravity (ﬁgure 15). A minimal example of such a cylinder consists of a conﬁguration in which eight balls sit at the corners of a cube. It would be fascinating to investigate the stability of such a cubic conﬁguration because, as Schönke et al. [15] recently demonstrated, its ground state is known to be energetically degenerate with a continuum of possible dipole orientations. What happens to the continuum of states at a bifurcation point? The sheet made from magnetic balls that is shown in ﬁgure 15 has a square packing and can therefore be locally deformed in numerous ways. For example, it can be sheared without rupturing the connections between the balls. 8. Discussion We investigated the linear stability of two interacting chains of magnetic balls, described as constrained mechanisms, subject to gravitational and magnetic forces. Our formulation yields the entries of the Hessian of the base conﬁguration in exact form. With the aid of a symbolic manipulation programme such as Mathematica, we obtained the eigenvalues and (eigen)modes of that Hessian up to machine precision. We found that the space of essential parameters N (number of balls), γ (dimensionless gap size) and α (strength of magnetic forces relative to gravity) has a rich structure with stable and unstable domains. That structure depends only on the applied boundary conditions for the endpoints of the chains which are in contact with the ground and the ceiling. For N sufﬁciently large, we conﬁrmed previous analyses showing that a single vertical chain of balls clamped at its base behaves like a classical heavy elastica cantilevered at its base. This supports the notion of an effective bending stiffness induced by the magnetic interactions for this system. Furthermore, we discovered two distinct families of (eigen)modes. While one family is conditionally stable, meaning that the modes lose stability for some non- zero values of α, the other family is unconditionally stable and its modes destabilize only if the magnetic interactions are fully switched off (α = 0). This can be viewed as a degeneracy that arises because the gravitational energy of the system does not depend on the degrees of freedom corresponding to the orientations of the magnetic dipole moments. The stability threshold for the buckling of the single chain was found to agree well with some simple experimental tests. For two chains separated by a ﬁnite gap, we encountered two instabilities distinct from the buckling instability of the lower chain that involve changes in the topology of the system. The upper chain can rupture due to gravity and/or the attractive force exerted by the lower chain. Moreover, the lower chain, if not ﬁxed at its base, can lift off due to the attractive force exerted by the upper chain. These additional instabilities can considerably reduce the sets of stable parameter combinations. 21 21 rspa.royalsocietypublishing.org Proc. R.Soc. A 473: 20160703 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A hexagonal packing in which each interior ball has six neighbours provides a fundamentally different way to pack the balls in a sheet. Maintaining the connections between the balls for such a sheet corresponds to a system which is locally incapable of extension or contraction and thus serves as a discrete model for approximately two- dimensional locally unstretchable materials like paper. Owing to the much reduced number of degrees of freedom of such assemblies, a cylinder with hexagonal packing is much more stable against buckling than is a cylinder with square packing. We therefore have the opportunity to investigate the structural stability of approximately two-dimensional materials with different properties. Since there is always a local orientation of the dipoles in a sheet made from magnetic (e) ( f ) (b) (a) (c) (d) (g) Figure 14. Stability of two horizontal clamped chains separated by a finite gap. Each chain contains 11 balls of diameter 5 mm, mass 0.5 g and magnetic flux density 1.19 T. In (a), the chains are nearly horizontal and are separated by approximately 1.7 mm andthedistancebetweenthecentresofthetwoclampedballsisapproximately106.7 mm.In(b–h),thegripontheleftisheld fixed and the grip on the right is progressively moved to the right. The approximate distances between the centres of the two clamped balls are (b) 109.7, (c) 112.7, (d) 115.1, (e) 115.6, (f) 116.1 and (g) 116.3 mm. 22 Figure 14. Stability of two horizontal clamped chains separated by a finite gap. Each chain contains 11 balls of diameter 5 mm, mass 0.5 g and magnetic flux density 1.19 T. In (a), the chains are nearly horizontal and are separated by approximately 1.7 mm andthedistancebetweenthecentresofthetwoclampedballsisapproximately106.7 mm.In(b–h),thegripontheleftisheld fixed and the grip on the right is progressively moved to the right. The approximate distances between the centres of the two clamped balls are (b) 109.7, (c) 112.7, (d) 115.1, (e) 115.6, (f) 116.1 and (g) 116.3 mm. (a) (b) Figure 15. Stability of cylindrical tubes made from square packings of magnetic balls of diameter 5 mm, mass 0.5 g and magnetic flux density 1.19 T. Each tube is formed by stacking circular rings consisting of 60 balls. While a stack of 10 such rings is stable (a), a stack of 12 such rings buckles under the influence of gravity (b). Figure 15. Stability of cylindrical tubes made from square packings of magnetic balls of diameter 5 mm, mass 0.5 g and magnetic flux density 1.19 T. Data accessibility. All data are contained within the text. Data accessibility. All data are contained within the text. ’ contributions. Both authors contributed to the conception of the theoretical framework and wrote t J.S. coded and ran the software programs used to generate the numerical data. paper. J.S. coded and ran the software programs used to generate the numerical data. Competing interests. We declare we have no competing interests. Funding. The authors gratefully acknowledge support from the Okinawa Institute of Science and Technology Graduate University with subsidy funding from the Cabinet Ofﬁce, Government of Japan. Acknowledgements. The authors thank Prof. Tadashi Tokieda for encouraging them to investigate the stability of chains with opposing dipole moments. nterests. We declare we have no competing interests. Competing interests. We declare we have no competing interests. Competing interests. We declare we have no competing interests. Funding. The authors gratefully acknowledge support from the Okinawa Institute of Science and Technology Graduate University with subsidy funding from the Cabinet Ofﬁce, Government of Japan. Funding. The authors gratefully acknowledge support from the Okinawa Institute of Science and Technology Graduate University with subsidy funding from the Cabinet Ofﬁce, Government of Japan. Acknowledgements. The authors thank Prof. Tadashi Tokieda for encouraging them to investigate the stability of chains with opposing dipole moments Acknowledgements. The authors thank Prof. Tadashi Tokieda for encouraging them to investigate the stability of chains with opposing dipole moments. Appendix A. Structure of the determinant of the Hessian for a chain clamped at its base In this appendix, we establish the relation In this appendix, we establish the relation det( ¯A−1 0 ¯C + αI2(N−1)) = αN−1 det[( ¯A−1 0 )tt ¯Ctt + αIN−1] (A 1) (A 1) and prove that P(α) = 0 has only real and positive roots in α. ¯ −1 ¯ and prove that P(α) = 0 has only real and positive roots in α. Si ¯A−1 d ¯C h bl k t t and prove that P(α) = 0 has only real and positive roots in α. Since ¯A−1 0 and ¯C have block structures and prove that P(α) 0 has only real and po Since ¯A−1 0 and ¯C have block structures ¯A−1 0 and ¯C have block structures ¯A−1 0 = ( ¯A−1 0 )tt ( ¯A−1 0 )ts ( ¯A−1 0 )st ( ¯A−1 0 )ss and ¯C = ¯Ctt 0 0 0 , (A 2) (A 2) with each block being a (N −1) × (N −1) matrix, their product is given by with each block being a (N −1) × (N −1) matrix, their product is given by ¯A−1 0 ¯C = ( ¯A−1 0 )tt ¯Ctt 0 ( ¯A−1 0 )st ¯Ctt 0 =: −¯B 0 −¯B′ 0 . (A 3) (A 3) It therefore follows from (A 1) that det( ¯A−1 0 ¯C + αI2(N−1)) = det −¯B + αIN−1 0 −¯B′ αIN−1 . (A 4) (A 4) We now expand the determinant along each of the N −1 columns starting from column N until the last column 2(N −1) of the matrix in (A 4). Each column gives another factor of α times the determinant of the reduced matrix which results from removing the corresponding column and the row of the same index, leading ﬁnally to We now expand the determinant along each of the N −1 columns starting from column N until the last column 2(N −1) of the matrix in (A 4). Each column gives another factor of α times the determinant of the reduced matrix which results from removing the corresponding column and the row of the same index, leading ﬁnally to det( ¯A−1 0 ¯C + αI2(N−1)) = αN−1 det(−¯B + αIN−1), (A 5) (A 5) which is (A 1). Since ¯A0 is positive-deﬁnite, so is ¯A−1 0 . Thus, by Sylvester’s criterion, ( ¯A−1 0 )tt is also positive-deﬁnite. Because ¯Ctt is negative-deﬁnite, it follows from Sylvester’s law of inertia that ¯B = −( ¯A−1 0 )tt ¯Ctt has only real and positive eigenvalues regardless of whether ¯B is symmetric (which it is not for N > 2). 21 rspa.royalsocietypublishing.org Proc. R.Soc. A 473: 20160703 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Each tube is formed by stacking circular rings consisting of 60 balls. While a stack of 10 such rings is stable (a), a stack of 12 such rings buckles under the influence of gravity (b). balls, we even have a macroscopic model system in which material anisotropy plays a role. The local bending stiffness, for example, should depend on direction. Moreover, if tearing occurs, it will occur preferentially in a direction parallel to the dipole direction of the magnetic balls. Beyond the linear, inﬁnitesimal analysis presented here, an important next step would be to conduct a ﬁnite amplitude analysis of the system, or even more generally, full dynamical simulations. This would allow for studies of the dynamical processes stemming from arbitrary initial conditions. Moreover, for time-dependent periodic boundary conditions, investigations of interesting phenomena like standing waves and of the frequency response of the system could be performed. For certain parameter regimes, it seems reasonable to expect chaotic behaviour from these dynamical systems. initial conditions. Moreover, for time-dependent periodic boundary conditions, investigations of interesting phenomena like standing waves and of the frequency response of the system could be performed. For certain parameter regimes, it seems reasonable to expect chaotic behaviour from these dynamical systems. 23 23 rspa.royalsocietypublishing.org Proc. R.Soc. A 473: 20160703 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Data accessibility. All data are contained within the text. Authors’ contributions. Both authors contributed to the conception of the theoretical framework and wrote the paper. J.S. coded and ran the software programs used to generate the numerical data. Competing interests. We declare we have no competing interests. Funding. The authors gratefully acknowledge support from the Okinawa Institute of Science and Technology Graduate University with subsidy funding from the Cabinet Ofﬁce, Government of Japan. Acknowledgements. The authors thank Prof. Tadashi Tokieda for encouraging them to investigate the stability of chains with opposing dipole moments. 24 rspa.royalsocietypublishing.org Proc. R.Soc. A 473: 20160703 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 αN=2 c (γ ) = 2(2 + γ )5 (1 + γ )9 12 i=0 aiγ i 31 j=0 biγ i , (B 1) where the coefﬁcients ai, i = 0, . . . , 12 and bi, i = 0, . . . , 31 are given by where the coefﬁcients ai, i = 0, . . . , 12 and bi, i = 0, . . . , 31 are given by i ai bi i bi 0 1 092 44 623 017 16 236 359 277 059 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 6 744 622 228 431 17 121 855 281 861 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 rspa.royalsocietypublishing.org Proc. R.Soc. A 473: 20160703 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 20 364 4 275 119 481 18 56 547 295 271 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 39 280 19 240 414 007 19 23 558 396 425 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 rspa.royalsocietypublishing.org Proc. R.Soc. A 473: 20160703 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 53 448 63 645 780 965 20 8 773 187 011 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 53 676 164 687 108 579 21 2 902 235 365 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 rspa.royalsocietypublishing.org Proc. 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A 473: 20160703 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 40 507 346 552 984 093 22 845 788 851 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 22 968 608 997 759 203 23 214 853 101 . . . . . . . . . . . 24 rspa.royalsocietypublishing.org Proc. R.Soc. A 473: 20160703 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 9 636 911 043 297 553 24 46 945 991 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 rspa.royalsocietypublishing.org Proc. R.Soc. A 473: 20160703 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 2 896 1 177 182 472 519 25 8 676 897 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 588 1 328 723 896 453 26 1 327 627 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 rspa.royalsocietypublishing.org Proc. R.Soc. A 473: 20160703 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 72 1 321 880 254 507 27 163 365 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 4 1 167 341 129 409 28 15 511 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 rspa.royalsocietypublishing.org Proc. R.Soc. A 473: 20160703 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 920 178 840 607 29 1 065 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 650 210 370 673 30 47 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References 1. 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A 473: 20160703 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 413 082 353 055 31 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Consistent with the results of §4a, it follows easily from (B 1) that αN=2 c (γ ) →4 = αN=2 c as γ →∞. Consistent with the results of §4a, it follows easily from (B 1) that αN=2 c (γ ) →4 = αN=2 c as γ →∞. x B. Critical behaviour of two clamped chains each consisting ll . . . . . . loses stability. That relation has the form loses stability. That relation has the form x B. Critical behaviour of two clamped chains each consisting ll For a pair of clamped chains, each consisting of N = 2 balls, separated by a gap γ (§5), it is possible to obtain an explicit relation for the critical value αc of α at which the position of the lower chain For a pair of clamped chains, each consisting of N = 2 balls, separated by a gap γ (§5), it is possible to obtain an explicit relation for the critical value αc of α at which the position of the lower chain 24 rspa.royalsocietypublishing.org Proc. R.Soc. A 473: 20160703 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . loses stability. That relation has the form αN=2 c (γ ) = 2(2 + γ )5 (1 + γ )9 12 i=0 aiγ i 31 j=0 biγ i , (B 1) where the coefﬁcients ai, i = 0, . . . , 12 and bi, i = 0, . . . , 31 are given by i ai bi i bi 0 1 092 44 623 017 16 236 359 277 059 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . x B. Critical behaviour of two clamped chains each consisting ll . . . . . . . . . . . . . . . . . . . 1 6 744 622 228 431 17 121 855 281 861 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 20 364 4 275 119 481 18 56 547 295 271 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . x B. Critical behaviour of two clamped chains each consisting ll . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 39 280 19 240 414 007 19 23 558 396 425 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 53 448 63 645 780 965 20 8 773 187 011 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . x B. Critical behaviour of two clamped chains each consisting ll . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 53 676 164 687 108 579 21 2 902 235 365 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 40 507 346 552 984 093 22 845 788 851 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . x B. Critical behaviour of two clamped chains each consisting ll . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 22 968 608 997 759 203 23 214 853 101 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 9 636 911 043 297 553 24 46 945 991 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . x B. Critical behaviour of two clamped chains each consisting ll . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 2 896 1 177 182 472 519 25 8 676 897 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 588 1 328 723 896 453 26 1 327 627 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . x B. Critical behaviour of two clamped chains each consisting ll . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 72 1 321 880 254 507 27 163 365 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 4 1 167 341 129 409 28 15 511 . . . . . . . . . . . . . . . . . . . . . . . . x B. Critical behaviour of two clamped chains each consisting ll . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 920 178 840 607 29 1 065 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 650 210 370 673 30 47 . . . . . x B. Critical behaviour of two clamped chains each consisting ll . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 413 082 353 055 31 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Hall C, Vella D, Goriely A. 2013 The mechanics of a chain or ring of spherical magnets. SIAM J. Appl. Math. 73, 2029–2054. (doi:10.1137/120897973) 11. Hall C, Vella D, Goriely A. 2013 The mechanics of a chain or ring of spherical magnets. SIAM J. Appl. Math. 73, 2029–2054. (doi:10.1137/120897973) 12. Boisson J, Rouby C, Lee J, Doaré O. 2015 Dynamics of a chain of permanent magnets. Europhys. Lett. 109, 34002. (doi:10.1209/0295-5075/109/34002) 13. Wang C. 1986 A critical review of the heavy elastica. Int. J. Mech. Sci. 28, 549–559. (doi:10.1016/ 0020-7403(86)90052-4) 14. Greenhill A. 1881 Determination of the greatest height consistent with stability that a vertical pole or mast can be made, and of the greatest height to which a tree of given proportions can grow. Proc. Camb. Philos. Soc. IV, 65–73. (doi:10.1051/jphystap:018820010033701) 15. Schönke J, Schneider T, Rehberg I. 2015 Inﬁnite geometric frustration in a cubic dipole cluster. Phys. Rev. B 91, 020410. (doi:10.1103/PhysRevB.91.020410) 12. Boisson J, Rouby C, Lee J, Doaré O. 2015 Dynamics of a chain of permanent magnets. Europhys. Lett. 109, 34002. (doi:10.1209/0295-5075/109/34002) References Kiani B, Faivre D, Klumpp S. 2015 Elastic properties of magnetosome chains. New J. Phys. 17, 043007. (doi:10.1088/1367-2630/17/4/043007) g y 9. Kiani B, Faivre D, Klumpp S. 2015 Elastic properties of magnetosome chains. New J. Phys. 17, 043007. (doi:10.1088/1367-2630/17/4/043007) ( ) 10. Vella D, du Pontavice E, Hall C, Goriely A. 2013 The magneto-elastica: from self-buckling to self-assembly. Proc. R. Soc. A 470, 20130609. (doi:10.1098/rspa.2013.0609) ( ) 10. Vella D, du Pontavice E, Hall C, Goriely A. 2013 The magneto-elastica: from self-buckling to self-assembly. Proc. R. Soc. A 470, 20130609. (doi:10.1098/rspa.2013.0609) 11. Hall C, Vella D, Goriely A. 2013 The mechanics of a chain or ring of spherical magnets. SIAM J. Appl. Math. 73, 2029–2054. (doi:10.1137/120897973) 25 12. Boisson J, Rouby C, Lee J, Doaré O. 2015 Dynamics of a chain of permanent magnets. Europhys. Lett. 109, 34002. (doi:10.1209/0295-5075/109/34002) 12. Boisson J, Rouby C, Lee J, Doaré O. 2015 Dynamics of a chain of permanent magnets. Europhys. Lett. 109, 34002. (doi:10.1209/0295-5075/109/34002) rspa.royalsocietypublishing.org Proc. R.Soc. A 473: 20160703 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13. Wang C. 1986 A critical review of the heavy elastica. Int. J. Mech. Sci. 28, 549–559. (doi:10.1016/ 0020-7403(86)90052-4) 13. Wang C. 1986 A critical review of the heavy elastica. Int. J. Mech. Sci. 28, 549–559. (doi:10.1016/ 0020-7403(86)90052-4) 14. Greenhill A. 1881 Determination of the greatest height consistent with stability that a vertical pole or mast can be made, and of the greatest height to which a tree of given proportions can grow. Proc. Camb. Philos. Soc. IV, 65–73. (doi:10.1051/jphystap:018820010033701) 14. Greenhill A. 1881 Determination of the greatest height consistent with stability that a vertical pole or mast can be made, and of the greatest height to which a tree of given proportions can grow. Proc. Camb. Philos. Soc. IV, 65–73. (doi:10.1051/jphystap:018820010033701) g ( jp y p ) 15. Schönke J, Schneider T, Rehberg I. 2015 Inﬁnite geometric frustration in a cubic dipole cluster. Phys. Rev. B 91, 020410. (doi:10.1103/PhysRevB.91.020410) g ( jp y p ) 15. Schönke J, Schneider T, Rehberg I. 2015 Inﬁnite geometric frustration in a cubic dipole cluster. Phys. Rev. B 91, 020410. 13. Wang C. 1986 A critical review of the heavy elastica. Int. J. Mech. Sci. 28, 549–559. (doi:10.1016/ 0020-7403(86)90052-4) 14. Greenhill A. 1881 Determination of the greatest height consistent with stability that a vertical pole or mast can be made, and of the greatest height to which a tree of given proportions can grow. Proc. Camb. Philos. Soc. IV, 65–73. (doi:10.1051/jphystap:018820010033701) References (doi:10.1103/PhysRevB.91.020410) g ( jp y p ) 15. Schönke J, Schneider T, Rehberg I. 2015 Inﬁnite geometric frustration in a cubic dipole cluster. Phys. Rev. B 91, 020410. (doi:10.1103/PhysRevB.91.020410)
https://openalex.org/W3105568539	https://hal.archives-ouvertes.fr/hal-02971961/document	English	null	The structure and characteristic scales of molecular clouds	Astronomy & astrophysics	2,020	cc-by	19,742	ABSTRACT The structure of molecular clouds holds important clues regarding the physical processes that lead to their formation and subsequent dynamical evolution. While it is well established that turbulence imprints a self-similar structure onto the clouds, other processes, such as gravity and stellar feedback, can break their scale-free nature. The break of self-similarity can manifest itself in the existence of characteristic scales that stand out from the underlying structure generated by turbulent motions. In this work, we investigate the structure of the Cygnus-X North and Polaris Flare molecular clouds, which represent two extremes in terms of their star formation activity. We characterize the structure of the clouds using the delta-variance (∆-variance) spectrum. In the Polaris Flare, the structure of the cloud is self-similar over more than one order of magnitude in spatial scales. In contrast, the ∆-variance spectrum of Cygnus-X North exhibits an excess and a plateau on physical scales of ≈0.5−1.2 pc. In order to explain the observations for Cygnus-X North, we use synthetic maps where we overlay populations of discrete structures on top of a fractal Brownian motion (fBm) image. The properties of these structures, such as their major axis sizes, aspect ratios, and column density contrasts with the fBm image, are randomly drawn from parameterized distribution functions. We are able to show that, under plausible assumptions, it is possible to reproduce a ∆-variance spectrum that resembles that of the Cygnus-X North region. We also use a “reverse engineering” approach in which we extract the compact structures in the Cygnus-X North cloud and reinject them onto an fBm map. Using this approach, the calculated ∆-variance spectrum deviates from the observations and is an indication that the range of characteristic scales (≈0.5−1.2 pc) observed in Cygnus-X North is not only due to the existence of compact sources, but is a signature of the whole population of structures that exist in the cloud, including more extended and elongated structures. ey words. stars: formation – ISM: clouds – ISM: general – ISM: structure – galaxies: star formation – galaxies: ISM This self-similarity can be broken on various scales. This can happen when speciﬁc physical processes dominate the injec- tion of energy and momentum in the ISM. ABSTRACT In galactic disks, various forms of feedback from massive stars (i.e., ionizing radiation, radiation pressure, stellar winds, and supernova explo- sions) impart signiﬁcant amounts of energy and momentum onto the ISM on intermediate scales, that is, ≈50−500 pc (e.g., Dib et al. 2006, 2011, 2013, 2017; Ostriker et al. 2010; Dib 2011; Hennebelle & Iffrig 2014; Hony et al. 2015; Padoan et al. 2016; Ntomousi et al. 2017; Seifried et al. 2020). Some of these scales could be detected as characteristic scales in the ISM. Dib et al. (2009) found that the orientations of molecular clouds in the outer Galaxy are correlated on spatial scales that are on the order of the expected sizes of supernova remnants, which are prevalent in those regions of the Galactic disk. On small scales, particu- larly within molecular clouds, the self-similarity can be broken on physical scales where the self-gravity of the gas becomes important and dictates the motions of the gas (e.g., Dib et al. 2008). When dynamically important, and due to their anisotropic nature, magnetic ﬁelds can also play a role in breaking the The structure and characteristic scales of molecular clouds Physikalisches Institut, Universität zu Köln, Zülpicher Straße 77, 50937 Köln, Germany 3 Istituto di Astroﬁsica e Planetologia Spazialli, INAF, Via Fosso del Cavaliere 100, Roma 00133, Ita y , , p , , y 3 Istituto di Astroﬁsica e Planetologia Spazialli, INAF, Via Fosso del Cavaliere 100, Roma 00133, Italy 4 Istituto di Astroﬁsica e Planetologia Spazialli, INAF, Via Fosso del Cavaliere 100, Roma 00133, Italy 4 Department of Physics, Faculty of Sciences, Golestan University, Gorgan 49138-15739, Iran 5 g p 4 Department of Physics, Faculty of Sciences, Golestan University, Gorgan 49138-15739, Ir Instituto de Astrofísica e Ciências do Espaço, Universidade do Porto, CAUP, Rua das Estrelas, 4150-762 Porto, Port Institut de Planétologie et d’Astrophysique de Grenoble Université Grenoble Alpes CNRS Grenoble France 5 Instituto de Astrofísica e Ciências do Espaço, Universidade do Porto, CAUP, Rua das Estrelas, 4150-762 Porto, Portugal 6 Institut de Planétologie et d’Astrophysique de Grenoble, Université Grenoble Alpes, CNRS, Grenoble, France 7 D f A U i i f M h A h MA 01003 USA Instituto de Astrofísica e Ciências do Espaço, Universidade do Porto, CAUP, Rua das Estrelas, 4150 762 Porto, Por 6 Institut de Planétologie et d’Astrophysique de Grenoble, Université Grenoble Alpes, CNRS, Grenoble, France 7 Institut de Planétologie et d Astrophysique de Grenoble, Université Grenoble Alpes, CNRS, Grenoble, France Department of Astronomy, University of Massachusetts, Amherst, MA 01003, USA p y y 8 Centre for Star and Planet Formation, the Niels Bohr Institute and the Natural History Museum of Denmark, University of Copenhagen, Øster Voldgade 5-7 1350, Denmark 9 Centre for Star and Planet Formation, the Niels Bohr Institute and the Natural History Museum of Denmark, University of Copenhagen, Øster Voldgade 5-7 1350, Denmark Received 5 July 2020 / Accepted 9 September 2020 Open Access article, published by EDP Sciences, under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4. which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Astronomy & Astrophysics Astronomy & Astrophysics Astronomy & Astrophysics A&A 642, A177 (2020) https://doi.org/10.1051/0004-6361/202038849 © S. Dib et al. 2020 The structure and characteristic scales of molecular clouds Sami Dib1, Sylvain Bontemps1, Nicola Schneider2, Davide Elia3, Volker Ossenkopf-Okada2, Mohsen Shadmehri4, Doris Arzoumanian5, Frédérique Motte6, Mark Heyer7, Åke Nordlund8, and Bilal Ladjelate Sami Dib1, Sylvain Bontemps1, Nicola Schneider2, Davide Elia3, Volker Ossenkopf-Okada2, Mohsen Shadmehri4, Doris Arzoumanian5, Frédérique Motte6, Mark Heyer7, Åke Nordlund8, and Bilal Ladjelate9 1 Laboratoire d’Astrophysique de Bordeaux, Université de Bordeaux, CNRS, B18N, allée Geoffroy Saint-Hilaire, 33615 Pessac, France e-mail: sami.dib@gmail.com 1 Laboratoire d’Astrophysique de Bordeaux, Université de Bordeaux, CNRS, B18N, allée Geoffroy Saint-Hilaire, 336 e-mail: sami.dib@gmail.com 1 Laboratoire d’Astrophysique de Bordeaux, Université de Bordeaux, CNRS, B18N, allée Geoffroy Saint-Hilaire, 33615 Pessac, France 2 I. Physikalisches Institut, Universität zu Köln, Zülpicher Straße 77, 50937 Köln, Germany 3 2 I. Physikalisches Institut, Universität zu Köln, Zülpicher Straße 77, 50937 Köln, Germany 3 I i di A ﬁi Pl l i S i lli INAF Vi F d l C li 100 R 00 I. 1. Introduction For the spectral energy distribution (SED) ﬁt, the speciﬁc dust opacity per unit mass (dust plus gas) is approximated by a power law κν = 0.1(ν/1000 GHz)βdust cm2 g−1 with βdust = 2 and the dust temper- ature and column density left as free parameters. The description of how high angular resolution maps were derived is detailed in Palmeirim et al. (2013). The concept is to employ a multi-scale decomposition of the ﬂux maps and assume a constant line-of- sight temperature. The ﬁnal map at 18.2′′ angular resolution is constructed from the difference maps of the convolved surface density SPIRE maps (at 500, 350, and 250 µm) and the temper- ature information from the color temperature derived from the 160 µm/250 µm ratio. p g g Using the delta-variance (∆-variance) spectrum (Stutzki et al. 1998), we analyzed the spatial structure of two Galac- tic molecular clouds that lie at the extreme ends of what can be found in the Galaxy in terms of their star formation activ- ity, namely the Cygnus-X North region and the Polaris Flare. The Cygnus-X North molecular cloud complex is an active region of star formation where many sub-regions of high-mass star formation can be found (e.g., Schneider et al. 2006, 2010; Motte et al. 2007; Reipurth & Schneider 2008; Bontemps et al. 2010; Csengeri et al. 2011; Hennemann et al. 2012; Kryukova et al. 2014; Maia et al. 2016). In contrast, the Polaris Flare is essentially a translucent, non-star forming cloud (e.g., Ward- Thompson et al. 2010; Miville-Deschênes et al. 2010). In Sect. 2, we brieﬂy summarize the Herschel satellite data that are ana- lyzed in this work, and in Sect. 3 we present and discuss the column density distribution functions of both regions. The ∆-variance method is discussed in Sect. 4, and its application to the Herschel satellite maps of Cygnus-X North and Polaris is presented in Sect. 5. In Sect. 6, we interpret our ﬁndings with the help of simulated synthetic observations and discuss the shape of the ∆-variance in models of increasing complexity. We start with models of pure fractal Brownian motion (fBm) images (Sect. 6.1) and continue to models where individual structures are superim- posed on an fBm (Sect. 6.2). 1. Introduction The interstellar medium (ISM), both in the Milky Way and in external galaxies, exhibits a scale-free nature that extends over many physical scales. This is observed both in the diffuse H I gas (e.g., Elmegreen et al. 2001; Dickey et al. 2001; Dib & Burkert 2005; Begum et al. 2006; Dutta et al. 2009, 2013; Zhang et al. 2012; Miville-Deschênes et al. 2016) and in the molecular phase (e.g., Stutzki et al. 1998; Heyer & Brunt 2004; Heyer et al. 2009; Schneider et al. 2011; Roman-Duval et al. 2011; Rebolledo et al. 2015; Panopoulou et al. 2017; Traﬁcante et al. 2018; Hirota et al. 2018; Kong et al. 2018; Dib & Henning 2019; Henschaw et al. 2020). This self-similarity is also observed in the spatial distri- bution of young clusters in galactic disks (e.g., Elmegreen et al. 2006; Gouliermis et al. 2017; Grasha et al. 2019). ) Turbulence is ubiquitously observed in all phases of the interstellar gas. It is thought to be the main regulator of the ISM structure and dynamics in cold, neutral gas and, hence, is responsible for setting a self-similar behavior in this regime (e.g., Elmegreen & Scalo 2004; Dib et al. 2008; Burkhart et al. 2013). A177, page 1 of 18 A&A 642, A177 (2020) self-similar nature of the gas (e.g., Soler 2019). Scales at which there might be a departure from self-similarity are the ones asso- ciated with the sizes of ﬁlaments and fragments within ﬁlaments (e.g., André et al. 2010; Arzoumanian et al. 2011; Hacar et al. 2013; Könyves et al. 2015) as well as scales on which ﬁlaments interact, with hubs and ridges forming at the intersection of two or more ﬁlaments (e.g., Schneider et al. 2012; Samal et al. 2015; Dewangan et al. 2017; Treviño-Morales et al. 2019; Clarke et al. 2019). Stellar feedback can also perturb the self-similarity of the gas on small scales. Russeil et al. (2013) ﬁnd, in a study of the massive star-forming region NGC6334, that characteristic scales around 1–10 pc can be caused by the injection of energy due to expanding H II regions. extended emission. All maps have an absolute ﬂux calibration using offset values determined in Bernard et al. (2010). 1. Introduction We ﬁnish with models in which an entire population of structures is superimposed on an fBm and which have sets of properties (such as the size of major axis, elongation, and column density contrast) that are described by parameterized probability distribution functions (Sect. 6.3). In Sect. 7, we discuss different caveats and limitations pertaining to the observations and the models, and in Sect. 8 we summarize our results and conclude. µ µ The molecular hydrogen (H2) column densities were trans- formed into visual extinction (AV) using the conversion formula N(H2)/AV = 0.94 × 1021 cm−2 mag−1 (Bohlin et al. 1978). The column density maps for the Cygnus-X North and Polaris Flare clouds are displayed in Fig. 1 (left- and right-hand pan- els, respectively). For Cygnus-X, column density maps were already presented in Hennemann et al. (2012) and Schneider et al. (2016a,b), and for Polaris in Robitaille et al. (2019). These maps have a lower angular resolution of 36.3′′ and cover differ- ent areas than those presented in the current study. The Polaris Flare cloud is located at a distance of 140 pc (Falgarone et al. 1998), and hence each pixel on the map corresponds to a spatial size of ≈0.002 pc. The Cygnus-X cloud is located at a distance of 1.7 kpc (Schneider et al. 2006), and, in this case, each pixel corresponds to a spatial size of ≈0.025 pc. The maps of Cygnus- X North and Polaris contain 5740 × 5740 pixels and 3538 × 3164 pixels, respectively. The total physical size covered by the maps of Cygnus-X North and Polaris is ≈143.5 pc × 143.5 pc and 7.07 pc × 6.32 pc, respectively. The full width at half maxi- mum (FHWM) of the beam is sampled with six pixels and thus the spatial resolution for the Cygnus-X North and Polaris maps are ≈0.15 pc and ≈0.012 pc, respectively. 3. Column density distribution functions Here, we only wish to highlight the differences between the Cygnus-X North and Polaris regions in terms of their column density distributions before analyzing the spatial structure of the clouds. Figure 2 displays the column density probability distribu- tion function (N-PDF) for Cygnus-X North (left-hand panel) and Polaris (right-hand panel). Both N-PDFs resemble those shown in Schneider et al. (2016b) for Cygnus-X and Schneider et al. (2013) for Polaris. The N-PDFs for both regions exhibit a log- normal behavior at low AV (2.5 ≤AV < 12 for Cygnus-X North and ≤1 for Polaris) with a signiﬁcant difference in the position of the peak between the two regions. A ﬁt to the lognormal part of the N-PDF yields AV,peak = 8.92+1.40 −1.21 in Cygnus-X North and AV,peak = 0.45+0.19 −0.13 in Polaris. The width of the lognormal is σAV = 1.14 ± 1.10 and σAV = 1.39 ± 1.25 in Cygnus-X North and Polaris, respectively. At larger column densities (AV ≥12 in Cygnus-X and AV ≥1 in Polaris), the N-PDF turns into a power- law distribution. In Cygnus-X, there are two distinct power laws: a steep power law with an exponent of −4.24 ± 0.15 in the AV range of [12, 30] and a shallower power law with an exponent of −1.85 ± 0.02 in the AV range [30, 300]. In contrast, the N-PDF for Polaris exhibits a single power-law tail (PLT) starting from AV ≳1 with an exponent of −3.97 ± 0.13. The exponent of the PLT we ﬁnd for Polaris matches the one found in other stud- ies for the same cloud (e.g., Alves et al. 2007; Schneider et al. 2013). The N-PDF parameters we ﬁnd for the two regions are 1 http://gouldbelt-herschel.cea.fr/archives S. Dib et al.: Characteristic scales in molecular clouds Fig. 1. Column density maps of the Cygnus-X North cloud (left) and the Polaris Flare cloud (right). Column densities are displayed in units of the visual extinction using the conversion NH2/AV = 0.94 × 1021 cm−2. Fig. 1. Column density maps of the Cygnus-X North cloud (left) and the Polaris Flare cloud (right). Column densities are displayed in units of the visual extinction using the conversion NH2/AV = 0.94 × 1021 cm−2. Fig. 2. Column density distribution function in the Cygnus-X North cloud (left panel) and the Polaris Flare cloud (right panel). The full red lines in both panels show a ﬁt by a lognormal function in the low column density regime (≲5 × 1021 cm−2 in Cygnus-X North and ≲1 × 1021 cm2 in Polaris). The dashed line is a ﬁt to the power-law regime that is observed in both regions in the intermediate column density regime, while the triple-dot dashed red line in the case of the Cygnus-X North region is a ﬁt to the shallower power law in the high column density regime. Fig. 2. Column density distribution function in the Cygnus-X North cloud (left panel) and the Polaris Flare cloud (right panel). The full red lines in both panels show a ﬁt by a lognormal function in the low column density regime (≲5 × 1021 cm−2 in Cygnus-X North and ≲1 × 1021 cm2 in Polaris). The dashed line is a ﬁt to the power-law regime that is observed in both regions in the intermediate column density regime, while the triple-dot dashed red line in the case of the Cygnus-X North region is a ﬁt to the shallower power law in the high column density regime. The second, shallower PLT is only associated with regions of the highest column densities in Cygnus-X. This was reported for the ﬁrst time in high-mass star-forming regions (Tremblin et al. 2014; Schneider et al. 2016b) and interpreted as aris- ing from gravitational collapse of cores with either internal sources (protostars, ultra-compact H II regions) that lead to inter- nal ionization compressions, or external compression from the associated H II region. However, the picture is probably more complicated since a second shallower PLT was also detected in low-mass star-forming regions (Schneider et al., in prep.). It is not within the scope of this paper to discuss the N-PDFs in extensive detail. 2. The data: Herschel maps of star-forming regions The observations that are analyzed in this work were performed using the Herschel space observatory (Pilbratt et al. 2010). In particular, we made use of data products from the Herschel Gould Belt Survey (HGBS1, André et al. 2010) for the Polaris Flare region and the Herschel imaging survey of OB Young Stellar objects (HOBYS, Motte et al. (2010)) program for the Cygnus-X North region. The column density maps were deter- mined from a pixel-to-pixel greybody ﬁt to the red wavelength of PACS (Poglitsch et al. 2010) observations at 160 µm (11.7′′ angular resolution), and the three SPIRE (Grifﬁn et al. 2010) wavelengths are 250, 350, and 500 µm at the resolutions of 18.2′′, 24.9′′, and 36.3′′, respectively. For the SPIRE data reduc- tion, we used the HIPE pipeline (versions 10 to 13), including the destriper task for SPIRE as well as HIPE and scanamorphos (Roussel 2013) for PACS. The SPIRE maps were calibrated for 1 http://gouldbelt-herschel.cea.fr/archives A177, page 2 of 18 S. Dib et al.: Characteristic scales in molecular clouds 5. Spatial structure of Cygnus-X North and Polaris ⊙L (r) = 4 πL2 e r2 (L/2)2 − 4 πL2(v2 −1) " e r2 (vL/2)2 −e r2 (L/2)2 # , (2) (2) We applied the ∆-variance method to the column density maps of Cygnus-X North and Polaris. As stated above, these two regions were selected because they are signiﬁcantly different, both in terms of their column density distribution (i.e., Fig. 2) and their star formation activity. While Polaris harbors a population of starless cores, it is still a region with no ongoing star formation and a modest contrast in column density. On the other hand, the Cygnus-X North cloud is a region with a much higher star for- mation rate and a much larger contrast in column densities (see Fig. 2, also Hennemann et al. 2012; Schneider et al. 2016b). The ∆-variance spectra for both clouds are displayed in Fig. 3. The ∆-variance spectrum of Polaris displays a self-similar behav- ior above the resolution limit, and this self-similarity extends for more than one order of magnitude in spatial scales (from ≈0.03 to ≈0.6 pc). A power-law ﬁt to the ∆-variance of Polaris in the range [0.035–0.6] pc yields a value of the power-law expo- nent of α = 0.4 ± 0.003, and this implies a value of β = 2.4. On scales larger than 0.6 pc, the self-similarity is perturbed, possibly due to the existence of a large ﬁlamentary structure (i.e., the MCLD 123.5+24.9 structure), though substructured, in the region. The scale-free nature of the ∆-variance spectrum of Polaris is consistent with earlier ﬁndings using the ∆-variance technique for the same cloud (Bensch et al. 2001; Ossenkopf- Okada & Stepanov 2019). However, Bensch et al. (2001) found larger values of β (≈3 from observations in the 12CO (J = 2−1) line and β ≈3.2 from observations in the 13CO (J = 1−0) line) when the ∆-variance spectrum is ﬁtted over a spatial range that is roughly similar to the one used in this study. The spatial resolu- tion of the observations they used are 2.2′ and 0.78′, respectively, and are lower than the resolution of the observations presented in this work (≈0.3′). Ossenkopf 2002 showed that the use of low-J CO isotopologues leads to somewhat steeper ∆-variance spectra than the one corresponding to the underlying column density structure. 4. Analysis: the ∆-variance method We quantiﬁed the structure of molecular clouds using the ∆- variance method. The method was originally introduced in Stutzki et al. (1998) and Zielinsky & Stützki (1999) and is a gen- eralization of the Allan variance (Allan 1966). In this work, we used an improved version of the method presented in Ossenkopf et al. (2008a)2. Here, we brieﬂy present a summary of the main steps and characteristics of the method. For a 2D ﬁeld A(x, y), the ∆- variance on a scale L is deﬁned as being the variance of the convolution of A with a ﬁlter function ⊙L such that σ2 ∆(L) = 1 2π⟨(A ∗⊙L)2⟩x,y. (1) (1) For the ﬁlter function, Ossenkopf et al. (2008a) recommend the use of a “Mexican hat” that is deﬁned as For the ﬁlter function, Ossenkopf et al. (2008a) recommend the use of a “Mexican hat” that is deﬁned as 5. Spatial structure of Cygnus-X North and Polaris The exact relative effect of the lower spatial resolution, which effectively smoothes the map and possibly increases the values of β, compared to the role of the optical depths of these molecular tracers in steepening the ∆-variance spectrum, is not yet entirely clear. where the two terms on the right side of Eq. (2) represent the core and the annulus of the Mexican hat function, respectively, and v is the ratio of their diameters (we used v = 1.5). For a faster and more efﬁcient computation of Eq. (1), Ossenkopf et al. (2008a) performed the calculation as a multiplication in Fourier space, and thus, the ∆-variance is given by σ2 ∆(L) = 1 2π Z Z P \| ¯⊙L\|2 dkxdky, (3) (3) where P is the power spectrum of A and ¯⊙L is the Fourier trans- form of the ﬁlter function. If P can be described by a power law, and if β is the exponent of the power spectrum, then a relation exists between the exponent of the power law that describes the ∆-variance (α) and β (Stutzki et al. 1998), and this is given by σ2 ∆(L) ∝Lα ∝Lβ−2. (4) σ2 ∆(L) ∝Lα ∝Lβ−2. (4) The value of α can be inferred from the range of spatial scales over which the ∆-variance displays a self-similar behav- ior and can be tied to the value of β. The error bars of the ∆-variance on a given scale are computed from the counting error determined by the ﬁnite number of statistically independent measurements in the ﬁltered map and the variance of the vari- ances (i.e., the fourth moment of the ﬁltered map). Characteristic scales are scales at which there is a break of the self-similarity and which show up in the ∆-variance spectra as break points, peaks, or inﬂection points. Any underlying self-similar behavior of the cloud can be entirely perturbed on many or all physical scales if there is a variety of structures that coexist in the cloud. The ∆-variance has been employed to analyze the structure of observed molecular clouds (e.g., Bensch et al. 2001; Campeggio et al. 2004; Sun et al. 2006; Ossenkopf et al. 2008b; Rowles & Froebrich 2011; Schneider et al. 2011; Russeil et al. 2013; Elia et al. S. Dib et al.: Characteristic scales in molecular clouds They propose that the physical process governing structure formation could be the scale at which either a large- scale supernova shock or an expanding H II region sweeping through the diffuse medium are broken at dense clouds, which turns the well-ordered velocity into turbulence. can be attributed to the formation of compact structures (ﬁla- ments or clumps) and the second, shallower PLT to the collapse of dense cores. Ossenkopf et al. 2001; Mac Low & Ossenkopf 2000; Ossenkopf & Mac Low 2002; Federrath et al. 2009; Bertram et al. 2015). In most cases, the ∆-variance has been used to investigate the self-similar nature of the clouds and examine whether the slope of the ∆-variance in the self-similar regime varies from cloud to cloud and, in the case of simulations, whether it depends on the properties of the turbulent motions that are generated in the clouds. However, it has already been demonstrated that the method is capable of detecting break points. Ossenkopf & Mac Low 2002 found, when applying the method to numeri- cal models of molecular clouds where turbulence is driven on various physical scales, that the ∆-variance departs from the self- similar regime on physical scales where turbulence is injected into the clouds. Using extinction maps, Schneider et al. (2011) found that low-mass star-forming clouds have a double-peak structure in the ∆-variance with characteristic size scales around ≈1 and ≈4 pc. They propose that the physical process governing structure formation could be the scale at which either a large- scale supernova shock or an expanding H II region sweeping through the diffuse medium are broken at dense clouds, which turns the well-ordered velocity into turbulence. 2 The IDL package for calculating the ∆-variance can be found at https://hera.ph1.uni-koeln.de/~ossk/Myself/ deltavariance.html S. Dib et al.: Characteristic scales in molecular clouds In summary, and despite the fact that grav- ity is suspected to be the primary culprit of the formation of both PLTs, we conclude that it currently is not straightfoward to explain the different parts of the PLT as the consequence of a hierarchical gravitational collapse, whereby the ﬁrst steep PLT only slightly different from what was obtained in earlier studies, and this difference is due to the fact that the considered areas of the clouds are different. A PLT in the N-PDF is connected to the existence of a power- law distribution in volume density and is commonly attributed to the effects of the self-gravity of the gas in generating dense struc- tures in the cloud (e.g., Klessen 2000; Dib 2005; Dib & Burkert 2005; Kainulainen et al. 2009; Kritsuk et al. 2011; Ward et al. 2014; Girichidis et al. 2014; Schneider et al. 2015; Donkov & Stefanov 2018; Corbelli et al. 2018; Veltchev et al. 2019). Another interpretation for the origin of the ﬁrst, steep PLT has been proposed by Auddy et al. (2018, 2019). These authors showed, using numerical simulations of molecular clouds with non-ideal magnetohydrodynamics (MHD), that in the case of a magneti- cally subcritical cloud, a steep PLT (slope ≈–4) can emerge as a result of gravitational contraction driven by ambipolar diffusion. A177, page 3 of 18 A177, page 3 of 18 A&A 642, A177 (2020) Ossenkopf et al. 2001; Mac Low & Ossenkopf 2000; Ossenkopf & Mac Low 2002; Federrath et al. 2009; Bertram et al. 2015). In most cases, the ∆-variance has been used to investigate the self-similar nature of the clouds and examine whether the slope of the ∆-variance in the self-similar regime varies from cloud to cloud and, in the case of simulations, whether it depends on the properties of the turbulent motions that are generated in the clouds. However, it has already been demonstrated that the method is capable of detecting break points. Ossenkopf & Mac Low 2002 found, when applying the method to numeri- cal models of molecular clouds where turbulence is driven on various physical scales, that the ∆-variance departs from the self- similar regime on physical scales where turbulence is injected into the clouds. Using extinction maps, Schneider et al. (2011) found that low-mass star-forming clouds have a double-peak structure in the ∆-variance with characteristic size scales around ≈1 and ≈4 pc. S. Dib et al.: Characteristic scales in molecular clouds S. Dib et al.: Characteristic scales in molecular clouds S. Dib et al.: Characteristic scales in molecular clouds Fig. 3. Delta-variance functions calculated for the Cygnus-X North cloud (left) and the Polaris Flare cloud (right). The vertical dashed black lines in both panels mark the position of the spatial resolution for each of these two regions. The vertical dashed red lines in the case of Polaris mark the spatial range over which the power-law ﬁt is performed. We do not attempt any ﬁt in Cygnus-X North because the underlying self-similar regime is heavily perturbed by the presence of structures (see Sect. 6.3). Fig. 3. Delta-variance functions calculated for the Cygnus-X North cloud (left) and the Polaris Flare cloud (right). The vertical dashed black lines in both panels mark the position of the spatial resolution for each of these two regions. The vertical dashed red lines in the case of Polaris mark the spatial range over which the power-law ﬁt is performed. We do not attempt any ﬁt in Cygnus-X North because the underlying self-similar regime is heavily perturbed by the presence of structures (see Sect. 6.3). resolution limit (dashed black lines in Fig. 3) and a broad peak at around ≈0.5−0.12 pc. A reasonable assumption to make is that the existence of many small-scale dense structures (e.g., cores, clumps, and ﬁlaments) in Cygnus-X North alters the underly- ing (i.e., primordial) self-similar structure of the gas that had existed before these structures formed. However, it remains an open question whether a massive star-forming region such as Cygnus-X North had, at an earlier stage, a spatial distribution of (column) density similar to that of Polaris. This is a plausible assumption given that, prior to the formation of massive stars in the region, turbulence in the Cygnus-X North cloud, like in Polaris and elsewhere in the ISM, must have been dominated by shearing motions. Large-scale converging ﬂows may be respon- sible for aggregating gas in speciﬁc regions that would be the parental structures of ridges and hubs. Compressive motions due to feedback from massive stars in speciﬁc regions of Cygnus-X North can also modify the spatial distribution of the (column) density ﬁeld. However, massive star formation is localized in Cygnus-X North and not distributed across the entire cloud (Beerer et al. 2010). S. Dib et al.: Characteristic scales in molecular clouds We speculate here that the underlying, “pri- mordial,” structure in the Cygnus-X North cloud resembled that of Polaris and use this as a working hypothesis. In what fol- lows, we focus our attention on the Cygnus-X North cloud and adopt the Polaris value of β = 2.4 as the exponent of the under- lying self-similar fBm structure in Cygnus-X. We explore, using synthetic data, if and how the addition of dense structures with speciﬁc properties on top of a cloud with a self-similar structure modiﬁes the ∆-variance spectrum. of the basic properties of fBm images that are known to pos- sess a self-similar structure. In a second step, we include discrete structures with speciﬁc characteristics on top of an existing fBm and investigate how the inclusion of these structures impacts the shape of the ∆-variance spectrum. Lastly, we investigate how the shape of the ∆-variance is modiﬁed in the presence of an entire population of structures that are characterized by distri- bution functions of their sizes, elongations, and column density contrasts. 6.1. Fractal Brownian motion maps Fractal Brownian motion images (Peitgen & Saupe 1988) are often used as a surrogate of ISM maps thanks to their visual similarity with cloud features (e.g., Stutzki et al. 1998; Bensch et al. 2001; Miville-Deschênes et al. 2003; Elia et al. 2014, 2018). A full description of their analytic properties is presented in Stutzki et al. (1998). Here, we simply review their basic prop- erties. Firstly, their radially averaged power spectrum exhibits a power-law behavior with an exponent β = E +2H, where E is the Euclidian dimension (E = 2 for 2D images) and H is the Hurst exponent whose value ranges from 0 to 1. For 2D maps, β can take values between 2 and 4. Secondly, the distribution of the phases of their Fourier transform is completely random. Thus, it is possible to generate fBm maps by deﬁning the value of β and a random phase distribution. If expressed in terms of the frac- tal dimension, the fractal dimension of an fBm image has been shown to be given by D = E + 1 −H, and this leads to a direct relation between D and β that is given by: 5. Spatial structure of Cygnus-X North and Polaris 2014) as well as simulated molecular clouds (e.g., 2 The IDL package for calculating the ∆-variance can be found at https://hera.ph1.uni-koeln.de/~ossk/Myself/ deltavariance.html In contrast to Polaris, the ∆-variance of Cygnus-X North dis- plays a more complex shape with a steep slope above the spatial A177, page 4 of 18 A177, page 4 of 18 6. Interpretation All maps are normalized by their own mean value, and the vertical offset between the three ∆-variance functions simply reﬂects the effect of this different normalization. Fig. 5. fBm images with values of β ranging from 2 to 4 in steps of 0.5. Fig. 5. fBm images with values of β ranging from 2 to 4 in steps of 0.5. All maps have a resolution of 1000 × 1000 pixels. The ∆-variance ﬁg- ures for all cases are compared in the bottom-right subpanel. All display a self-similar regime with an exponent of the power law of α = β −2 (i.e., Eq. (4)). All maps are normalized by their own mean value. The random number series generating the phases has been kept the same in all maps. Fig. 5. fBm images with values of β ranging from 2 to 4 in steps of 0.5. All maps have a resolution of 1000 × 1000 pixels. The ∆-variance ﬁg- ures for all cases are compared in the bottom-right subpanel. All display a self-similar regime with an exponent of the power law of α = β −2 (i.e., Eq. (4)). All maps are normalized by their own mean value. The random number series generating the phases has been kept the same in all maps. and 3D (E = 3) fractal dimensions. This will be given by: and 3D (E = 3) fractal dimensions. This will be given by: D2 = D3 −3 2. (6) D2 = D3 −3 2. (6) In this paper, we used fBm images as a reference for self- similar structures since they can be obtained with preconditioned statistical properties (e.g., Stutzki et al. 1998; Shadmehri & Elmegreen 2011; Elia et al. 2018). Figure 4 displays three fBm images generated with a value of β = 2.5 and for three resolu- tions: 250 × 250 pixels (top left), 500 × 500 pixels (top right), and 1000 × 1000 pixels (bottom left)3. The bottom-right panel in Fig. 4 displays the ∆-variance functions calculated for these three fBm images. The self-similar regime is observed in all cases and extends to larger spatial scales for cases with a higher spatial resolution. The fBm images are periodic, and, if we were using a periodic analysis, the ∆-variance spectra would be perfect power laws. 6. Interpretation D = 3E + 2 −β 2 . (5) D = 3E + 2 −β 2 . (5) While one of our aims is to understand the structure of the Cygnus-X North region as revealed by its ∆-variance spectrum, a broader goal is to investigate how the existence of compact and dense structures (cores, clumps, and ﬁlaments) with diverse char- acteristics can alter the self-similar nature of a molecular cloud and modify the ∆-variance spectrum. We ﬁrst summarize some Stutzki et al. (1998) showed that the power spectrum of the (E −1) projection of an E-dimensional fBm is also a power law with the same spectral index (i.e., the same β). Using this prop- erty, it is possible to establish the link between the 2D (E = 2) A177, page 5 of 18 A177, page 5 of 18 A&A 642, A177 (2020) Fig. 5. fBm images with values of β ranging from 2 to 4 in steps of 0.5. All maps have a resolution of 1000 × 1000 pixels. The ∆-variance ﬁg- ures for all cases are compared in the bottom-right subpanel. All display a self-similar regime with an exponent of the power law of α = β −2 (i.e., Eq. (4)). All maps are normalized by their own mean value. The random number series generating the phases has been kept the same in all maps. Fig. 4. fBm images with β = 2.5 and with resolutions of 250 × 250 pix- els (top left), 500 × 500 pixels (top right), and 1000 × 1000 pixels (bottom left). The ∆-variance spectra for all three cases are compared in the bottom-right subpanel. All display a self-similar regime with an exponent of the power law of α = β −2 (i.e., Eq. (4)). All maps are normalized by their own mean value, and the vertical offset between the three ∆-variance functions simply reﬂects the effect of this different normalization. Fig. 5. fBm images with values of β ranging from 2 to 4 in steps of 0.5. Fig. 4. fBm images with β = 2.5 and with resolutions of 250 × 250 pix- els (top left), 500 × 500 pixels (top right), and 1000 × 1000 pixels (bottom left). The ∆-variance spectra for all three cases are compared in the bottom-right subpanel. All display a self-similar regime with an exponent of the power law of α = β −2 (i.e., Eq. (4)). 3 By construction, the mean value of the fBm is zero. We applied an arbitrary offset to the maps in order to insure that all values were posi- tive. The maps were then normalized by their mean value. The addition of a constant offset for the whole map does not alter the shape of the ∆- variance spectrum since the relative differences between pixels remain the same. 6. Interpretation However, in order to compare them to the observational data, we performed the calculations of the ∆-variance with a cut at the map boundaries. This cut has two effects. First, there is a natural limit to the size of any structure so that the ∆-variance spectrum ﬂattens at the largest scales. Second, the statistical signiﬁcance of the structures close to the map boundaries is reduced. This changes the denominator in the normalization of the ∆-variance when computing mean properties of the map according to the area-to-boundary ratio of maps of different sizes so that the absolute scale of the ∆-variance is only comparable in the limit of very large maps. Figure 5 displays the same type of fBm images, but in this case all images have a ﬁxed resolu- tion of 1000 × 1000 pixels and the value of β is varied between 2 and 4 (in steps of 0.5). While in Fig. 4 the phase distribution varies from image to image, in Fig 5 the same distribution of phases is kept, so that the basic “shape” of the image remains the same. Increasing the value of β produces a gradual smooth- ing of the image due to the transfer of power from high to low spatial frequencies. The bottom-right panel in Fig. 5 displays the corresponding ∆-variance functions for each of these cases. As expected, the ∆-variance functions are scale-free power-law functions whose exponent is given by α = β −2. 6.2. Fractal Brownian motion maps with additional structure We now explore the effect of discrete structures on the ∆-variance spectrum. The structures we superimpose on top of fBm images are generalized 2D Gaussian functions that are given by: NG(x, y) =Npeakexp[−a(x−x0)2 +2b(x−x0)(y−y0)+c(y−y0)2)], (7) A177, page 6 of 18 A177, page 6 of 18 S. Dib et al.: Characteristic scales in molecular clouds Fig. 6. 2D Gaussian structures injected on top of an fBm image with β = 2.4. The 2D Gaussian functions have an aspect ratio ( f = σ1/σ2) that is varied in the range [1–10], and all have a value of δc = 5 and a ﬁxed size of σ1 = 50 pixels. All maps are normalized to their mean value. Bottom-right ﬁgure: corresponding ∆-variance functions calculated for each case, and these are compared to the ∆-variance function of the underlying fBm image. where NG is the added column density of the 2D Gaussian struc- ture, Npeak is its peak value, x0 and y0 are the coordinates of the peak, and the terms a, b, and c are given by: a =cos2 (θ) 2σ2 1 + sin2 (θ) 2σ2 2 b =sin(2θ) 4σ2 1 −sin(2θ) 4σ2 2 c =sin2 (θ) 2σ2 1 + cos2 (θ) 2σ2 2 . (8) a =cos2 (θ) 2σ2 1 + sin2 (θ) 2σ2 2 b =sin(2θ) 4σ2 1 −sin(2θ) 4σ2 2 c =sin2 (θ) 2σ2 1 + cos2 (θ) 2σ2 2 . (8) a =cos2 (θ) 2σ2 1 + sin2 (θ) 2σ2 2 b =sin(2θ) 4σ2 1 −sin(2θ) 4σ2 2 c =sin2 (θ) 2σ2 1 + cos2 (θ) 2σ2 2 . (8) In Eq. (8), σ1 and σ2 are the standard deviations of the 2D Gaussian along the major and minor axes, respectively, and θ is the angle between the Gaussian function major axis and the x-axis, deﬁned in the counterclockwise direction. We generated a very large number of synthetic maps on which we superim- posed one or several 2D Gaussian structures over fBm images in a controlled manner. All fBm images have a value of β = 2.4, similar to the value found in the Polaris cloud, and a resolution of 1000 × 1000 pixels. For individual structures, we varied the aspect ratio of the 2D Gaussian, f = (σ1/σ2), over a range of 1 to 10. 6.2. Fractal Brownian motion maps with additional structure The peak value of the 2D Gaussians is expressed in terms of the mean value of the fBm image, Npeak = δc ⟨NfBm⟩, and the column density contrast between the peak of the 2D Gaussian and the mean value of the fBm, δc, is varied between 1 and 10. We also explored the effect of varying the absolute size of the Gaussian function with respect to the image size, as well as the effect of including multiple Gaussian functions in the fBm images. Figure 6 displays ﬁve realizations of an fBm with superim- posed Gaussian structures. The 2D Gaussians all have δc = 5, σ1 = 50 pixels, and an aspect ratio f = (σ1/σ2) that is varied between 1 and 10. The ∆-variance functions of these maps are displayed in the bottom-right panel of Fig. 6 and are compared to the ∆-variance function of a pure fBm image with β = 2.4. The inclusion of an additional structure in the fBm image increases the value of σ2 ∆on all spatial scales. The increment of the ∆-variance function with respect to the ∆-variance of the pure fBm reaches a maximum on a scale that is on the order of the equivalent diameter of the injected structure. Figure 6 shows that as the aspect ratio is reduced, the position of the point where the deviation from the fBm is maximized in the ∆-variance func- tion moves to larger spatial scales. If we approximate the surface of the 2D Gaussian by the area that lies within [2σ1, 2σ2] (i.e., where most of the signal lies), the equivalent diameter is then given by Deq ≈4 √σ1σ2. The measured positions of the points of maximum deviation in the ∆-variance functions in Fig. 6 do indeed conﬁrm that the position of maximum deviation is well approximated by Deq. A deviation from this value (by up to ≈30%) can be observed for smaller structures (i.e., in this case, the most elongated) as they are less well resolved on the grid. Fig. 6. 2D Gaussian structures injected on top of an fBm image with β = 2.4. The 2D Gaussian functions have an aspect ratio ( f = σ1/σ2) that is varied in the range [1–10], and all have a value of δc = 5 and a ﬁxed size of σ1 = 50 pixels. All maps are normalized to their mean value. 6.2. Fractal Brownian motion maps with additional structure A177, page 7 of 18 A177, page 7 of 18 A177, page 7 of 18 A&A 642, A177 (2020) Fig. 7. 2D Gaussian structures injected on top of an fBm image with β = 2.4. The Gaussian functions have an aspect ratio f = 5, a value of σ1 = 50 pixels, and a column density contrast between the peak of the 2D Gaussian and the mean value of the fBm, δc, that is varied between 1 and 5. All maps are normalized to their mean value. Bottom-right ﬁgure: corresponding ∆-variance functions calculated for each case, and these are compared to the ∆-variance function calculated for the underlying fBm image Fig. 8. One or several similar 2D Gaussian structures injected on top of an fBm image with β = 2.4. The Gaussian functions have an aspect ratio f = 5, a value σ1 = 50 pixels, and δc = 3. All maps are normalized to their mean value. Bottom-right ﬁgure: corresponding ∆-variance func- tions calculated for each case, and these are compared to the ∆-variance function calculated for the underlying fBm image. Fig. 8. One or several similar 2D Gaussian structures injected on top of an fBm image with β = 2.4. The Gaussian functions have an aspect ratio f = 5, a value σ1 = 50 pixels, and δc = 3. All maps are normalized to their mean value. Bottom-right ﬁgure: corresponding ∆-variance func- tions calculated for each case, and these are compared to the ∆-variance function calculated for the underlying fBm image. Fig. 8. One or several similar 2D Gaussian structures injected on top of an fBm image with β = 2.4. The Gaussian functions have an aspect ratio f = 5, a value σ1 = 50 pixels, and δc = 3. All maps are normalized to their mean value. Bottom-right ﬁgure: corresponding ∆-variance func- tions calculated for each case, and these are compared to the ∆-variance function calculated for the underlying fBm image. Fig. 7. 2D Gaussian structures injected on top of an fBm image with β = 2.4. The Gaussian functions have an aspect ratio f = 5, a value of σ1 = 50 pixels, and a column density contrast between the peak of the 2D Gaussian and the mean value of the fBm, δc, that is varied between 1 and 5. All maps are normalized to their mean value. 6.2. Fractal Brownian motion maps with additional structure Bottom-right ﬁgure: corresponding ∆-variance functions calculated for each case, and these are compared to the ∆-variance function calculated for the underlying fBm image. self-gravitating molecular clouds. However, the parameter space can be very large, namely, models with or without gravity, with various magnetic ﬁeld strengths, and with various driving schemes, Mach numbers, and turbulence driving scales. When gravity is included, the extracted information will also unavoid- ably depend on the time evolution of the simulated clouds. This remains a valuable approach that has in fact been explored to a certain extent (e.g., Ossenkopf et al. 2001; Ossenkopf 2002) and deserves to be explored further with more reﬁned models. In this work, we prefer to generate models whose parameters can be easily controlled and for which we can easily understand and disentangle the effects on the ∆-variance function. As in Sect. 6.2, we superimposed 2D Gaussians on top of predeﬁned fBm images. However, instead of including individual structures or structures that are set apart from each other, we now include 2D Gaussians with speciﬁc distribution functions that character- ize their properties. The parameters we varied are the number of 2D Gaussian structures, Ns, the distribution function of the size of the major axis (dN/dL1), the distribution function of the aspect ratios (dN/d f), and the distribution function of the structures column density contrast (dN/dδc). Each structure is assigned a randomly drawn orientation on the map, and the struc- tures are allowed to overlap. Keeping in mind that clumps, cores, self-gravitating molecular clouds. However, the parameter space can be very large, namely, models with or without gravity, with various magnetic ﬁeld strengths, and with various driving schemes, Mach numbers, and turbulence driving scales. When gravity is included, the extracted information will also unavoid- ably depend on the time evolution of the simulated clouds. This remains a valuable approach that has in fact been explored to a certain extent (e.g., Ossenkopf et al. 2001; Ossenkopf 2002) and deserves to be explored further with more reﬁned models. In this work, we prefer to generate models whose parameters can be easily controlled and for which we can easily understand and disentangle the effects on the ∆-variance function. As in Sect. 6.2, we superimposed 2D Gaussians on top of predeﬁned fBm images. 6.2. Fractal Brownian motion maps with additional structure However, instead of including individual structures or structures that are set apart from each other, we now include 2D Gaussians with speciﬁc distribution functions that character- ize their properties. The parameters we varied are the number of 2D Gaussian structures, Ns, the distribution function of the size of the major axis (dN/dL1), the distribution function of the aspect ratios (dN/d f), and the distribution function of the structures column density contrast (dN/dδc). Each structure is assigned a randomly drawn orientation on the map, and the struc- tures are allowed to overlap. Keeping in mind that clumps, cores, underlying fBm is maximized on scales that are on the order of the equivalent diameter of the structure, Deq ≈4 √σ1σ2. Given the expression of the ∆-variance in Eq. (3), one thus expects that the amplitude of the maximum deviation of σ2 ∆in the presence of structures from the σ2 ∆of an fBm (deﬁned here- after as ∆(σ2 ∆)max) and which occurs on spatial scales that are equal to the equivalent diameter of the structures, to scale with Aδ2 c, where A is the total area covered by the structures. We ver- ify whether this scaling holds for all cases displayed in Figs. 6–9. We calculate the area as being A = Nsπ2σ1σ2, where Ns is the number of structures present on the map. Figure 10 displays the value of ∆(σ2 ∆)max as a function of Aδ2 c. A linear scaling between these quantities is found, even though we observe a small devi- ation from linearity for smaller values of Aδ2 c. This is due to the fact that when structures are small, there are larger uncertainties associated with the determination of their surface. 6.2. Fractal Brownian motion maps with additional structure Bottom-right ﬁgure: corresponding ∆-variance functions calculated for each case, and these are compared to the ∆-variance function of the underlying fBm image. In Fig. 8, we investigate the effect of the surface area by increasing the number of structures that are superimposed onto the underlying fBm image. In this ﬁgure, one or several simi- lar structures (all with δc = 3, f = 5, and σ1 = 50 pixels) are superimposed onto the fBm images (all with 1000 × 1000 pixels resolution and β = 2.4). The lower-right panel in Fig. 8 shows that increasing the surface area of the injected structures has a signiﬁcant impact (i.e., linear with the number) on the increase of the σ2 ∆on spatial scales that are on the order of the size of the structures. We also note an increase in the width of the excess of the ∆-variance spectrum up to scales of 250 pixels (i.e., larger than the sizes of the individual structures themselves) when there are several structures. This is the direct signature of the 250 pixel separation between the structures. We also explore the effect of changing the size of the structure with respect to the image size while ﬁxing the aspect ratio and column density contrast. Figure 9 displays ﬁve realizations with f = 5, δc = 3 but where σ1 is varied between 150 and 16.67 pixels. The ∆-variance func- tions for these realizations are displayed in the lower-right panel of Fig. 9. Here as well, the increment in σ2 ∆with respect to the We explored the effect of varying the contrast between the injected 2D Gaussian structure and the underlying fBm image. Figure 7 displays ﬁve realizations where the value of δc = Npeak/ ⟨NfBm⟩is varied between 1 and 5. For all cases, the other parameters are ﬁxed to f = 5 and σ1 = 50 pixels. All images have a resolution of 1000 × 1000 pixels, and the underlying fBm has an exponent of β = 2.4. The lower-right panel in Fig. 7 dis- plays the corresponding ∆-variance functions, which, here once again, are compared to the ∆-variance of the fBm image. Figure 7 shows that higher contrasts (δc) between the self-similar fBm and the injected structure lead to higher values of the σ2 ∆on spatial scales equal to Deq. 4 The Hi-GAL sources were extracted with CuTEx (Molinari et al. 2016), which is designed to identify relatively roundish sources. In prin- ciple, during the detection step, it keeps only structures with both minor and major axes ranging from 1 to 3 instrumental point spread functions. Subsequently, starting from this initial guess, the 2D Gaussian ﬁt that is used to determine the ﬂux as well as the ﬁnal estimate of the two axes has an additional tolerance to adjust itself on the source proﬁle, so that one can ﬁnd one (or both) of the two axes shorter than 1 RMS or longer than 3 RMS. Usually, no large differences are found between the two axes, so that the ratio is never larger than ≈4. On the contrary, algo- rithms used to extract sources from CO surveys, such as the one used in Heyer et al. (2001) that is based on a friend-of-friend approach in position-position-velocity space, do not have any constraint on source size. If the CO emission is kinematically connected over an elongated area, such a structure might be identiﬁed as a single source with a large aspect ratio. 6.3. Toward more realistic conﬁgurations The Gaussian functions all have an aspect ratio f = 0.2, a col- umn density contrast between the peak of the 2D Gaussian and the mean value of the fBm, δc = 3, and a value of σ1 that is varied between 150 and 16.67 pixels. Bottom-right ﬁgure: corresponding ∆-variance func- tions calculated for each case, and these are compared to the ∆-variance function calculated for the underlying fBm image. Fig. 10. Maximum deviation of the ∆-variance function in the presence of structures from that of a pure fBm as a function of the quantity Aδ2 c, where A is the area covered by the discrete structure(s) and δc is the column density contrast between the peak of the structure and the mean value of the underlying fBm. The dashed line has a slope of one. roundish than the ones detected in molecular line observations4. The distribution functions in Fig. 11 are normalized and are thus transformed into probability distribution functions. For the aspect ratio distributions of the Hi-GAL and FCRAO HCS clouds and clumps, (dN/df)norm (Fig. 11, middle panel), we ﬁnd that these distributions are best approximated by the following function: Fig. 9. 2D Gaussian structures injected on top of an fBm image with β = 2.4. The Gaussian functions all have an aspect ratio f = 0.2, a col- umn density contrast between the peak of the 2D Gaussian and the mean value of the fBm, δc = 3, and a value of σ1 that is varied between 150 and 16.67 pixels. Bottom-right ﬁgure: corresponding ∆-variance func- tions calculated for each case, and these are compared to the ∆-variance function calculated for the underlying fBm image. log dN d f ! norm = η f + Af . (9) (9) Fitting the distributions of aspect ratios for the Hi-GAL (for f > 1) and HCS (for f > 2.5) clouds yields [η = −1.15 ± 0.04, Af = 0.75±0.13] and [η = −0.37±0.01, Af = 0.41±0.10], respectively. The results of the corresponding ﬁts are shown in Fig. 11 (dashed red lines, middle panel). For any other chosen value of η, the corresponding value of Af can be calculated by requiring that R fmax fmin (dN/d f)norm d f = 1, and where fmin and fmax are the lower and upper limits on f, respectively. 6.3. Toward more realistic conﬁgurations In principle, the generation of realistic column density maps could rely on numerical simulations of turbulent and A177, page 8 of 18 A177, page 8 of 18 S. Dib et al.: Characteristic scales in molecular clouds Fig. 9. 2D Gaussian structures injected on top of an fBm image with β = 2.4. The Gaussian functions all have an aspect ratio f = 0.2, a col- umn density contrast between the peak of the 2D Gaussian and the mean value of the fBm, δc = 3, and a value of σ1 that is varied between 150 and 16.67 pixels. Bottom-right ﬁgure: corresponding ∆-variance func- tions calculated for each case, and these are compared to the ∆-variance function calculated for the underlying fBm image. Fig. 10. Maximum deviation of the ∆-variance function in the presence of structures from that of a pure fBm as a function of the quantity Aδ2 c, where A is the area covered by the discrete structure(s) and δc is the column density contrast between the peak of the structure and the mean value of the underlying fBm. The dashed line has a slope of one. roundish than the ones detected in molecular line observations4. The distribution functions in Fig. 11 are normalized and are thus transformed into probability distribution functions. For the aspect ratio distributions of the Hi-GAL and FCRAO HCS clouds and clumps, (dN/df)norm (Fig. 11, middle panel), we ﬁnd that these distributions are best approximated by the following function: log dN d f ! norm = η f + Af . (9) Fitting the distributions of aspect ratios for the Hi-GAL (for f > 1) and HCS (for f > 2 5) clouds yields [η = 1 15 ± Fig. 10. Maximum deviation of the ∆-variance function in the presenc Fig. 10. Maximum deviation of the ∆-variance function in the presence of structures from that of a pure fBm as a function of the quantity Aδ2 c, where A is the area covered by the discrete structure(s) and δc is the column density contrast between the peak of the structure and the mean value of the underlying fBm. The dashed line has a slope of one. Fig. 9. 2D Gaussian structures injected on top of an fBm image with β = 2.4. 6.3. Toward more realistic conﬁgurations In the same vain, we ﬁtted the normalized distribution function of the size of and ﬁlaments such as those found in the column density map of Cygnus-X North may have a more complex internal struc- ture than 2D Gaussian functions, we aim to understand which combination of the parameters leads to ∆-variance functions that are similar to that of the Cygnus-X North region. More broadly, our aim is also to understand the sensitivity of the ∆-variance to the choice of the distribution functions that characterize the statistical properties of the structures. The distribution of sizes and aspect ratios of cores and clumps in molecular clouds is likely to depend on the density tracer as well as on the clump identiﬁcation algorithm. To illus- trate this, in Fig. 11 we compare the size (i.e., major axis; L1, left panel) and aspect ratio (f, middle panel) distributions of structures found in the Herschel infrared Galactic Plane survey (Hi-GAL; Molinari et al. 2016; Elia et al. 2017) and in the Five College Radio Astronomy Observatory (FCRAO) CO survey of the outer Galaxy (HCS; Heyer et al. 1998; Dib et al. 2009). Struc- tures in the Hi-GAL survey are extracted from 250 µm emission maps (78 952 objects in total), whereas the HCS survey is based on the (1–0) transition in 12CO molecular line observations, in which 10 156 discrete structures were identiﬁed. The clouds and clumps reported in the Hi-GAL survey are ostensibly more A177, page 9 of 18 A177, page 9 of 18 A&A 642, A177 (2020) Fig. 11. Fractional probability distribution functions of the size of the major axis (left) and aspect ratio of clouds (middle) found in the 250 µm maps of the Hi-GAL survey (Molinari et al. 2016; Elia et al. 2017; full line) and in the 12CO FCRAO HCS survey (Heyer et al. 2001; Dib et al. 2009; dashed line). The distribution of column density contrast is assumed to be a power law. The distributions of L1 and f are ﬁtted with parameterized functions. The values of the parameters of the ﬁt are reported in the main text. Fig. 11. Fractional probability distribution functions of the size of the major axis (left) and aspect ratio of clouds (middle) found in the 250 µm maps of the Hi-GAL survey (Molinari et al. 2016; Elia et al. 2017; full line) and in the 12CO FCRAO HCS survey (Heyer et al. 6.3. Toward more realistic conﬁgurations 2001; Dib et al. 2009; dashed line). The distribution of column density contrast is assumed to be a power law. The distributions of L1 and f are ﬁtted with parameterized functions. The values of the parameters of the ﬁt are reported in the main text. the major axis, (dN/dL1)norm. Here again, we ﬁnd that the data is best ﬁtted with a function that is given by: self-gravitating structures will have, statistically, higher column density contrasts at time goes by). As stated above, we would like to understand the sensitivity of the ∆-variance spectrum in relation to the underlying distribu- tion functions of the different parameters, and, as a by-product, understand which particular set of parameters can help generate a ∆-variance spectrum that resembles the one found in Cygnus-X North. In principle, the parameter space is relatively large with four free parameters to probe (Ns, η, ξ, and ψ), and even larger if the lower and upper limits on f, L1, and δc are also varied. As a ﬁrst step, we explore below models of synthetic clouds whose properties are inspired from the Hi-GAL and the HCS samples. In a second step, we expand this “forward modeling” approach and present a broader parameter study where we vary, in a more systematic fashion, the parameters of the distributions functions. log dN dL1 ! norm = ξ L1 + AL1. (10) (10) Using Eq. (10) the ﬁt to the data of the Hi-GAL clouds for values of L1 in the range 1.5 pc ≥L1 ≥5 pc and for the HCS clouds using values of L1 in the range 4 pc ≥L1 ≥70 pc yields values of the parameters ξ and AL1 of [ξ = −0.89 ± 0.02, AL1 = 0.50 ± 0.06] and [ξ = −0.04 ± 0.002, AL1 = −0.91 ± 0.09], respectively. The ﬁt functions are displayed with the dashed red lines in Fig. 11 (left-hand panel). For any other value of ξ, the corresponding values of AL1 can be obtained by requiring that R L1,max L1,min (dN/dL1)norm dL1 = 1, where L1,min and L1,max are the lower and upper limits on L1. 6.3. Toward more realistic conﬁgurations y p For any given choice of (Ns, η, ξ, ψ), and owing to the fact that the orientations and positions of the injected structures are random, it is important that, for each choice of the parameters, a statistically signiﬁcant number of realizations is performed in order to capture the mean behavior and standard deviation around the mean of the ∆-variance spectrum. We chose to perform 25 realizations with any given set of the parameters. Fur- thermore, and owing to computational limitations, we performed the synthetic calculations on maps with 1000 × 1000 pixels, whereas the map of Cygnus-X North has 5740 × 5740 pixels. We assigned the same physical size to each pixel in the synthetic maps as in the observations (≈0.025 pc), and we matched the mean AV in the synthetic maps to the mean AV of the Cygnus-X North cloud. Because we are comparing the results from the syn- thetic maps to the observations of the Cygnus-X North region, all synthetic maps (i.e., both the reference fBm and the fBm plus structures maps) were convolved with a beam similar to that of the observations. The beam is represented by a Gaussian function whose FWHM = 18.2′′. We compared the ∆-variance spectra of the synthetic maps and the observations on scales that are larger than the beam size, namely scales that are ≥0.15 pc. The distribution of column density contrasts of dense struc- tures in nearby molecular clouds is not yet fully established. Recent work by Arzoumanian et al. (2019) derived the contrast between the average column density on ﬁlament crests and their local background for ﬁlaments detected in a number of nearby molecular clouds. Roy et al. (2019) constructed the distribution function of the contrast between ﬁlaments and their local back- ground and found that it scales as dN/dlogδc ∝δ−1.5 c for δc > 1. The exact scaling found in Arzoumanian et al. (2019) and Roy et al. (2019) may not apply directly to our synthetic models since we deﬁne the contrast as being the one between the peak column density of the structure and the mean value over the entire map. 6.3. Toward more realistic conﬁgurations With this in mind, we parameterized the distribution of column density contrasts as being a power law of the form: dN dδc = Acδ−ψ c , (11) dN dδc = Acδ−ψ c , (11) where Ac is a normalization coefﬁcient that is given by R δc,max δc,min (dN/dδc) dδc = 1, and δc,min and δc,max are the lower and upper limits on δc. We took δc,min = 1 in all cases and varied δc,max between 3 and 10. This is consistent with the range of val- ues found by Arzoumanian et al. (2019) for ﬁlaments and with the range of column densities that are present in the high den- sity tail of the N-PDF of the Cygnus-X North cloud. We chose three values of ψ, of 2, 2.5, and 3, which, as an extrapolation of the results presented in Arzoumanian et al. (2019), should cover both variations due to differences in the clouds environ- mental conditions and variations due to temporal evolution (i.e., 6.3.1. Hi-GAL- and HCS-like clumps The ∆-variance spectra for the synthetic maps are compared to the spectrum of the under- lying fBm (open diamonds) and to that of the Cygnus-X North cloud (ﬁlled stars). As observed earlier in the case where individ- ual structures are injected (in Sect. 6.2), the ∆-variance spectrum in the presence of structures shows a departure from that of the underlying fBm, and, in the case of an entire population of struc- tures, the point of maximum departure from the underlying fBm case corresponds to the characteristic scale of the ensemble of structures that are injected onto the map. Figure 12 shows that a better agreement is obtained for Ns = 300. We followed the same procedure and generated structures similar to those found in the HCS survey. Three examples of such maps with Ns = 50, 100, and 200 are displayed in Fig. 13 (top panel). The HCS- like structures have a shallower spectrum of major axis sizes, and the corresponding ∆-variance spectrum peaks at higher spa- tial scales than their Hi-GAL-like counterparts (Fig. 13, bottom panel). Figures 12 and 13 show that while the Hi-GAL-like structures provide a better match to the observations of Cygnus- X, neither of these two cloud samples ﬁt the observations of Cygnus-X North well. However, it is useful to compare these two cases to Cygnus-X in order to highlight how structures with fun- damentally different statistical properties impact the ∆-variance spectrum. These cases also provide a starting point for a more detailed exploration of the parameter space. the HCS survey are both larger and more elongated than struc- tures detected in the Hi-GAL survey. The lower and upper limits on the aspect ratios for the submm-like and CO-like clouds are taken to be (fmin = 1, fmax = 4) and ( fmin = 3, fmax = 12), as per the observational constraints (Fig. 11, middle panel). For the lower limits on the major axis size, we extrapolated the major axis size distributions for both the submm-like and CO-like clouds down to the resolution limit such that L1,min = 0.025 pc5. For the upper limit on L1, we adopted a common value of L1,max = 5 pc for both the Hi-GAL-like and HCS-like clouds. 6.3.1. Hi-GAL- and HCS-like clumps As illustrative examples, we ﬁrst explored the resulting ∆-variance spectra corresponding to population of clumps and cores similar to the ones found in the Hi-GAL (submm) and HCS (CO) surveys. We recall that the values of ξ and η are [−0.04, −0.37] and [−0.89,−1.15] for the Hi-GAL clouds and the CO clouds, respectively, which implies that the CO structures in A177, page 10 of 18 S. Dib et al.: Characteristic scales in molecular clouds Fig. 12. Top: examples of 200 (left), 300 (middle), and 400 (right) 2D Gaussian structures injected on top of an fBm image with β = 2.4. The Gaussian structures are randomly sampled using the distribution functions of the major axis size and aspect ratio distributions of the Hi-GAL clumps. The column density contrasts are randomly sampled from a distribution with ψ = 2.5. All synthetic maps are convolved with a beam whose FWHM = 18.2′′. Bottom: ∆-variance spectrum of the synthetic models for cases with Ns = 200 (left), Ns = 300 (middle), and Ns = 400 (right) injected structures. Each synthetic spectrum is the average over 25 realizations, and the full lines represent the 1σ dispersion around the mean. The synthetic ∆-variance spectra are compared to those of the Cygnus-X North region and an fBm with β = 2.4. Fig. 12. Top: examples of 200 (left), 300 (middle), and 400 (right) 2D Gaussian structures injected on top of an fBm image with β = 2.4. The Gaussian structures are randomly sampled using the distribution functions of the major axis size and aspect ratio distributions of the Hi-GAL clumps. The column density contrasts are randomly sampled from a distribution with ψ = 2.5. All synthetic maps are convolved with a beam whose FWHM = 18.2′′. Bottom: ∆-variance spectrum of the synthetic models for cases with Ns = 200 (left), Ns = 300 (middle), and Ns = 400 (right) injected structures. Each synthetic spectrum is the average over 25 realizations, and the full lines represent the 1σ dispersion around the mean. The synthetic ∆-variance spectra are compared to those of the Cygnus-X North region and an fBm with β = 2.4. panel in Fig. 12 displays the corresponding ∆-variance spectra, which are calculated, in each case, as the mean spectrum from the 25 realizations (blue triangles). 5 In Cygnus-X North, the real lower limit on the sizes of the struc- tures and the true shape of the size distribution are very uncertain in the regime of small sizes. The choice of L1,min = 0.025 pc basically means no lower limit (as it is the smallest resolved structure), while all other higher values would be questionable. 6.3.1. Hi-GAL- and HCS-like clumps Adopting a larger value of L1,max (up to ≈70 pc) for the HCS- like clouds would be excessive on the grounds that Cygnus-X North does not contain any such large structures, and this is fur- ther motivated by the fact that we are considering a region that is 5.74 smaller, in each direction, than the real map. In the absence of additional information from the surveys, we imposed, in both cases, a value of ψ = 2.5 and lower and upper bounds on δc of 1 and 3, respectively. The top panel in Fig. 12 displays three examples of the Hi-GAL-like maps generated with Ns = 200 (left), 300 (mid- dle), and 400 structures (right) out of a total of 25 realizations performed for each case. The size of the major axis, aspect ratio, and column density contrast for each individual structure are ran- domly sampled from the corresponding distribution functions, and the structures are assigned random positions and orientations and overlaid on top of an fBm image with β = 2.4. The bottom 6.3.2. Parameter study Furthermore, we ﬁxed here the lower and upper bounds of the structures sizes (i.e., size of the major axis) to L1,min = 0.025 pc and L1,max = 5 pc, respectively. The lower and upper bounds on the aspect ratios are ﬁxed in all cases to the values of fmin = 3 and fmax = 12, and the lower and upper limits on the column den- sity contrasts are δc,min = 1 and δc,max = 3. Examples of maps generated with each permutation of these parameters are dis- played in Fig. 14. The ∆-variance spectra for all of these cases are displayed in Fig. 15. Here again, we performed 25 real- izations with each set of parameters and computed the mean value and standard deviation on each spatial scale. What Fig. 15 reveals is that the ∆-variance spectrum is more sensitive to the shape of the distribution of the major axis sizes, character- ized here by the parameter ξ, than to the distribution of aspect ratios (parameter η). Shallower distributions of the major axis (ξ = −0.2) lead to an over abundance of larger structures on the map and to a noticeable mismatch of the ∆-variance for those cases with the ∆-variance spectrum of the Cygnus-X North region, irrespective of the value of η. In contrast, steeper dis- tribution functions of the major axis (e.g., ξ = −0.8) lead to signiﬁcantly less variance than what is observed in Cygnus- X North on scales ≳0.2 pc. For intermediate values of ξ in the range [−0.6,−0.4], there is a good agreement between the ∆-variance spectrum of the synthetic models and the spectrum of Cygnus-X North. Models with ξ = −0.4 present the best ﬁt, but one can reasonably argue that cases with ξ = −0.6 could still be considered a good ﬁt to the data if higher values of Ns were employed. From the grid of models shown in Fig. 15, the case with ξ = −0.4 and η = −0.35 represents the best ﬁt to the observations. This corresponds to a mean value of the size of the major axis of ¯L1 = R 5 0.025 (dN/dL1) L1dL1/ R 5 0.025 (dN/dL1) dL1 ≈ 1.05 pc and to a mean value of the aspect ratio of ¯f = R 12 3 (dN/d f) fdf/ R 12 3 (dN/df) df ≈4.23. 6.3.2. Parameter study In this section, we perform a broader parameter study and inves- tigate how the ∆-variance spectrum is affected by variations in A177, page 11 of 18 A177, page 11 of 18 A&A 642, A177 (2020) Fig. 13. Top: examples of 50 (left), 100 (middle), and 200 (right) 2D Gaussian structures injected on top of an fBm image with β = 2.4. The Gaussian structures are randomly sampled using the distribution functions of the major axis size and aspect ratio distributions of the HCS clouds. The column density contrasts are randomly sampled from a distribution with ψ = 2.5. All synthetic maps are convolved with a beam whose FWHM = 18.2′′. Bottom: ∆-variance spectrum of the synthetic models for cases with Ns = 50 (left), Ns = 100 (middle), and Ns = 200 (right) injected structures. Each synthetic ∆-variance spectrum is the average over 25 realizations and the full lines represent the 1σ dispersion around the mean. The synthetic ∆-variance spectra are compared to those of the Cygnus-X North region and an fBm with β = 2.4. Fig. 13. Top: examples of 50 (left), 100 (middle), and 200 (right) 2D Gaussian structures injected on top of an fBm image with β = 2.4. The Gaussian structures are randomly sampled using the distribution functions of the major axis size and aspect ratio distributions of the HCS clouds. The column density contrasts are randomly sampled from a distribution with ψ = 2.5. All synthetic maps are convolved with a beam whose FWHM = 18.2′′. Bottom: ∆-variance spectrum of the synthetic models for cases with Ns = 50 (left), Ns = 100 (middle), and Ns = 200 (right) injected structures. Each synthetic ∆-variance spectrum is the average over 25 realizations and the full lines represent the 1σ dispersion around the mean. The synthetic ∆-variance spectra are compared to those of the Cygnus-X North region and an fBm with β = 2.4. the distribution functions of the structure sizes, aspect ratios, and column density contrasts. It is nearly impossible to cover the entire parameter space for the four parameters (Ns, η, ξ, ψ). We therefore ﬁxed Ns = 300, and adopted, in the ﬁrst instance, a value of ψ = 2.5. We varied the shapes of the major axis and aspect ratio distribution functions and considered values of ξ = [−0.8, −0.6, −0.4, −0.2] and η = [−1.15, −0.75, −0.35]. 6.3.2. Parameter study This implies a mean value of the minor axis ¯L2 = 0.25 pc, and, taking σ1 = L1/3 and σ2 = L2/3, this yields a value of the effective size Deﬀ= 4 √σ1σ2 ≈0.70 pc, which is very close to the mid position of the plateau in the ∆-variance spectrum of the Cygnus-X North cloud. the distribution functions of the structure sizes, aspect ratios, and column density contrasts. It is nearly impossible to cover the entire parameter space for the four parameters (Ns, η, ξ, ψ). We therefore ﬁxed Ns = 300, and adopted, in the ﬁrst instance, a value of ψ = 2.5. We varied the shapes of the major axis and aspect ratio distribution functions and considered values of ξ = [−0.8, −0.6, −0.4, −0.2] and η = [−1.15, −0.75, −0.35]. Furthermore, we ﬁxed here the lower and upper bounds of the structures sizes (i.e., size of the major axis) to L1,min = 0.025 pc and L1,max = 5 pc, respectively. The lower and upper bounds on the aspect ratios are ﬁxed in all cases to the values of fmin = 3 and fmax = 12, and the lower and upper limits on the column den- sity contrasts are δc,min = 1 and δc,max = 3. Examples of maps generated with each permutation of these parameters are dis- played in Fig. 14. The ∆-variance spectra for all of these cases are displayed in Fig. 15. Here again, we performed 25 real- izations with each set of parameters and computed the mean value and standard deviation on each spatial scale. What Fig. 15 reveals is that the ∆-variance spectrum is more sensitive to the shape of the distribution of the major axis sizes, character- ized here by the parameter ξ, than to the distribution of aspect ratios (parameter η). Shallower distributions of the major axis (ξ = −0.2) lead to an over abundance of larger structures on the map and to a noticeable mismatch of the ∆-variance for those cases with the ∆-variance spectrum of the Cygnus-X North region, irrespective of the value of η. In contrast, steeper dis- tribution functions of the major axis (e.g., ξ = −0.8) lead to signiﬁcantly less variance than what is observed in Cygnus- X North on scales ≳0.2 pc. 6.3.2. Parameter study All models shown here share the same values of δc = 2.5, (L1,min = 0.025 pc, L1,max = 5 pc), (fmin = 3, fmax = 12), and (δc,min = 1, δc,max = 3). All synthetic maps are convolved with a beam whose FWHM = 18.2′′. In summary, we are able to show that it is possible to repro- duce ∆-variance spectra that resemble that of the Cygnus-X North region under reasonable assumptions of the size distri- butions of structures, their aspect ratios, and column density contrasts. Broadly speaking, reproducing the ∆-variance spec- trum of the Cygnus-X North region requires a size distribution that is steeper than the size distribution of structures detected in CO surveys, such as the HCS survey, and shallower than the one inferred from the Hi-GAL submm survey. We also show that the observations are best ﬁtted when structures are allowed to have aspect ratios that are predominantly ≳3. bottom panel) show that the existence of more roundish struc- tures (i.e., larger structures for the same major axis size) results in ∆-variance spectra that peak at higher spatial scales, and those cases present a poor ﬁt to the observations of Cygnus-X North. We now explore the effect of varying the distribution func- tion of the column density contrast. In addition to the ﬁducial case with ψ = 2.5, we constructed synthetic maps with ψ = 2 and ψ = 3. We also generated additional maps in which the value of ψ is ﬁxed to 2.5 and varied the values of the lower and upper limits on the column density contrast, δc. We considered cases with (δc,min = 1, δc,max = 3; ﬁducial case shown earlier) and cases with (δc,min = 1, δc,max = 5) and (δc,min = 1, δc,max = 10). The remaining parameters were ﬁxed to their ﬁducial values, namely ξ = −0.4, η = −0.35, (L1,min = 0.025 pc, L1,max = 5 pc), and ( fmin = 3, fmax = 12). Figure 18 (top panel) displays selected realizations of maps generated with various values of ψ, and Fig. 19 (top panel) displays examples of maps generated with different values of δc,max and with a ﬁxed value of ψ = 2.5. The ∆-variance spectra for various cases of ψ are shown in Fig. 18 (bottom panel) and are always calculated as being the mean val- ues from 25 realizations. 6.3.2. Parameter study For intermediate values of ξ in the range [−0.6,−0.4], there is a good agreement between the ∆-variance spectrum of the synthetic models and the spectrum Figure 16 shows the individual ∆-variance spectra for the 25 individual realizations (i.e., gray lines) when structures are randomly drawn from the distribution functions with the best ﬁt- ting set of parameters, namely Ns = 300, ξ = −0.4, η = −0.35, and ψ = 2.5. We adopted these values of Ns, ξ, and η and further investigated the effect of the remaining parameters. In order to further explore the effect of the aspect ratio, f, we performed additional tests in which we varied its lower and upper bounds. In addition to the ﬁducial case in which fmin = 3 and fmax = 12, we considered models with ( fmin = 1, fmax = 6) and ( fmin = 1, fmax = 12). All other parameters were ﬁxed to those of the best ﬁtting model in Fig. 15, namely ξ = −0.4, η = −0.35, ψ = 2.5, and (L1,min = 0.025 pc, L1,max = 5 pc), (δc,min = 1, δc,max = 3). Figure 17 (top panel) displays exam- ples of the maps for each one of the considered cases. The calculations of the ∆-variance spectra in those cases (Fig. 17, A177, page 12 of 18 S. Dib et al.: Characteristic scales in molecular clouds Fig. 14. Examples of synthetic maps generated by overlaying structures (2D Gaussians, Ns = 300) on top of an fBm image with β = 2.4. The properties of the structures are randomly sampled from distribution functions of the aspect ratio (Eq. (9)), size of the major axis (Eq. (10)), and column density contrast (Eq. (11)). All models shown here share the same values of δc = 2.5, (L1,min = 0.025 pc, L1,max = 5 pc), (fmin = 3, fmax = 12), and (δc,min = 1, δc,max = 3). All synthetic maps are convolved with a beam whose FWHM = 18.2′′. Fig. 14. Examples of synthetic maps generated by overlaying structures (2D Gaussians, Ns = 300) on top of an fBm image with β = 2.4. The properties of the structures are randomly sampled from distribution functions of the aspect ratio (Eq. (9)), size of the major axis (Eq. (10)), and column density contrast (Eq. (11)). 6.3.2. Parameter study Overall, the ∆-variance spectrum is less impacted by variations in ψ, even though values of ψ ≥2.5 lead to a better agreement with the observations. On the other hand, allowing for higher values of the maximum column density con- trast, δc,max, has an impact on the amplitude of the deviation from the ∆-variance of the underlying fBm, but it has no effect on the position of the point of maximum deviation (Fig. 19, bottom panel), in agreement with our ﬁndings in Sect. 6.2. We ﬁnd that a value of δc,max = 3 ﬁts the observations better. 6.3.3. Contribution of compact sources to the ∆-variance plateau in Cygnus-X We also generated other models with different values of the fBm exponent, in the range β = [2, 3] (ﬁgures not shown for redundancy). The ∆-variance spectrum of the mod- els (i.e., Fig. 20) exhibits a peak at ≈0.6−0.8 pc, which is at the lower end of the plateau found in the observations. However, there is no agreement between the models and the observations, neither in terms of the width of the ∆-variance spectrum nor its amplitude. What Fig. 20 reveals is that the compact sources taken alone, despite having an important contribution to the signal at ≈0.6−0.8 pc, cannot explain the full extent of the plateau that is observed in the ∆-variance spectrum of Cygnus-X North; it further reveals that there is a need to consider a distribution of structures that includes both larger and more elongated objects in order to explain the observations. A broader distribution of sizes is required in order to reproduce the broad ∆-variance spectrum in Cygnus-X North as well as to adjust the amplitude of the spec- trum to the observed values since more extended structures can provide the intermediate column densities between the compact sources and the underlying fBm structure. of the background emission, thanks to a recently developed minimization, to interpolate an empty space (the footprint of a detected source) in a 2D map. Applying this code to the map of Cygnus-X North, we were able to detect a total of 1242 com- pact sources. The mean values of σ1 and aspect ratio (f) for this sample of compact sources are 0.23 pc and 1.32, respec- tively. This implies a mean effective size for the compact sources of Deﬀ= 4 √σ1σ2 ≈0.82 pc, which corresponds, roughly, to where the ∆-variance of Cygnus has its peak. Because the syn- thetic maps we are using have 1000 × 1000 pixels and are thus ≈33 times smaller than the map of Cygnus-X North, we injected a total of (1242/33) ≈38 compact sources onto each synthetic map. Unlike synthetic maps generated earlier, only the underly- ing fBm is convolved with the Gaussian beam since the compact sources extracted from the Cygnus-X North map are already affected by beam smearing. 6.3.3. Contribution of compact sources to the ∆-variance plateau in Cygnus-X In this section, we examine the contribution of compact sources to the observed plateau in the ∆-variance spectrum of Cygnus- X North. In the previous section, we generated populations of structures whose properties were sampled from parameterized distribution functions and overlaid these structures on an fBm image. Here, we follow a different approach and extract the com- pact sources from the map of Cygnus-X North before reinjecting them onto the fBm image. To this purpose, we used a newly developed clump ﬁnding algorithm. The details of the code will be presented in a forthcoming paper (Bontemps et al., in prep.). Here we simply summarize its basic concepts. This code uses second-order spatial derivatives in order to recognize high curvature peaks where 2D Gaussian ﬁts are applied, after sub- tracting a local background. It uses an improved determination A177, page 13 of 18 A177, page 13 of 18 A&A 642, A177 (2020) 15. ∆-variance spectra related to the models presented in Fig. 14. Each synthetic spectrum (blue triangles) is the average over 25 realizat he maps with the same set of parameters. In all models, Ns = 300 and ψ = 2.5. The full line is the 1σ dispersion around the mean. The synth ariance spectra are compared to that of the Cygnus-X North region and that of an fBm image with β = 2.4. In the grid of models, the best ﬁ data of Cygnus North is for the case with ξ = −0.4 and η = −0.35. Fig. 15. ∆-variance spectra related to the models presented in Fig. 14. Each synthetic spectrum (blue triangles) is the average over 25 realizations of the maps with the same set of parameters. In all models, Ns = 300 and ψ = 2.5. The full line is the 1σ dispersion around the mean. The synthetic ∆-variance spectra are compared to that of the Cygnus-X North region and that of an fBm image with β = 2.4. In the grid of models, the best ﬁt to the data of Cygnus North is for the case with ξ = −0.4 and η = −0.35. using this approach, and the mean ∆-variance spectrum calcu- lated from the 50 realizations is shown in the right-hand panel of Fig. 20. The exponent of the fBm in this case is also taken to be β = 2.4. A177, page 14 of 18 8. Conclusions The present study relies on the comparison of observations with synthetic maps that are generated using 2D fBm images with superimposed structures. As stated above, the structures are injected at random positions and are not necessarily asso- ciated with existing higher column density regions in the fBm images. Additionally, the random position and orientation that are assigned to each structure imply that the structures are not spatially correlated, and this may affect the signal on spa- tial scales on the order of the structures’ effective separation. Furthermore, fBm images, albeit a good proxy for self-similar structures, are known to differ from real molecular clouds in terms of their multifractal nature, or, more speciﬁcally, their lack thereof (e.g., see details in Elia et al. 2018). There is, however, no reason to believe that any of these assumptions or simpliﬁ- cations are critical to the analysis. Our results demonstrate that the ∆-variance spectrum of a complex region such as Cygnus-X North can be reproduced reasonably well using realistic distri- bution functions of the characteristics of these structures (size, contrast, aspect ratio). Nonetheless, it is important to stress that a more physical model is still needed in order to tie the existence of these structures to the physical conditions that prevail in the gas and to the initial conditions of the gas when the molecular cloud has started to assemble. While some reﬁnements can be made to the empirical models presented in this work, it is prob- ably safe to state that numerical models that incorporate most or all of the necessary physics – and that preferably simulate galac- tic scales larger than the clouds themselves while resolving the internal structure of the clouds – constitute the next step for com- paring models to the observations. Complex features, such as striations that are observed in molecular clouds (e.g., Heyer et al. 2016; Tritsis et al. 2018), can naturally emerge self-consistently in numerical models and are harder to implement in empirical models. The internal structure of molecular clouds holds important clues regarding the physical processes that lead to their formation and their subsequent dynamical evolution. 6.3.3. Contribution of compact sources to the ∆-variance plateau in Cygnus-X We generated 50 synthetic maps such that each core is statis- tically selected at least once and, for each map, the 38 structures that are injected are randomly sampled from the list of structures that are extracted from the observational map and are assigned random positions and orientations on the map. Figure 20 (left- hand panel) displays one of the realizations of the synthetic maps A177, page 14 of 18 S. Dib et al.: Characteristic scales in molecular clouds S. Dib et al.: Characteristic scales in molecular clouds by a factor of two in order to match the resolution of the 250 µm maps. This was done using the method detailed in Palmeirim et al. (2013). This approach has the advantage of increasing the dynamical range of the maps, but it may have introduced addi- tional signal on small scales; this, in turn, may have contributed to worsening the agreement between the synthetic models and the observations of Cygnus-X on these scales. While revisiting this correction is well beyond the scope of this paper, this effect is possibly what is causing the ∆-variance spectrum in Cygnus- X North to fall less sharply at smaller spatial scales than what is expected from the effects of beam smearing (i.e., the slope of the ∆-variance spectrum in the ﬁrst bin is shallower than the slope in the second bin of the spectrum). Another possible issue relates to the existence of an underlying self-similar regime in Cygnus-X. The simple experiments presented in Figs. 6–9 when a single (or a few similar) structure(s) is (are) superimposed onto the fBm image show that the self-similarity in the ∆-variance spectrum is preserved on scales that are either smaller or larger than the effective diameter of the structure(s). In the case of mul- tiple structures with different sizes, contrasts, and elongations, the underlying self-similarity is perturbed on a larger range of spatial scales. Thus, the identiﬁcation of a self-similar regime in Cygnus-X North, if it exists, would in principle require higher resolution observations in order to probe the shape of the ∆- variance at smaller spatial scales and/or a larger map, possibly connecting to the H I gas at the outer edges of the cloud in order to probe the shape of the spectrum at larger spatial scales. Fig. 16. 6.3.3. Contribution of compact sources to the ∆-variance plateau in Cygnus-X Same as the ﬁgure with ξ = −0.4, η = −0.35, ψ = 2.5, and Ns = 300 displayed in Fig. 15 but additionally showing the ∆-variance spectra of individual realizations with this set of parameters. Fig. 16. Same as the ﬁgure with ξ = −0.4, η = −0.35, ψ = 2.5, and Ns = 300 displayed in Fig. 15 but additionally showing the ∆-variance spectra of individual realizations with this set of parameters. 8. Conclusions While the overall mor- phology of a molecular cloud can be linked to its star formation activity (Dib & Henning 2019) and thus provide hints about the cloud’s assembly mechanism, the internal structure of the cloud also holds important information about the fragmentation pro- cess and the competition between different physical processes that redistribute matter within the cloud. Using the ∆-variance spectrum, we have characterized the structure of the Cygnus-X North and Polaris Flare molecular clouds. These two clouds rep- resent two extremes in terms of their star formation activity in the Milky Way. In Polaris, the structure of the cloud as revealed by the ∆-variance is self-similar over more than one order of mag- nitude in spatial scales. In contrast, the ∆-variance spectrum of Cygnus-X North exhibits an excess (compared to Polaris) and a plateau in the range of physical scales of ≈0.5−1.2 pc. The depar- ture from self-similarity in a region such as Cygnus-X North is due to the existence of over-dense structures, including compact sources (i.e., hubs and ridges), and more elongated clumps and ﬁlaments. In such a region, these structures may arise as a result of large-scale compressions (i.e., converging ﬂows) before being dominated by their own self-gravity. They are also likely to be affected by the mechanical and radiative feedback from massive stars that form in the cloud. In order to explain the observations of Cygnus-X North, we built synthetic maps in which we overlaid a population of dis- crete structures (i.e., 2D Gaussians) on top of an fBm image. The properties of these structures, such as their major axis sizes, aspect ratios, and column density contrasts, are randomly drawn from parameterized probability distribution functions of these quantities. We show that the inclusion of discrete structures “on top” of a self-similar image increases the ∆-variance, and this increment has its maximum on spatial scales that are equal to On the observational side, we recall that the Herschel maps presented in this work have been resampled to a higher resolution A177, page 15 of 18 A177, page 15 of 18 A&A 642, A177 (2020) A&A 642, A177 (2020) Fig. 17. Top: examples of 2D Gaussian structures injected on top of an fBm image with β = 2.4. The maps only differ in the values of the lower and upper bounds of the aspect ratios, fmin and fmax, respectively. 8. Conclusions All other parameters have the same values (see text for details). All synthetic maps are convolved with a beam whose FWHM = 18.2′′. Bottom: ∆-variance spectrum of the synthetic models for the three cases with the considered sets of fmin and fmax. Each synthetic spectrum is the average over 25 realizations, and the full lines represent the 1σ dispersion around the mean. The synthetic ∆-variance spectra are compared to that of the Cygnus-X North region and to that of an fBm image with β = 2.4. A&A 642, A177 (2020) Fig. 17. Top: examples of 2D Gaussian structures injected on top of an fBm image with β = 2.4. The maps only differ in the values of the lower and upper bounds of the aspect ratios, fmin and fmax, respectively. All other parameters have the same values (see text for details). All synthetic maps are convolved with a beam whose FWHM = 18.2′′. Bottom: ∆-variance spectrum of the synthetic models for the three cases with the considered sets of fmin and fmax. Each synthetic spectrum is the average over 25 realizations, and the full lines represent the 1σ dispersion around the mean. The synthetic ∆-variance spectra are compared to that of the Cygnus-X North region and to that of an fBm image with β = 2.4. Fig. 18. Top: examples of 2D Gaussian structures injected on top of an fBm image with β = 2.4. The maps only differ in the values of the exponent ψ. All other parameters have the same values (see text for details). All synthetic maps are convolved with a beam whose FWHM = 18.2′′. Bottom: ∆-variance spectrum spectra of the synthetic models for the three cases with ψ = 2, 2.5, and 3. Each synthetic spectrum is the average over 25 realizations, and the full lines represent the 1σ dispersion around the mean. The synthetic ∆-variance spectra are compared to that of the Cygnus-X North region and that of an fBm image with β = 2.4. Fig. 18. Top: examples of 2D Gaussian structures injected on top of an fBm image with β = 2.4. The maps only differ in the values of the exponent ψ. All other parameters have the same values (see text for details). All synthetic maps are convolved with a beam whose FWHM = 18.2′′. 8. Conclusions Bottom: ∆-variance spectrum spectra of the synthetic models for the three cases with ψ = 2, 2.5, and 3. Each synthetic spectrum is the average over 25 realizations, and the full lines represent the 1σ dispersion around the mean. The synthetic ∆-variance spectra are compared to that of the Cygnus-X North region and that of an fBm image with β = 2.4. A177, page 16 of 18 S. Dib et al.: Characteristic scales in molecular clouds Fig. 19. Top: examples of 2D Gaussian structures injected on top of an fBm image with β = 2.4. The maps only differ in the values of the lower and upper bounds of column density contrast δc,min and δc,max, respectively. All other parameters have the same values (see text for details). All synthetic maps are convolved with a beam whose FWHM = 18.2′′. Bottom: ∆-variance spectra of the synthetic models for the three cases with the considered sets of δc,min and δc,max. Each synthetic spectrum is the average over 25 realizations, and the full lines represent the 1σ dispersion around the mean. The synthetic ∆-variance spectra are compared to that of the Cygnus-X North region and to that of an fBm image with β = 2.4. Fig. 19. Top: examples of 2D Gaussian structures injected on top of an fBm image with β = 2.4. The maps only differ in the values of the lower and upper bounds of column density contrast δc,min and δc,max, respectively. All other parameters have the same values (see text for details). All synthetic maps are convolved with a beam whose FWHM = 18.2′′. Bottom: ∆-variance spectra of the synthetic models for the three cases with the considered sets of δc,min and δc,max. Each synthetic spectrum is the average over 25 realizations, and the full lines represent the 1σ dispersion around the mean. The synthetic ∆-variance spectra are compared to that of the Cygnus-X North region and to that of an fBm image with β = 2.4. Fig. 20. Left: synthetic column density map generated by reinjecting the compact source extracted from the Cygnus-X North map on top of an fBm image (convolved with a beam with FWHM = 18.2′′) with β = 2.4. Right: ∆-variance spectrum of the synthetic models using this approach. The ∆-variance spectrum of the models is the average over 50 realiza- tions, and the full lines represent the 1σ dispersion around the mean. Acknowledgements. We thank the referee for a careful reading of the paper and for useful suggestions. We would also like to thank Shantanu Basu, Bruce Elmegreen, Jan Palouš, João Alves, Nanda Kumar, and Alessio Traﬁcante for their feedback and useful discussions on an early version of the paper. S.D., 8. Conclusions The synthetic ∆-variance spectra are compared to that of the Cygnus-X North region and to the case of an fBm image with β = 2.4. not only due to the existence of compact sources, but is a signa- ture of the whole population of structures that exist in the cloud, including more extended and more elongated structures such as ridges and hubs. Such structures are required in order to broaden the peak of the ∆-variance spectrum and also because they pro- vide the required intermediate column densities that reduce the contrast between the compact sources and the potentially under- lying fBm, bringing the amplitude of the ∆-variance in line with the observations. At present, it is relatively difﬁcult to ascertain which physical process leads to the formation of structures with scales in the range 0.5−1.2 pc. While gravity is the suspected culprit because its effect can precede from an evolutionary point of view over those of stellar feedback, an analysis of the pil- lars and globules in the Cygnus OB2 association has shown that these structures have typical sizes of ≈0.6 pc (Schneider et al. 2016a). This indicates that feedback may be responsible, at least partially, for generating the peak in the ∆-variance spectrum that is observed in the entire Cygnus-X North region. Further work should shed more light on the possible correlation between the shape of the ∆-variance spectrum and star formation activity, such as the surface density of star formation and the intensity of the radiation ﬁeld in different parts of the cloud. Independently, the application of the ∆-variance method to numerical simula- tions of self-gravitation clouds with and without feedback effects will also help explain the dominant physical processes that can generate a structure similar to the one observed in the Cygnus-X North molecular cloud. Fig. 20. Left: synthetic column density map generated by reinjecting the compact source extracted from the Cygnus-X North map on top of an fBm image (convolved with a beam with FWHM = 18.2′′) with β = 2.4. Right: ∆-variance spectrum of the synthetic models using this approach. The ∆-variance spectrum of the models is the average over 50 realiza- tions, and the full lines represent the 1σ dispersion around the mean. The synthetic ∆-variance spectra are compared to that of the Cygnus-X North region and to the case of an fBm image with β = 2.4. 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https://openalex.org/W2948331556	https://europepmc.org/articles/pmc3343832?pdf=render	English	null	Poly[di-μ<sub>3</sub>-chlorido-di-μ<sub>2</sub>-chlorido-{μ<sub>4</sub>-<i>N</i>,<i>N</i>,<i>N</i>′,<i>N</i>′-tetrakis[(diphenylphosphanyl)methyl]benzene-1,4-diamine-κ<sup>4</sup><i>P</i>:<i>P</i>′:<i>P</i>′′:<i>P</i>′′′}tetracopper(II)]	Acta crystallographica. Section E	2,012	cc-by	3,745	Related literature For the structures and properties of CuI complexes containing polyphosphine ligands, see: Li et al. (2009); Kohl et al. (2006); Wang et al. (2008); Hou et al.(2011); Ni et al. (2011). For the synthesis of Cu(I) complexes with diphosphine ligands, see: Saravanabharathi et al. (2002); Sivasankar et al. (2004). Supplementary data and ﬁgures for this paper are available from the IUCr electronic archives (Reference: ZQ2150). Table 1 In the title complex, [Cu4Cl4(C58H52N2P4)]n, four CuII atoms are held together via two doubly bridging and two triply bridging chlorides, forming a stair-like Cu4Cl4 core having crystallographically imposed inversion symmetry, while the benzene-1,4-diamine ligand (with a crystallographic inversion center at the centroid) acts in a tetradentate coordination mode, bridging two adjacent Cu4Cl4 cores, resulting in a chain along the a-axis direction. One Cu atom has a distorted tetrahedral geometry, coordinated by one P atom, one 2-Cl and two 3-Cl atoms, while the second Cu atom adopts a trigonal geometry, coordinated by one P atom, one 2-Cl and one 3-Cl atoms. Data collection: SMART (Bruker, 1998); cell reﬁnement: SAINT (Bruker, 1998); data reduction: SAINT; program(s) used to solve structure: SHELXS97 (Sheldrick, 2008); program(s) used to reﬁne structure: SHELXL97 (Sheldrick, 2008); molecular graphics: SHELXTL (Sheldrick, 2008); software used to prepare material for publication: SHELXTL. This work was supported by the Key Program of Xihua University (E0913305, E0913307). metal-organic compounds metal-organic compounds metal-organic compounds Experimental Crystal data [Cu4Cl4(C58H52N2P4)] Mr = 1296.86 Monoclinic, P21=c a = 10.298 (7) A˚ b = 17.649 (12) A˚ c = 18.009 (9) A˚ = 123.94 (3) V = 2715 (3) A˚ 3 Z = 2 Mo K radiation = 1.90 mm1 T = 296 K 0.20 0.15 0.13 mm Data collection Bruker SMART CCD area-detector diffractometer Absorption correction: multi-scan (SADABS; Bruker, 1998) Tmin = 0.852, Tmax = 1.000 15819 measured reﬂections 5333 independent reﬂections 3089 reﬂections with I > 2(I) Rint = 0.085 Reﬁnement R[F 2 > 2(F 2)] = 0.054 wR(F 2) = 0.143 S = 0.94 5333 reﬂections 325 parameters H-atom parameters constrained max = 0.70 e A˚ 3 min = 0.62 e A˚ 3 Acta Crystallographica Section E Structure Reports Online ISSN 1600-5368 Acta Crystallographica Section E Structure Reports Online ISSN 1600-5368 Supplementary data and ﬁgures for this paper are available from the IUCr electronic archives (Reference: ZQ2150). Poly[di-l3-chlorido-di-l2-chlorido-{l4- N,N,N000,N000-tetrakis[(diphenylphosphan- yl)methyl]benzene-1,4-diamine- j4P:P000:P000000:P000000000}tetracopper(II)] Received 12 January 2012; accepted 12 March 2012 Key indicators: single-crystal X-ray study; T = 296 K; mean (C–C) = 0.009 A˚; R factor = 0.054; wR factor = 0.143; data-to-parameter ratio = 16.4. Table 1 Selected bond lengths (A˚ ). Cu1—P1 2.1998 (19) Cu1—Cl2 2.3975 (19) Cu1—Cl1 2.4140 (17) Cu1—Cl1i 2.565 (2) Cu2—P2ii 2.188 (2) Cu2—Cl2 2.3062 (18) Cu2—Cl1 2.3255 (18) Symmetry codes: (i) x 1; y; z þ 1; (ii) x; y; z þ 1. Comment Recently, CuI complexes containing multiphosphine ligands have received much attention so far due to their special structures, novel reactivity, as well as catalytic and luminescent properties (Kohl et al., 2006; Wang et al., 2008; Hou et al., 2011; Ni et al., 2011). However, synthesis of CuI complexes with tetraphosphine ligands have been virtually not explored, though a great number of Cu(I) complexes with diphosphines were reported (Saravanabharathi et al., 2002; Sivasankar et al., 2004; Li et al., 2009). Herein, we report the synthesis and crystal structure of a new CuI complex with the tetrakis[(diphenylphosphanyl)methyl]benzene-1,4-diamine ligand (dpppda), the title complex [Cu4Cl4(dpppda)]n. The asymmetric unit of the title complex, [Cu2Cl2(C29H26NP2)]n, contains two copper ions, two chlorine atoms and one half of the N,N,N′,N′-tetrakis[(diphenylphosphanyl)methyl]benzene-1,4-diamine ligand (dpppda). Four copper atoms are held together via two doubly bridging and two triply-bridging chlorides to form a stair-like Cu4Cl4 core having a crystallographically imposed centrosymmetry, while the N,N,N′,N′-tetrakis[(diphenylphosphanyl)methyl]benzene-1,4-di- amine ligand (with a crystallographic inversion center at the midpoint of the central phenyl ring) acts as a tetradentate coordination mode to bridge two adjacent Cu4Cl4 cores resulting in a one-dimentional chain. The structure of the title complex is anologous to the reported complex [Cu4I4(dpppda)] (Li et al., 2009). Cu1 has a distorted tetrahedral geometry, coordinated by one P atom, one µ2-Cl and two µ3-Cl atoms, while Cu2 adopts a trigonal geometry, coordinated by one P atom, one µ2-Cl and one µ3-Cl atoms. The mean Cu—Cl and Cu—P bond distances are 2.40 (1) and 2.19 (2) Å, respectively. Recently, CuI complexes containing multiphosphine ligands have received much attention so far due to their special structures, novel reactivity, as well as catalytic and luminescent properties (Kohl et al., 2006; Wang et al., 2008; Hou et al., 2011; Ni et al., 2011). However, synthesis of CuI complexes with tetraphosphine ligands have been virtually not explored, though a great number of Cu(I) complexes with diphosphines were reported (Saravanabharathi et al., 2002; Sivasankar et al., 2004; Li et al., 2009). Herein, we report the synthesis and crystal structure of a new CuI complex with the tetrakis[(diphenylphosphanyl)methyl]benzene-1,4-diamine ligand (dpppda), the title complex [Cu4Cl4(dpppda)]n. The asymmetric unit of the title complex, [Cu2Cl2(C29H26NP2)]n, contains two copper ions, two chlorine atoms and one half of the N,N,N′,N′-tetrakis[(diphenylphosphanyl)methyl]benzene-1,4-diamine ligand (dpppda). Refinement All hydrogen atoms were generated geometrically with C—H distances of 0.93Å (aromatic H atoms) and 0.97Å (methyl- ene H atoms) and refined with a riding model with Uiso(H) = 1.2Ueq(C). Comment Four copper atoms are held together via two doubly bridging and two triply-bridging chlorides to form a stair-like Cu4Cl4 core having a crystallographically imposed centrosymmetry, while the N,N,N′,N′-tetrakis[(diphenylphosphanyl)methyl]benzene-1,4-di- amine ligand (with a crystallographic inversion center at the midpoint of the central phenyl ring) acts as a tetradentate coordination mode to bridge two adjacent Cu4Cl4 cores resulting in a one-dimentional chain. The structure of the title complex is anologous to the reported complex [Cu4I4(dpppda)] (Li et al., 2009). Cu1 has a distorted tetrahedral geometry, coordinated by one P atom, one µ2-Cl and two µ3-Cl atoms, while Cu2 adopts a trigonal geometry, coordinated by one P atom, one µ2-Cl and one µ3-Cl atoms. The mean Cu—Cl and Cu—P bond distances are 2.40 (1) and 2.19 (2) Å, respectively. References Bruker (1998). SMART, SAINT and SADABS. Bruker AXS Inc., Madison, Wisconsin, USA. Bruker (1998). SMART, SAINT and SADABS. Bruker AXS Inc., Madison, Wisconsin, USA. Hou, R., Huang, T.-H., Wang, X.-J., Jiang, X.-F., Ni, Q.-L., Gui, L.-C., Fan, Y.-J. & Tan, Y.-L. (2011). Dalton Trans. 40, 7551–7558. Hou, R., Huang, T.-H., Wang, X.-J., Jiang, X.-F., Ni, Q.-L., Gui, L.-C., Fan, Y.-J. & Tan, Y.-L. (2011). Dalton Trans. 40, 7551–7558. ( ) Kohl, S. W., Heinemann, F. W., Hummert, M., Bauer, W. & Grohmann, A. (2006). Dalton Trans. pp. 5583–5592. Li, N.-Y., Ren, Z.-G., Liu, D., Wang, J., Dai, M., Li, H.-X. & Lang, J.-P. (2009). Inorg. Chem. Commun. 12, 1031–1034. g Ni, Q.-L., Jiang, X.-F., Gui, L.-C., Wang, X.-J., Yang, K.-G. & Bi, X.-S. (2011). New J. Chem. 35, 2471–2476. Saravanabharathi, D., Monika,Venugopalan, P. & Samuelson, A. G. (2002). Polyhedron, 21, 2433–2443. Sheldrick, G. M. (2008). Acta Cryst. A64, 112–122. Sivasankar, C., Nethaji, M. & Samuelson, A. G. (2004). Inorg. Chem. Commun. 7, 238–240. Wang, X.-J., Gui, L.-C., Ni, Q.-L., Liao, Y.-F., Jiang, X.-F., Tang, L.-H., Zhang, Z. & Wu, Q. (2008). CrystEngComm, 10, 1003–1010. m430 Liu et al. Acta Cryst. (2012). E68, m430 doi:10.1107/S1600536812010860 supplementary materials Experimental CuCl (0.0198 g, 0.2 mmol) was added with stirring to a solution of N,N,N′,N′-tetrakis[(diphenylphosphanyl)methyl]- benzene-1,4-diamine (0.0900 g, 0.10 mmol) in DMF (5 ml), and the resulting solution was allowed to stir for 1 h at room temperature. Slow diffusion of diethyl ether into the solution give colourless block crystals suitable for X-ray analysis after three days. Poly[di-µ3-chlorido-di-µ2-chlorido-{µ4-N,N,N′,N′-tetrakis[(diphenyl- phosphanyl)methyl]benzene-1,4-diamine-κ4P:P′:P′′:P′′′}tetracopper(II)] Jia-Qin Liu, Yan Zhang, Ya-Jing Lü and Zhen-Jü Jiang Poly[di-µ3-chlorido-di-µ2-chlorido-{µ4-N,N,N′,N′-tetrakis[(diphenyl- phosphanyl)methyl]benzene-1,4-diamine-κ4P:P′:P′′:P′′′}tetracopper(II)] Jia-Qin Liu, Yan Zhang, Ya-Jing Lü and Zhen-Jü Jiang Poly[di-µ3-chlorido-di-µ2-chlorido-{µ4-N,N,N′,N′-tetrakis[(diphenyl- phosphanyl)methyl]benzene-1,4-diamine-κ4P:P′:P′′:P′′′}tetracopper(II)] supplementary materials Acta Cryst. (2012). E68, m430 [doi:10.1107/S1600536812010860] Acta Cryst. (2012). E68, m430 [doi:10.1107/S1600536812010860] Computing details Data collection: SMART (Bruker, 1998); cell refinement: SAINT (Bruker, 1998); data reduction: SAINT (Bruker, 1998); program(s) used to solve structure: SHELXS97 (Sheldrick, 2008); program(s) used to refine structure: SHELXL97 (Sheldrick, 2008); molecular graphics: SHELXTL (Sheldrick, 2008); software used to prepare material for publication: Acta Cryst. (2012). E68, m430 sup-1 supplementary materials SHELXTL (Sheldrick, 2008). Figure 1 The structure of the title compound with displacement ellipsoids drawn at the 20% probability level. Hydrogen atoms are omitted for clarity. [symmetry code: (A) 1-x, -y, 1-z; (B) -x, -y, 1-z; (C) -1+x, y, z.] SHELXTL (Sheldrick, 2008). Refinement Refinement Refinement on F2 Least-squares matrix: full R[F2 > 2σ(F2)] = 0.054 wR(F2) = 0.143 S = 0.94 5333 reflections 325 parameters 0 restraints Primary atom site location: structure-invariant direct methods Secondary atom site location: difference Fourier map Hydrogen site location: inferred from neighbouring sites H-atom parameters constrained w = 1/[σ2(Fo2) + (0.P)2] where P = (Fo2 + 2Fc2)/3 (Δ/σ)max = 0.001 Δρmax = 0.70 e Å−3 Δρmin = −0.62 e Å−3 Secondary atom site location: difference Fourier map Hydrogen site location: inferred from neighbouring sites H-atom parameters constrained w = 1/[σ2(Fo2) + (0.P)2] where P = (Fo2 + 2Fc2)/3 (Δ/σ)max = 0.001 Δρmax = 0.70 e Å−3 Δρmin = −0.62 e Å−3 Hydrogen site location: inferred from Data collection 15819 measured reflections 5333 independent reflections 3089 reflections with I > 2σ(I) Rint = 0.085 θmax = 26.0°, θmin = 1.8° h = −12→9 k = −19→21 l = −21→22 Figure 1 g The structure of the title compound with displacement ellipsoids drawn at the 20% probability level. Hydrogen atoms are omitted for clarity. [symmetry code: (A) 1-x, -y, 1-z; (B) -x, -y, 1-z; (C) -1+x, y, z.] g The structure of the title compound with displacement ellipsoids drawn at the 20% probability level. Hydrogen atoms are omitted for clarity. [symmetry code: (A) 1-x, -y, 1-z; (B) -x, -y, 1-z; (C) -1+x, y, z.] The structure of the title compound with displacement ellipsoids drawn at the 20% probability level. Hydrogen atoms are omitted for clarity. [symmetry code: (A) 1-x, -y, 1-z; (B) -x, -y, 1-z; (C) -1+x, y, z.] Poly[di-µ3-chlorido-di-µ2-chlorido-{µ4- N,N,N′,N′- tetrakis[(diphenylphosphanyl)methyl]benzene-1,4-diamine- κ4P:P′:P′′:P′′′}tetracopper(II)] Poly[di-µ3-chlorido-di-µ2-chlorido-{µ4- N,N,N′,N′- tetrakis[(diphenylphosphanyl)methyl]benzene-1,4-diamine- κ4P:P′:P′′:P′′′}tetracopper(II)] Crystal data [Cu4Cl4(C58H52N2P4)] Mr = 1296.86 Monoclinic, P21/c Hall symbol: -P 2ybc a = 10.298 (7) Å b = 17.649 (12) Å c = 18.009 (9) Å β = 123.94 (3)° V = 2715 (3) Å3 Z = 2 sup-2 Acta Cryst. (2012). E68, m430 supplementary materials F(000) = 1316 Dx = 1.586 Mg m−3 Mo Kα radiation, λ = 0.71073 Å Cell parameters from 5682 reflections θ = 2.8–26.3° µ = 1.90 mm−1 T = 296 K Block, colourless 0.20 × 0.15 × 0.13 mm Data collection Bruker SMART CCD area-detector diffractometer Radiation source: fine-focus sealed tube Graphite monochromator ω scans Absorption correction: multi-scan (SADABS; Bruker, 1998) Tmin = 0.852, Tmax = 1.000 15819 measured reflections 5333 independent reflections 3089 reflections with I > 2σ(I) Rint = 0.085 θmax = 26.0°, θmin = 1.8° h = −12→9 k = −19→21 l = −21→22 µ = 1.90 mm−1 T = 296 K Block, colourless 0.20 × 0.15 × 0.13 mm Special details Geometry. All e.s.d.'s (except the e.s.d. in the dihedral angle between two l.s. planes) are estimated using the full covariance matrix. The cell e.s.d.'s are taken into account individually in the estimation of e.s.d.'s in distances, angles and torsion angles; correlations between e.s.d.'s in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell e.s.d.'s is used for estimating e.s.d.'s involving l.s. planes. pp ( p ) g g p Refinement. Refinement of F2 against ALL reflections. The weighted R-factor wR and goodness of fit S are based on F2, conventional R-factors R are based on F, with F set to zero for negative F2. The threshold expression of F2 > σ(F2) is used only for calculating R-factors(gt) etc. and is not relevant to the choice of reflections for refinement. R-factors based on F2 are statistically about twice as large as those based on F, and R- factors based on ALL data will be even larger. Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) x y z Uiso/Ueq Cu1 −0.45394 (8) 0.04466 (4) 0.43168 (4) 0.0540 (2) Cu2 −0.15464 (7) 0.07255 (4) 0.62081 (4) 0.0487 (2) Cl1 −0.30501 (14) −0.03659 (7) 0.56069 (8) 0.0417 (3) Cl2 −0.32846 (15) 0.15805 (7) 0.51454 (9) 0.0526 (4) P1 −0.52339 (15) 0.01608 (8) 0.29527 (8) 0.0402 (3) P2 −0.07631 (15) −0.08176 (7) 0.25371 (8) 0.0367 (3) N1 −0.2438 (4) −0.0268 (2) 0.3202 (2) 0.0397 (10) C1 −0.1055 (6) 0.0562 (3) 0.4500 (3) 0.0370 (12) H1A −0.1767 0.0948 0.4172 0.044 C2 −0.1233 (5) −0.0133 (3) 0.4094 (3) 0.0336 (11) C3 −0.0155 (6) −0.0698 (3) 0.4619 (3) 0.0364 (11) H3A −0.0251 −0.1175 0.4372 0.044* Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) x y z Uiso/Ueq Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) sup-3 Acta Cryst. (2012). E68, m430 supplementary materials supplementary materials su Acta Cryst. (2012). Acta Cryst. (2012). E68, m430 supplementary materials C4 −0.3712 (5) 0.0258 (3) 0.2699 (3) 0.0393 (12) H4A −0.4193 0.0173 0.2064 0.047 H4B −0.3304 0.0771 0.2835 0.047* C5 −0.5742 (6) −0.0830 (3) 0.2617 (3) 0.0430 (13) C6 −0.6482 (7) −0.1100 (4) 0.1736 (4) 0.0658 (18) H6A −0.6962 −0.0752 0.1269 0.079* C7 −0.6531 (8) −0.1842 (4) 0.1535 (5) 0.077 (2) H7A −0.7037 −0.1999 0.0943 0.092* C8 −0.5818 (8) −0.2361 (4) 0.2222 (5) 0.087 (2) H8A −0.5802 −0.2870 0.2094 0.104* C9 −0.5123 (8) −0.2134 (4) 0.3103 (4) 0.088 (2) H9A −0.4684 −0.2490 0.3562 0.105* C10 −0.5089 (7) −0.1380 (3) 0.3292 (4) 0.0647 (17) H10A −0.4619 −0.1230 0.3884 0.078* C11 −0.6787 (6) 0.0772 (3) 0.2085 (3) 0.0481 (14) C12 −0.7157 (8) 0.0836 (4) 0.1220 (4) 0.078 (2) H12A −0.6607 0.0545 0.1053 0.094* C13 −0.8307 (9) 0.1312 (4) 0.0603 (4) 0.099 (3) H13A −0.8583 0.1319 0.0015 0.118* C14 −0.9051 (8) 0.1783 (4) 0.0868 (5) 0.086 (2) H14A −0.9836 0.2108 0.0454 0.103* C15 −0.8645 (7) 0.1776 (4) 0.1730 (5) 0.0743 (19) H15A −0.9118 0.2110 0.1910 0.089* C16 −0.7513 (7) 0.1263 (3) 0.2341 (4) 0.0647 (17) H16A −0.7245 0.1253 0.2927 0.078* C17 −0.2353 (5) −0.0931 (3) 0.2727 (3) 0.0402 (12) H17A −0.3346 −0.0990 0.2156 0.048* H17B −0.2165 −0.1385 0.3077 0.048* C18 −0.1096 (5) −0.1626 (2) 0.1820 (3) 0.0352 (11) C19 −0.2520 (6) −0.1996 (3) 0.1284 (3) 0.0516 (14) H19A −0.3385 −0.1823 0.1271 0.062* C20 −0.2680 (7) −0.2612 (3) 0.0773 (4) 0.0551 (15) H20A −0.3647 −0.2849 0.0418 0.066* C21 −0.1420 (7) −0.2879 (3) 0.0785 (4) 0.0525 (14) H21A −0.1526 −0.3296 0.0441 0.063* C22 −0.0008 (7) −0.2525 (3) 0.1307 (4) 0.0548 (15) H22A 0.0849 −0.2699 0.1312 0.066* C23 0.0160 (6) −0.1911 (3) 0.1827 (3) 0.0453 (13) H23A 0.1138 −0.1685 0.2190 0.054* C24 −0.1553 (6) 0.0012 (3) 0.1797 (3) 0.0430 (13) C25 −0.1172 (7) 0.0719 (3) 0.2188 (4) 0.0545 (15) H25A −0.0423 0.0769 0.2798 0.065* C26 −0.1918 (8) 0.1364 (3) 0.1661 (5) 0.0701 (19) H26A −0.1672 0.1840 0.1928 0.084* C27 −0.2981 (8) 0.1301 (4) 0.0776 (4) 0.0710 (19) H27A −0.3477 0.1732 0.0435 0.085* C28 −0.3334 (8) 0.0610 (4) 0.0378 (4) 0.0714 (19) H28A −0.4057 0.0570 −0.0237 0.086* C29 −0.2629 (7) −0.0035 (3) 0.0880 (4) 0.0607 (17) sup-4 Acta Cryst. (2012). E68, m430 supplementary materials supplementary materials s Acta Cryst (2012) E68 m430 Cu2—Cl1 2.3255 (18) C13—H13A 0.9300 Cl1—Cu1i 2.565 (2) C14—C15 1.364 (9) P1—C5 1.828 (5) C14—H14A 0.9300 P1—C11 1.837 (5) C15—C16 1.399 (7) P1—C4 1.869 (5) C15—H15A 0.9300 P2—C18 1.824 (5) C16—H16A 0.9300 P2—C24 1.836 (5) C17—H17A 0.9700 P2—C17 1.861 (5) C17—H17B 0.9700 P2—Cu2ii 2.188 (2) C18—C23 1.381 (7) N1—C2 1.397 (5) C18—C19 1.389 (6) N1—C4 1.442 (6) C19—C20 1.374 (7) N1—C17 1.479 (6) C19—H19A 0.9300 C1—C3ii 1.386 (6) C20—C21 1.368 (8) C1—C2 1.387 (6) C20—H20A 0.9300 C1—H1A 0.9300 C21—C22 1.364 (7) C2—C3 1.396 (6) C21—H21A 0.9300 C3—C1ii 1.386 (6) C22—C23 1.378 (7) C3—H3A 0.9300 C22—H22A 0.9300 C4—H4A 0.9700 C23—H23A 0.9300 C4—H4B 0.9700 C24—C25 1.378 (7) C5—C10 1.400 (7) C24—C29 1.386 (7) C5—C6 1.405 (7) C25—C26 1.404 (7) C6—C7 1.352 (8) C25—H25A 0.9300 C6—H6A 0.9300 C26—C27 1.342 (8) C7—C8 1.377 (9) C26—H26A 0.9300 C7—H7A 0.9300 C27—C28 1.358 (8) C8—C9 1.387 (9) C27—H27A 0.9300 C8—H8A 0.9300 C28—C29 1.380 (7) C9—C10 1.368 (8) C28—H28A 0.9300 C9—H9A 0.9300 C29—H29A 0.9300 P1—Cu1—Cl2 127.78 (6) C16—C11—C12 117.7 (5) P1—Cu1—Cl1 125.05 (7) C16—C11—P1 117.4 (4) Cl2—Cu1—Cl1 93.71 (7) C12—C11—P1 124.5 (4) P1—Cu1—Cl1i 109.03 (6) C13—C12—C11 122.2 (6) Cl2—Cu1—Cl1i 102.38 (6) C13—C12—H12A 118.9 Cl1—Cu1—Cl1i 91.68 (6) C11—C12—H12A 118.9 P2ii—Cu2—Cl2 134.50 (6) C12—C13—C14 118.9 (7) P2ii—Cu2—Cl1 126.84 (6) C12—C13—H13A 120.5 Cl2—Cu2—Cl1 98.57 (7) C14—C13—H13A 120.5 Cu2—Cl1—Cu1 81.73 (6) C15—C14—C13 120.7 (6) Cu2—Cl1—Cu1i 115.71 (6) C15—C14—H14A 119.6 Cu1—Cl1—Cu1i 88.32 (6) C13—C14—H14A 119.6 Cu2—Cl2—Cu1 82.49 (7) C14—C15—C16 119.5 (6) C5—P1—C11 109.2 (2) C14—C15—H15A 120.2 C5—P1—C4 97.5 (2) C16—C15—H15A 120.2 C11—P1—C4 100.9 (2) C11—C16—C15 120.8 (6) C5—P1—Cu1 116.30 (18) C11—C16—H16A 119.6 C11—P1—Cu1 113.65 (18) C15—C16—H16A 119.6 2.3255 (18) C13—H13A 0.9300 2.565 (2) C14—C15 1.364 (9) 1.828 (5) C14—H14A 0.9300 1.837 (5) C15—C16 1.399 (7) 1.869 (5) C15—H15A 0.9300 1.824 (5) C16—H16A 0.9300 1.836 (5) C17—H17A 0.9700 1.861 (5) C17—H17B 0.9700 2.188 (2) C18—C23 1.381 (7) 1.397 (5) C18—C19 1.389 (6) 1.442 (6) C19—C20 1.374 (7) 1.479 (6) C19—H19A 0.9300 1.386 (6) C20—C21 1.368 (8) 1.387 (6) C20—H20A 0.9300 0.9300 C21—C22 1.364 (7) 1.396 (6) C21—H21A 0.9300 1.386 (6) C22—C23 1.378 (7) 0.9300 C22—H22A 0.9300 0.9700 C23—H23A 0.9300 0.9700 C24—C25 1.378 (7) 1.400 (7) C24—C29 1.386 (7) 1.405 (7) C25—C26 1.404 (7) 1.352 (8) C25—H25A 0.9300 0.9300 C26—C27 1.342 (8) 1.377 (9) C26—H26A 0.9300 0.9300 C27—C28 1.358 (8) 1.387 (9) C27—H27A 0.9300 0.9300 C28—C29 1.380 (7) 1.368 (8) C28—H28A 0.9300 0.9300 C29—H29A 0.9300 127.78 (6) C16—C11—C12 117.7 (5) 125.05 (7) C16—C11—P1 117.4 (4) 93.71 (7) C12—C11—P1 124.5 (4) 109.03 (6) C13—C12—C11 122.2 (6) 102.38 (6) C13—C12—H12A 118.9 91.68 (6) C11—C12—H12A 118.9 134.50 (6) C12—C13—C14 118.9 (7) 126.84 (6) C12—C13—H13A 120.5 98.57 (7) C14—C13—H13A 120.5 81.73 (6) C15—C14—C13 120.7 (6) 115.71 (6) C15—C14—H14A 119.6 88.32 (6) C13—C14—H14A 119.6 82.49 (7) C14—C15—C16 119.5 (6) 109.2 (2) C14—C15—H15A 120.2 97.5 (2) C16—C15—H15A 120.2 100.9 (2) C11—C16—C15 120.8 (6) 116.30 (18) C11—C16—H16A 119.6 113.65 (18) C15—C16—H16A 119.6 sup-6 Acta Cryst. supplementary materials E68, m430 H29A −0.2878 −0.0505 0.0599 0.073* Atomic displacement parameters (Å2) U11 U22 U33 U12 U13 U23 Cu1 0.0651 (5) 0.0619 (5) 0.0341 (4) 0.0056 (3) 0.0270 (4) 0.0001 (3) Cu2 0.0389 (4) 0.0554 (4) 0.0406 (4) −0.0038 (3) 0.0154 (3) −0.0054 (3) Cl1 0.0392 (7) 0.0407 (7) 0.0396 (7) −0.0005 (5) 0.0187 (6) 0.0031 (5) Cl2 0.0503 (8) 0.0430 (8) 0.0577 (9) 0.0049 (6) 0.0259 (7) 0.0008 (6) P1 0.0376 (8) 0.0506 (9) 0.0318 (7) 0.0042 (6) 0.0190 (6) 0.0011 (6) P2 0.0355 (7) 0.0419 (8) 0.0322 (7) −0.0021 (6) 0.0185 (6) −0.0041 (6) N1 0.038 (2) 0.043 (2) 0.035 (2) 0.0018 (19) 0.018 (2) −0.0083 (18) C1 0.040 (3) 0.038 (3) 0.032 (3) 0.001 (2) 0.020 (2) 0.004 (2) C2 0.035 (3) 0.039 (3) 0.029 (2) −0.002 (2) 0.019 (2) −0.001 (2) C3 0.046 (3) 0.031 (3) 0.040 (3) −0.006 (2) 0.030 (3) −0.006 (2) C4 0.041 (3) 0.048 (3) 0.026 (2) −0.002 (2) 0.017 (2) 0.001 (2) C5 0.035 (3) 0.055 (3) 0.047 (3) −0.004 (2) 0.027 (3) −0.008 (3) C6 0.066 (4) 0.079 (5) 0.044 (3) −0.022 (3) 0.026 (3) −0.010 (3) C7 0.083 (5) 0.070 (5) 0.087 (5) −0.034 (4) 0.053 (4) −0.037 (4) C8 0.068 (5) 0.058 (4) 0.119 (7) −0.011 (4) 0.044 (5) −0.022 (5) C9 0.088 (5) 0.063 (5) 0.066 (5) 0.000 (4) 0.014 (4) 0.005 (4) C10 0.064 (4) 0.058 (4) 0.054 (4) −0.006 (3) 0.021 (3) 0.000 (3) C11 0.044 (3) 0.064 (4) 0.034 (3) 0.010 (3) 0.021 (3) 0.003 (2) C12 0.093 (5) 0.090 (5) 0.047 (4) 0.045 (4) 0.037 (4) 0.017 (3) C13 0.123 (7) 0.107 (6) 0.053 (4) 0.055 (5) 0.041 (5) 0.022 (4) C14 0.065 (5) 0.106 (6) 0.064 (5) 0.027 (4) 0.021 (4) 0.022 (4) C15 0.075 (5) 0.072 (5) 0.083 (5) 0.027 (4) 0.048 (4) 0.016 (4) C16 0.064 (4) 0.070 (4) 0.057 (4) 0.018 (3) 0.032 (3) 0.015 (3) C17 0.040 (3) 0.047 (3) 0.036 (3) −0.001 (2) 0.023 (2) −0.002 (2) C18 0.038 (3) 0.034 (3) 0.034 (3) 0.000 (2) 0.020 (2) −0.001 (2) C19 0.043 (3) 0.058 (4) 0.053 (3) −0.005 (3) 0.026 (3) −0.017 (3) C20 0.052 (4) 0.061 (4) 0.056 (4) −0.018 (3) 0.033 (3) −0.022 (3) C21 0.068 (4) 0.043 (3) 0.056 (4) −0.006 (3) 0.040 (3) −0.011 (3) C22 0.052 (4) 0.053 (4) 0.065 (4) 0.005 (3) 0.036 (3) −0.007 (3) C23 0.040 (3) 0.048 (3) 0.050 (3) 0.000 (2) 0.027 (3) −0.007 (2) C24 0.043 (3) 0.046 (3) 0.041 (3) −0.004 (2) 0.025 (3) −0.004 (2) C25 0.062 (4) 0.048 (3) 0.053 (3) −0.011 (3) 0.032 (3) −0.009 (3) C26 0.090 (5) 0.035 (3) 0.085 (5) −0.005 (3) 0.048 (4) −0.002 (3) C27 0.077 (5) 0.056 (4) 0.068 (4) 0.006 (3) 0.033 (4) 0.021 (3) C28 0.077 (5) 0.067 (5) 0.046 (4) 0.000 (3) 0.020 (3) 0.008 (3) C29 0.074 (4) 0.048 (4) 0.046 (3) −0.003 (3) 0.025 (3) −0.004 (3) Geometric parameters (Å, º) Cu1—P1 2.1998 (19) C10—H10A 0.9300 Cu1—Cl2 2.3975 (19) C11—C16 1.381 (7) Cu1—Cl1 2.4140 (17) C11—C12 1.386 (8) Cu1—Cl1i 2.565 (2) C12—C13 1.369 (8) Cu2—P2ii 2.188 (2) C12—H12A 0.9300 Cu2—Cl2 2.3062 (18) C13—C14 1.382 (9) −0.2878 H29A sup-5 supplementary materials Symmetry codes: (i) −x−1, −y, −z+1; (ii) −x, −y, −z+1. supplementary materials (2012). E68, m430 supplementary materials C4—P1—Cu1 117.26 (16) N1—C17—P2 111.4 (3) C18—P2—C24 106.1 (2) N1—C17—H17A 109.4 C18—P2—C17 102.1 (2) P2—C17—H17A 109.4 C24—P2—C17 98.1 (2) N1—C17—H17B 109.4 C18—P2—Cu2ii 117.10 (16) P2—C17—H17B 109.4 C24—P2—Cu2ii 118.43 (17) H17A—C17—H17B 108.0 C17—P2—Cu2ii 112.24 (17) C23—C18—C19 117.1 (4) C2—N1—C4 121.7 (4) C23—C18—P2 118.3 (4) C2—N1—C17 119.9 (4) C19—C18—P2 124.6 (4) C4—N1—C17 118.2 (4) C20—C19—C18 121.5 (5) C3ii—C1—C2 121.7 (4) C20—C19—H19A 119.2 C3ii—C1—H1A 119.1 C18—C19—H19A 119.2 C2—C1—H1A 119.1 C21—C20—C19 120.2 (5) C1—C2—C3 117.0 (4) C21—C20—H20A 119.9 C1—C2—N1 121.9 (4) C19—C20—H20A 119.9 C3—C2—N1 121.1 (4) C22—C21—C20 119.3 (5) C1ii—C3—C2 121.2 (4) C22—C21—H21A 120.3 C1ii—C3—H3A 119.4 C20—C21—H21A 120.3 C2—C3—H3A 119.4 C21—C22—C23 120.6 (5) N1—C4—P1 112.3 (3) C21—C22—H22A 119.7 N1—C4—H4A 109.1 C23—C22—H22A 119.7 P1—C4—H4A 109.1 C22—C23—C18 121.2 (5) N1—C4—H4B 109.1 C22—C23—H23A 119.4 P1—C4—H4B 109.1 C18—C23—H23A 119.4 H4A—C4—H4B 107.9 C25—C24—C29 118.2 (5) C10—C5—C6 116.0 (5) C25—C24—P2 117.9 (4) C10—C5—P1 117.8 (4) C29—C24—P2 123.6 (4) C6—C5—P1 125.0 (4) C24—C25—C26 119.8 (5) C7—C6—C5 123.2 (6) C24—C25—H25A 120.1 C7—C6—H6A 118.4 C26—C25—H25A 120.1 C5—C6—H6A 118.4 C27—C26—C25 120.8 (5) C6—C7—C8 118.8 (6) C27—C26—H26A 119.6 C6—C7—H7A 120.6 C25—C26—H26A 119.6 C8—C7—H7A 120.6 C26—C27—C28 120.1 (6) C7—C8—C9 120.8 (6) C26—C27—H27A 120.0 C7—C8—H8A 119.6 C28—C27—H27A 120.0 C9—C8—H8A 119.6 C27—C28—C29 120.5 (6) C10—C9—C8 119.4 (6) C27—C28—H28A 119.8 C10—C9—H9A 120.3 C29—C28—H28A 119.8 C8—C9—H9A 120.3 C28—C29—C24 120.6 (5) C9—C10—C5 121.7 (6) C28—C29—H29A 119.7 C9—C10—H10A 119.1 C24—C29—H29A 119.7 C5—C10—H10A 119.1 Symmetry codes: (i) −x−1, −y, −z+1; (ii) −x, −y, −z+1. sup-7 Acta Cryst. (2012). E68, m430
https://openalex.org/W2049170752	http://www.scielo.cl/pdf/eatacam/n28/art11.pdf	Spanish; Castilian	null	Tierras comestibles de la Cuenca del Titicaca: Geofagia en la prehistoria boliviana	Estudios atacameños	2,004	cc-by	6,157	TIERRAS COMESTIBLES Estudios Atacameños N° 28, pp. 133-141 (2004) TIERRAS COMESTIBLES Estudios Atacameños N° 28, pp. 133-141 (2004) 1 Depto. de Antropología C. B. 1114, Washington University, Saint Louis, Missouri 63130-4899, ESTADOS UNIDOS. Email: dlbrowma@artsci.wustl.edu ABSTRACT This paper summarizes the evidence for 24 different earths utilized as food supplements or medicines by peoples of the Central Andes, based on current market samples and archaeological specimens. The earths include a phyllosillicate cluster, a sodium and calcium earth clus- ter, a sulfur mineral cluster, and an iron and copper salt cluster. Archaeological evidence for usage of the main minerals dates back at least five millennia. Mi hipótesis, presentada anteriormente con ma- yor detalle (Browman y Gunderson 1993: 422- 423), plantea que la práctica de geofagia en los Andes Centrales se desarrolló o quizás evolucio- nó en conjunto con la práctica del pastoreo de camélidos. Los minerales que los camélidos sil- vestres lamían (así como lo hicieron posteriormen- te sus congéneres domesticados) son ricos en filosilicatos hidratados (esmécticas, caolinitas, cloritos e illitas), que son precisamente aquellas tierras comestibles de mayor importancia: p’asa o ch’aqo. Estas tierras alivian los problemas gastrointestinales relacionados con las fitotoxinas (glicoalcaloides, taninos, entre otros) encontradas en todas las plantas domesticadas más importan- tes de la sierra (como la solanina en las papas, saponina en la quinoa, lunatina en frijoles, etc.). Igualmente proveen de complementos minerales esenciales. Asimismo, es muy probable que du- rante este período inicial se haya llevado a cabo el descubrimiento, desarrollo e integración de las diferentes propiedades medicinales de otras tie- rras comestibles. Aún hoy en día, en algunos lu- Key words: geophagy – comestible earths – Titicaca – p’asa – ch’aqo. Recibido: abril 2004. Manuscrito revisado aceptado: noviembre 2004. Recibido: abril 2004. Manuscrito revisado aceptado: noviembre 2004. RESUMEN las excavaciones arqueológicas ya que son difíci- les de diferenciar del resto de los sedimentos excavados. El objetivo de este artículo es hacer un breve resumen de la historia del uso de estas tierras, ofrecer una revisión sucinta de los con- textos arqueológicos conocidos, y entregar una sinopsis del trabajo que he venido realizando re- cientemente con diferentes tipos de tierras obte- nidas de vendedores especializados en los merca- dos rurales. El presente artículo resume la evidencia que existe sobre el uso que se le da a 24 tipos de tierras por los poblado- res de los Andes Centrales, ya sea como complemento alimenticio o como medicina. Las muestras analizadas provienen de contextos arqueológicos y de muestras ad- quiridas en los mercados locales. Las tierras incluyen un grupo de filosilicatos, otro grupo se compone de tierras sódicas y de calcio, otro de minerales de azufre, y el últi- mo corresponde a un grupo compuesto por hierro y sales de cobre. La evidencia arqueológica del uso de los prin- cipales minerales se remonta a por lo menos cinco milenios. Creo que los primeros habitantes andinos no prac- ticaron la geofagia. Al llegar, encontraron un me- dio ambiente totalmente nuevo y, por lo tanto, las plantas adecuadas a las cuales se recurre ahora y que tienen relación con el uso de arcillas co- mestibles, habrían sido completamente descono- cidas por ellos. Asimismo, habría tomado un tiem- po considerable determinar la ubicación de fuen- tes geológicas que contengan los minerales ade- cuados. Palabras claves: geofagia – tierras comestibles – Titicaca – p’asa – ch’aqo. Tierras comestibles de la Cuenca del Titicaca: Geofagia en la prehistoria boliviana DAVID L. BROWMAN1 Palabras claves: geofagia – tierras comestibles – Titicaca – p’asa – ch’aqo. Muestras arqueológicas La evidencia más temprana de geofagia que co- nozco proviene del estudio de coprolitos humanos del Período Arcaico. Bonavía y colaboradores (1993: 437) informaron sobre la presencia de una insospechada alta frecuencia de tierra y arena en coprolitos hallados durante sus trabajos en Los Gavilanes, sitio ocupado hace unos 4500 a 3500 años. Estos autores atribuyeron este hallazgo a prác- ticas de geofagia en donde se habrían consumido ciertos tipos de tierra como las vermífugas con el fin de ayudar a erradicar las lombrices intestinales y los parásitos, así como para tratar infecciones estomacales. Además, señalaron que en un infor- me inédito escrito por Glendon Weir y Vaughn Bryant también se detalla evidencia similar encon- trada en restos de 5000 o más años de antigüedad documentados por el proyecto de MacNeish en Ayacucho. Por otro lado, Jones (1988: 86) ha he- cho un estudio intensivo de los desechos en los sitios de La Paloma, Chilca, Asia y El Paraíso en la costa central del Perú, para un período que abar- ca aproximadamente entre 6000 a 3000 años. Sin embargo, Jones no ha encontrado ninguna eviden- cia de geofagia en estos sitios. Figura 1. Mapa de la Cuenca del Titicaca y áreas adyacentes, que muestra los principales sitios mencionados en el texto. 491) ha informado sobre el hallazgo de restos de “lejía” o llipt’a. Estos fueron encontrados dentro de cántaros recuperados en Pucara fechados sólo unos siglos después que nuestras muestras de Chiripa. Asimismo, y casi contemporáneo con los hallazgos en Pucara, Kroeber y Collier (1998: 259) informaron sobre el hallazgo de taku y yeso (o makaya) en sus excavaciones de tumbas de la cultura Nazca. Si enfocamos nuestra atención a la cuenca del Titicaca y áreas adyacentes, podemos informar sobre evidencia disponible para, por lo menos, los últimos tres milenios (Figura 1). En Chiripa recu- peramos un espécimen que un trabajador de cam- po local identificó preliminarmente como q’atawi o como “lejía”. Estas muestras están asociadas a dos grupos de fechados de C14 que señalan fe- chas de 510 años AC y 470 años AC. Análisis posteriores de difracción de Rayos X (DRX) in- dicaron que, en efecto, la sustancia se trataba de q’atawi. También hay otros dos tipos de evidencia de prác- tica continua de geofagia durante la subsiguiente cultura Tiwanaku. Introducción Las tierras comestibles son, por lo general, utili- zadas para proveer de complementos minerales, contrarrestar componentes perjudiciales en la dieta (como las fitotoxinas) y/o para ciertos tratamien- tos médicos. A pesar de que existen casos docu- mentados en algunos sitios prehistóricos en los Andes, estas tierras rara vez son recuperadas en 133 DAVID L. BROWMAN Figura 1. Mapa de la Cuenca del Titicaca y áreas adyacentes, que muestra los principales sitios mencionados en el texto. gares remotos donde regularmente transitan cara- vanas de camélidos a lo largo de rutas de inter- cambio (como en Susques en el noroeste de Ar- gentina o en Lipez en Bolivia), encontramos refe- rencias sobre la obtención de estas tierras a través de la explotación de fuentes minerales o por in- tercambio a cargo de los arrieros de llamas. Estas tierras tienen tanta importancia que los arrieros las cambian por otros alimentos que tengan un peso igual al de la tierra a intercambiar. Muestras del mercado p’asa. Este hallazgo fue hecho en el sitio Az-6, con una antigüedad quizá de hasta 400 años DC, y asociado con materiales de las fases Azapa, Cabuza y San Miguel. Igualmente, Rivera (1994: 226) tiene una muestra obtenida en el mismo si- tio de Tiwanaku, la cual está asociada a contextos Tiwanaku IV y fue sometida también a un análi- sis de DRX, el cual indicó que los componentes eran los de la p’asa. Asimismo, en Iwawi, un si- tio cercano a la capital Tiwanaku y ubicado a ori- llas del lago (posiblemente uno de los principales puertos de esa capital), O’Brien (1997: 166) ha identificado un patrón de desgaste dental en en- tierros humanos, el cual considera que está clara- mente asociado con prácticas de geofagia. Con el fin de saber qué tipo de tierras debemos buscar en el registro arqueológico, he revisado la literatura etnográfica tratando de identificar las tierras utilizadas por los habitantes del altiplano del siglo XX. Durante este proceso he descubier- to que existió una considerable diversidad de opi- niones entre los etnógrafos acerca de cuál era la composición química de las tierras. Pocos inves- tigadores han hecho análisis químicos de ellas. Por el contrario, he encontrado que otras “autorida- des” en la materia simplemente adivinaron los componentes de las tierras a partir de su aparien- cia física, lo que resultó algunas veces en identi- ficaciones geológicas muy divergentes. Luego de preparar una lista de nombres de unos 30 tipos de tierras usados por los habitantes quechua y aymara del altiplano, conseguí fondos para hacer análisis de DRX de 70 muestras, y fondos adicionales para hacer un corto viaje a Bolivia y recolectar las muestras pertinentes. La geofagia continuó siendo practicada posterior- mente en culturas costeñas adyacentes a la cuen- ca del Titicaca. Clark (1993: 630, 632) obtuvo muestras que tienen una antigüedad de aproxima- damente 1300-1450 DC procedentes tanto de Estuquiña (Moquegua, sur del Perú) como de Azapa en el norte de Chile. Análisis de DRX en la muestra de Estuquiña indicaron que ésta se tra- taba de p’asa mientras que la de Azapa fue q’atawi. Santoro (1995:346, 349) ha reportado el hallazgo de millu y sirsukena en sus trabajos en Molle Pampa Medio, Molle Pampa Este y Vila Vila Sur en el norte de Chile, dentro de unidades que han sido fechadas en 1100-1350 años DC, 1250-1400 DC y 1000-1450 DC. Muestras del mercado Finalmente, Rowe (1946: 217) indicó medio siglo atrás que en el Imperio Inca “una cantidad de arcillas co- mestibles fue recolectada e intercambiada con cier- ta frecuencia en la sierra sur”. Hicimos un viaje de 10 días desde St. Louis (Missouri) hasta Bolivia especialmente para tratar de comprar muestras en el mercado de Huari en Oruro, un mercado sólo de hierbas y medicinas que se lleva a cabo una vez al año, por lo general alre- dedor del “Domingo de Ramos”, en Semana San- ta. Lamentablemente, debido a problemas políti- cos y climáticos, el mercado fue postergado por una semana, por lo que perdimos la oportunidad de hablar directamente con los principales abas- tecedores. Por este motivo recolectamos muestras del puesto de un vendedor en el mercado de Oruro y también de cinco diferentes vendedores ubicados a lo largo de la famosa Calle Linares en La Paz. Posteriormente, las muestras fueron analizadas en los laboratorios de Ciencias Planetarias y de la Tie- rra de Washington University, St. Louis. Se hicie- ron análisis de DRX para caracterizar los compo- nentes mineralógicos. Asimismo, recolectamos du- plicados de muestras de varios de los minerales, originarios de áreas diferentes, para determinar con certeza si la terminología empleada por los pobla- dores locales aymara o quechua era específica, o si era posible que cubriera o incluyera más de un tipo geológico de tierra. Sin embargo, el listado que sigue a continuación no entra en tal detalle y sola- mente es una síntesis de todos los datos. Los arqueólogos no nos hemos mostrado particu- larmente interesados en la búsqueda de estas ar- cillas y, además, éstas se mezclan de manera casi invisible con la matriz de los sedimentos en nues- tras excavaciones. Sin embargo, a pesar de esta realidad tenemos evidencia física durante la épo- ca pre-Inca del uso de al menos media docena de las dos docenas de tierras que se describen más adelante: p’asa, q’atawi, “lejía”, makaya, millu y sirsukena. Creo que la totalidad de tierras que se discutirán en este artículo fueron posiblemente empleadas desde el Período Formativo Temprano en la cuenca del lago Titicaca. Con un poco de suerte y mayor atención sobre su presencia du- rante los trabajos de campo, espero que dentro de las próximas dos décadas estemos en condiciones de demostrar este hecho. He dividido las dos docenas de tierras bajo discu- sión en cuatro grupos. Muestras arqueológicas Paz Soria (2000) informa so- bre los resultados de un análisis de DRX sobre una muestra obtenida en ACH-10, un sitio rural Tiwanaku. Los componentes identificados seña- lan que la muestra se trata de p’asa, tierra que es consumida junto con las papas y otros tubérculos. En el valle de Azapa en el norte de Chile, Focacci (1993: 78) ha informado del hallazgo de una tie- rra conocida como “upi”, la cual es la misma que Tampoco hubiera sorprendido si se trataba de “le- jía” si tenemos en cuenta que S. Chávez (1992: 134 TIERRAS COMESTIBLES DE LA CUENCIA DEL TITICACA: GEOFAGIA EN… TIERRAS COMESTIBLES DE LA CUENCIA DEL TITICACA: GEOFAGIA EN… Muestras del mercado La mayoría de tierras que 135 DAVID L. BROWMAN fines dietéticos. Sin embargo, también es usada para otras funciones como aliviar úlceras y dolo- res estomacales, tratar quemaduras y hemorragias persistentes, y aún para lavar ropa. de hecho son consumidas por razones nutricionales están incluidas dentro del primer grupo, el grupo de los filosilicatos. Estas son las que más apro- piadamente podrían llamarse “tierras geofágicas”. La clasificación de los otros grupos, además de sus características “nutricionales”, se centra en lo que pienso son los elementos principales de cada grupo: categorías de tierras a partir de sus fuentes de azufre, sodio y calcio, o sobre la base de sus componentes metálicos (o de su color). Estos úl- timos tres grupos son utilizados con mayor fre- cuencia para tratamientos externos, pero algunas veces también son consumidos de manera inter- na, y por lo general tienen principalmente usos medicinales. Todas las tierras discutidas son po- tencialmente recuperables en contextos arqueoló- gicos. b) ch’aqo, ch’aqu, chaco, ch’ako, ch’aquo, chhacco, ch’akko, chachakko. Por lo general, esta tierra es conocida como el equivalente quechua de p’asa. En lengua Uru es conocida como qhiqhiche y en Chile se le llama upi. Sin embargo, los vende- dores en el mercado de La Paz ofrecen grupos de p’asa y ch’aqo por separado. Por un lado, las mues- tras que analizamos fueron muy similares a las muestras de p’asa en cuanto a su naturaleza quí- mica. Por otro lado, eran ligeramente diferentes en cuanto a sus componentes geológicos en don- de, además de las esmectitas, había cantidades importantes de caolinitas, cloritos e illitas. La mayoría de muestras de ch’aqo estaban en polvo o se trataba de terrones de tierra natural y de for- ma irregular. No obstante, dos de las muestras de Oruro sí eran en forma de barras y cada una tenía un logotipo que las identificaba como de marcas diferentes. Nuestras muestras fueron identificadas como procedentes de los departamentos de Puno, La Paz y Oruro. Las tierras de ch’aqo son utiliza- das de manera idéntica a las de p’asa. 1. Grupo de los filosilicatos a) p’asa, pasa, ppassa, pahsa, phasa, p’asalla, phasalla. Todas las muestras eran esmectitas, es decir, silicato de aluminio hidratado de K, Mg y Fe, procedentes de dos lugares en el departamen- to de Oruro y dos de La Paz. Por lo general, p’asa se obtiene de minas que tienen hasta 3 m de pro- fundidad. En el pasado fue un objeto importante que era movilizado por los arrieros de las carava- nas de llamas como parte de sus relaciones de intercambio. Aún hoy en día los comerciantes de Oruro a veces entregan un poco de p’asa como una yapa o regalo a su contraparte de intercam- bio. Todas nuestras muestras estaban pulveriza- das o se trataban de pedazos de tierra de forma irregular. Sin embargo, otros investigadores han encontrado p’asa en forma compacta a manera de barras u otras formas para ser comidas como si fueran caramelos o chocolates. Incluso se ha vis- to que éstas tienen grabadas en uno de los extre- mos de la barra la cantidad que se necesita con- sumir. La presencia de p’asa es particularmente común en zonas donde hay un alto consumo de papas amargas como en los departamentos de La Paz y Oruro. También es preparada a manera de salsa que se vierte sobre los tubérculos cocinados y que se consume varias veces a la semana. Como se ha indicado líneas arriba se trata de una arcilla disponible en la naturaleza cuyos cationes se di- suelven con una tendencia a juntarse con las com- plejas moléculas de las fitotoxinas, y cuyos aniones proveen a la dieta de esenciales comple- mentos minerales. Así, su uso más importante es el de una arcilla comestible principalmente con c) alchata, llampu, mitu, mittu, mito. La muestra de esta tierra era una mezcla de caolinita, illita y calcita, es decir, una composición química muy similar a las de p’asa y ch’aqo. Se identificó que la muestra procedía de la provincia de Sicasica (Bolivia). Otros investigadores han informado de composiciones de esmécticas y caolinitas para estas tierras, las que son casi idénticas a las nues- tras. A diferencia de las otras tierras descritas lí- neas arriba, esta preparación no fue utilizada como un complemento en la alimentación sino solamen- te para tratar el susto. Se puede decir entonces que una posibilidad es que p’asa o ch’aqo es uti- lizada para fines medicinales pero identificada con nombres diferentes. mesas, y algunas veces como complemento de sal para animales y como fertilizante agrícola. (calcita y natrón los más comunes), y nitratos (nitrito el más común). Teniendo en cuenta el tipo de uso que se le da a las tierras y sales, he consi- derado que es más útil enfatizar los aspectos de sodio y calcio de las sales en el grupo del salitre, y considerar al yeso y otras sales de sulfato en el siguiente grupo del azufre. g) hakemasa, jakemasa, jaqimasi, hakemasi, haquimasi, haquemasa, haquimaasa, haqque, waripa tullpa, huaripa tullu. Esta tierra ha sido descrita en la literatura como calcárea o toba rica en carbonato de calcio (Girault 1988: 239-240; Losa 1983 [1780]: 64, 269; Valdizan y Maldonado1922:19). La muestra que obtuvimos era principalmente un carbonato de calcio con pequeñas cantidades de magnesio y algo de silvita, procedente de las salinas de Potosí. Su uso medi- cinal es para tratar problemas menstruales, dete- ner hemorragias nasales, curar heridas persisten- tes y heridas sangrantes, y para tratar irritaciones menores como picaduras de pulga; también es usada para curar el susto. Igualmente, analizamos otras dos muestras (álcali fijo y chali) que pare- cerían pertenecer o ser parte de esta categoría. Se determinó que: d) q’atawi o isku: catahui, cataui, catawi, katawi, katahua, katawa, qatawi, isku, ishku, yschu, yzcu, ischu, iso, toqra. Todas las muestras eran de cal o calcita con cantidades a nivel de elementos tra- za de complejos como illita o silvita. Es prepara- da como salsa y consumida con quinoa o cañihua como en el caso de la p’asa o ch’aqo. Preferen- temente es usada varias veces a la semana en al- gunas aldeas ubicadas a lo largo de la orilla oes- te del lago. También es usada por sus cualidades de astringente para tratar enfermedades de la piel, detener hemorragias y como desinfectante. Ob- viamente, también es un componente principal en la elaboración de blanqueadores para los enlucidos. h) álcali fijo. Es una calcita o básicamente una tierra rica en carbonato de calcio empleada para tratar dolores y hemorragias. Asimismo se deter- minó que: Una fuente (Girault 1988: 242) sugiere que cuan- do es usada como mordiente o como pigmento recibe el nombre de: i) chali. También es una tierra rica en carbonato de calcio procedente de las salinas de Potosí y empleada en el tratamiento del susto. La caracte- rización química de estas dos últimas es la misma que la de hakemasa. 2. Grupo de sodio y calcio Muchas de las tierras de este grupo se derivan de los salitres que son producto de la desecación de lagunas y salinas del Pleistoceno. Existen más de tres docenas de sales identificadas procedentes de estos salitrales, principalmente haluros (siendo la halita o cloruro de sodio la más común), sulfatos (yeso y mirabilita los más comunes), carbonatos 136 TIERRAS COMESTIBLES DE LA CUENCIA DEL TITICACA: GEOFAGIA EN… mesas, y algunas veces como complemento de sal para animales y como fertilizante agrícola. 3. Grupo de minerales de azufre n) sirsukina, sirsukena, sirsuquima, sirsuquena, choque quesima, sirpukiena, salli, sallina, sillana, sillita. Por lo general, esta tierra es azufre puro, lo que se confirmó en la muestra que procede de Potosí. Como en varios otros casos de tierras com- puestas de azufre, existen dos variedades: una buena, blanca o amarilla, y una negra, mala o sal- vaje. La amarilla es usada en diversos tratamien- tos médicos; por el contrario, la variedad negra es usada principalmente para la “magia negra” aun- que también se emplea en ceremonias de cura- ción. La variedad amarilla es quemada a manera de incienso y se usa para fumigar. También es usada para curar la inflamación del ojo, tratar erup- ciones en la piel y dolores de cabeza; igualmente se usa para curar la demodicosis, sarna, y otras enfermedades de la piel en el ganado. Igualmente es utilizada para combatir problemas de hongos en las papas, plantas de ají, así como para curar enfermedades causadas por el mal de aire. l) jaintilla, haintilla, limpa colla, piedra de bezoar. Las piedras bezoares fueron de gran importancia durante el Período Incaico. Por tal motivo he es- tado particularmente interesado en verificar si es- tas “piedras” (concreciones calculosas digestivas de los camélidos) aún son usadas. Lamentable- mente, no fue sino hasta el final de nuestro breve viaje que descubrimos que la preparación conoci- da como “jaintilla” se hace sobre la base de pie- dras bezoares molidas. La muestra que analiza- mos procedía de un rebaño que pastaba en el alti- plano en el límite entre Oruro y Cochabamba. Esta se trata principalmente de fosfato de calcio, como así era de esperarse, teniendo en cuenta análisis previos de piedras bezoares de camélidos. Estas piedras bezoares son recetadas a mujeres gestantes y también para curar el susto. o) millu, millo, millay, millo-kollpa, qullpa, ccollpa, kkollpa, q’ullpa, q’ollpa, qollpa, collpa, cullpa. Se trata de alumbre que se presenta de manera natural. Las muestras que recogimos pro- vienen de las salinas en el departamento de Oruro y son sulfatos ferrosos de aluminio hidratado. Es preferible usar el término millu y así evitar con- fusiones (sobre todo de parte de investigadores foráneos) entre los términos q’ullpa y coipa. Los pobladores del altiplano no tienen dificultades en hacer esta distinción y la mayoría de los vende- dores en el mercado lo ofrecen como una varian- te de q’ullpa. mesas, y algunas veces como complemento de sal para animales y como fertilizante agrícola. Su utilización es también idéntica, por lo que éstas podrían ser fácilmente incluidas como nombres alternativos de esta tierra medicinal local. Debido a que las caracterizacio- nes químicas fueron hechas recién al regresar a Estados Unidos, no fuimos conscientes de esta su- perposición de nombres y usos. Por la misma ra- zón no nos fue posible preguntar a los vendedores por qué las venden con nombres diferentes. Este es un tema que se necesita clarificar en el futuro. e) qontoy, kontoya, contuya, kontai, kuntayu, qojtoy, cachima, cachina. Algunas fuentes están de acuerdo en que estos términos se refieren a un mordiente o a su uso como pigmento. Sin embar- go las mismas fuentes están en desacuerdo en cuanto a sus elementos constitutivos, identifican- do alguno de ellos como nitrato de sodio o aún como alumbre (Petersen 1970:12; Weiss 1953: 20). No nos fue posible obtener una muestra para po- der evaluar esta identificación. f) colpa, coipa, callpa, kollpa, q’olpa. Esta tierra es una halita y la muestra viene de Uyuni en Oruro. Fue fabricada a manera de una pequeña bola de color blanco y era principalmente sulfato de potasio sódico y carbonato de sodio. Otras fuentes han identificado a coipa como un bicar- bonato de sodio o como un nitrato de sodio. Se debe tener mucho cuidado al tratar de diferenciar coipa de qullpa o millu, ambos discutidos líneas abajo. Uno de los usos de coipa más frecuente- mente mencionados es su utilización como jabón especialmente para lavar la lana de los camélidos trasquilados. Asimismo también es usado como mordiente al teñir la lana, como ofrenda en las j) mat’aqi, matake, mataka, matakque. Nuestra muestra de mat’aqi resultó ser una halita. Tam- bién podría presentar otras composiciones ya que según dos fuentes en la literatura (Bertonio 1984 [1612] Segunda Parte, p. 219; Browman y Gunderson 1993: 418) le dan una identificación ligeramente diferente: una como hueso pulveriza- do y composición de halita, y la otra como un derivado del guano. Se emplea para tratar con- gestiones del pecho, resfríos y tos, y a veces tam- bién como lavado o baño de sal. 137 DAVID L. BROWMAN k) taku, taco, thaco, llonqhe-taku, puka llampu. Las muestras que analizamos (una procedente de La Paz y otra de Oruro) estaban principalmente compuestas, para sorpresa mía, de caolinita con menores cantidades de esmectita. mesas, y algunas veces como complemento de sal para animales y como fertilizante agrícola. Previamente, pensaba que se trataba de hematita, debido a su color rojo y a que estaba convencido de lo que se informaba en la literatura etnográfica (Valdizan y Maldonado 1922, 2: 29; Brougere 1980: 113; Girault 1988: 232; Tomoeda 1993: 291, y otros). Taku, además de ser utilizado como pintura o pig- mento para marcar el ganado, también es emplea- da para curar la demodicosis en los animales. Se le da forma de pan de molde o bolas y es vendido para tratar pequeñas molestias del pecho, proble- mas cardíacos, fiebres, ataques de dolor, dolor en los costados, úlceras, mal de aire y depresión. También es usado como astringente para curar cortes. bas en dos muestras para ver cómo podrían verse desde un punto de vista arqueológico. Estas mues- tras tenían una composición variada con K, Ca, Mg, Al, Fe, fosfatos, sulfatos, cloruro y amonio, entre otros componentes. Esta variabilidad nos señala claramente que llipta o “lejía” serían muy difíciles de identificar en contextos prehistóricos a menos que se encuentren guardados en recipien- tes especiales. 4. Grupo de hierro y cobre t) llipi, ancas llimpi, llimpiyaj, piedra lipes, alca- parrosa, caparrosa, cardenillo, copajira, kopakiri, coravari, lahas. Esta categoría puede incluir una variedad de minerales definidos principalmente sobre la base del color de su mineral de cobre, el cual presenta una coloración o tinte verdoso. Tres de nuestras muestras eran sustancias de cobre, es decir, sulfatos cúpricos (dos eran de Santa Cruz y una de Puno). La cuarta muestra, color de agua- marina, resultó ser un sulfato férrico. Otras fuen- tes sugieren que a los minerales de este grupo se les pueden sumar los acetatos y carbonatos cúpricos. Estos productos minerales son sólo para tratamientos externos. Se les utiliza para cauteri- zar heridas, tratar cortes y úlceras en la piel, con- tra parásitos externos, matar pulgas y como bactericida. Debido a su color también es usado como ofrenda en las mesas de curandería. p) makaya, macaya, macay. La muestra que con- seguimos era un sulfato de bario procedente de Oruro. Otros autores (Cobo 1956 [1653]: 128; Losa Avila 1983 [1780]: 269; Valdizan y Maldonado 1922, 2: 22) creen que también podría incluir sulfuros de arsénico. Muchas de las sales de bario son tóxicas, por lo que evidentemente la mayoría de los usos de esta tierra es sólo para tratamiento externo. Sin embargo, se dice que es empleada para tratar dolores de cabeza, de estómago, y de mue- las. Por lo general, se les fabrica en forma de pe- queños panecillos. Preparaciones sobre la base de esta tierra se usan para hacer cataplasmas, para tra- tar flujo menstrual persistente, pequeñas molestias de la piel, y como sustancia depilatoria. Una mues- tra que nos fue vendida como: q) llink’i. El término usualmente hace referencia a tierra pura, pero resultó ser sulfuro de arsénico. Se nos informó que esta tierra era buena para tra- tar dolencias cardíacas, hemorragias y para curar enfermedades causadas por el mal aire. Este tipo de utilización parece muy similar al atribuido a la makaya , uso que se necesita constatar. ¿Puede entonces la makaya ser tanto sulfato/sulfuro de bario como sulfuro de arsénico? u) iman kala, pieda iman, iman rumi, kichi-rumi, qhichicalla, quisu cala, aputuri cala, achutiri. Este mineral se presenta en dos variedades, una negra y otra amarilla, usadas en ceremonias de magia blanca y negra. Las dos muestras en nuestro in- ventario eran de hematita y magnetita, una de ellas procedente del área de Puno. TIERRAS COMESTIBLES DE LA CUENCIA DEL TITICACA: GEOFAGIA EN… TIERRAS COMESTIBLES DE LA CUENCIA DEL TITICACA: GEOFAGIA EN… la preparación de blanqueadores para los enlucidos y como fertilizante de granos y tubérculos. como coagulante y purgante, para tratar inflamaciones del ojo, molestias en la piel, fie- bres y para el dolor de muelas. Asimismo, se la emplea para tratar las semillas y así prevenir las enfermedades de las plantas. Se usa como mordien- te para varios tipos de colorantes (Ravinés 1978: 267). Inclusive en ciertas ocasiones, según un in- formante –autoridad en la materia–, se le agrega a la p’asa o ch’aqo a manera de salsa para los tu- bérculos; no obstante, creo que esto último es muy probablemente un uso que rara vez ocurre. s) kkhakkya chunta, khuya chunta, kaka chunta, khakya chunta, kkakya chunta. No poseemos una muestra. También es identificada como yeso en la literatura (Girault 1988: 242) y podría ser otro grupo de nombres alternativos de mullu. 3. Grupo de minerales de azufre Otras fuentes refieren que esta tie- rra podría también contener sulfato ferroso de potasio y sales de sulfato de plomo. Es usada en mesas de curandería, para diagnosticar y curar ciertas enfermedades, en rituales de limpieza, para curar pérdidas de almas, y para proteger a los pacientes de maldiciones, robo, malos espíritus y magia negra. Se usa como sustancia cáustica, m) llipta, llipht’a, llikta, llichta, llijt’a, lloita, llinta, llujt’a, lukta, llucta, lejía, toqra, t’oqura, toqro, y muchos otros términos. Se trata de un compuesto muy conocido que se usa mientras se mastica la hoja de coca. Generalmente, en la cos- ta está compuesto de cal calcinada, mientras que en la sierra y la puna está compuesto por ceniza de plantas quemadas. Entre las plantas que son utilizadas de esta manera están la quinoa, cañihua, especies de baccharis (Asteracea o Compositae), las amarantáceas, vainas de cacao, cactus, tola, y varias hierbas aromáticas. Durante la época incaica la preparación de esta sustancia fue de tal impor- tancia que existían los llipta camayocs encarga- dos de su fabricación y distribución. Hicimos prue- 138 Comentarios finales análisis indicaron que, por el contrario, se trataba de hematita, posiblemente una lógica confusión debido a la coloración rojiza en ambas. Es princi- palmente utilizado en ofrendas y como un trata- miento para el susto. He procedido a tratar con cierto detalle las mues- tras modernas ya que éstas deben ser los tipos de preparaciones minerales que son potencialmente recuperables en sitios prehistóricos. Como se ha señalado en mi breve discusión de los trabajos pre- vios, al menos seis de las 24 tierras habían sido ya identificadas en contextos prehistóricos, algunas de ellas con una antigüedad de miles de años. Estos estudios pueden potencialmente contribuir con nue- va información sobre etnomedicina del Período Formativo y de culturas posteriores. Igualmente pueden ofrecer algo de información adicional a ser incluida en nuestros estudios nutricionales y en nuestros intentos por recrear la cosmología y las formas autóctonas de ver el mundo. x) suca, jali sebario, siwairo, siwairu, sihuayru, siwairu, siwayru. Siwayru o sebario es un nom- bre algo genérico que se usa para denominar mi- nerales pulverizados empleados en ceremonias medicinales en el altiplano; se presentan en va- riedades tanto masculinas como femeninas. Una de las muestras que obtuvimos era hematina, mien- tras que la otra resultó ser yeso. Otros investiga- dores (Girault 1984: 526) han indicado que los minerales pulverizados de sebario/siwayru inclu- yen al plomo y otros óxidos metálicos. Es usado para tratar cólicos, flujos menstruales fuertes, para combatir el susto y el aire y para proteger al ganado. Reconocimiento Este artículo fue traducido por Jorge Montenegro. Reconocimiento Este artículo fue traducido por Jorge Montenegro. 4. Grupo de hierro y cobre En general, son usa- das como objetos de buena suerte, como ofrendas para la Pachamama y los campos de cultivo, y en ceremonias de curación. r) mullu, piedra berenguela, pachas, pachach, pachacha, pahachi, poke lomanake. Los vende- dores del mercado usaron el termino mullu para referirse a yeso; no obstante, otras fuentes ofre- cen otros nombres como alternativa. El análisis de DRX de nuestra muestra (procedente de Oruro) la identificó como un típico hidrato de sulfato de calcio. Se puede presentar como una preparación de color blanco (femenino) o una de color negro (masculino). Se utiliza para hacer illas, para pro- teger y curar, para tratar males relacionados con el susto y el aire, para proteger de los rayos, mal- diciones y espíritus malignos, así como para de- tener hemorragias fuertes. Igualmente se usa en v) llimpi, lliphi. Como el mismo nombre local lo sugería, esta muestra resultó ser mica. Es emplea- da externamente en ceremonias de diagnóstico y curación, para tratar el susto y curar maldiciones, y como ofrenda a varios espíritus de la naturaleza. w) paria, pari, pucca llimpi, llimpe, llimphi, ychma, ichma. Este mineral tiene una variedad roja y otra blanca, y se suponía que se trataba de un óxido de mercurio. La muestra que compramos fue ofrecida por el vendedor como si se tratase esencialmente de cinabrio. Sin embargo, nuestros 139 DAVID L. BROWMAN REFERENCIAS CITADAS FOCACCI, G., 1993. Excavaciones arqueológicas en el ce- menterio Az-6, valle de Azapa. Chungara 24/25: 69-123. BERTONIO, L., 1984 [1612]. Vocabulario de la lengua aymara. Travaux de l’Institut Français d’Etudes Andines, T. 26. Instituto Francés de Estudios Andinos, Lima. GIRAULT, L., 1984. Kallawaya: Guérisseurs itinérants des Andes. Recherches sur les practiques médicinales et magiques. Institut Français de Recherche Scientifique pour le Développement en Coopération, Collection Mémoires 107, París. BONAVIA, D., L. JOHNSON, E. REITZ, E. WING y G. WEIR, 1993. Un sitio precerámico de Huarmey (PV35- 6) antes de la introducción del maíz. Bulletin de l’Institute Français d’Etudes Andines 22 (2): 409-442. ——1988. Rituales en las regiones andinas de Bolivia y Perú. CERES-MUSEF-QUIPUS, La Paz. BROUGERE, A. M., 1980. Traditions, changements et écologie dans des communautés paysannes andines. Te- sis presentada para obtener el grado de Doctor en Etno- logía. Departamento de Etnología. Universidad de París, París. JONES, J. G., 1988. Middle to Late Preceramic (6000-3000 BP) subsistence patterns on the central coast of Peru: The coprolite evidence. M. A. Thesis. Department of Anthropology, Texas A & M University, College Station, Texas. BROWMAN, D. L. y J. N. GUNDERSON, 1993. Altiplano comestible earths: Prehistoric and historic geophagy of highland Peru and Bolivia. Geoarchaeology 8 (5): 413-425. KROEBER, A. L. y D. COLLIER, 1998. The archaeology and pottery of Nazca,Peru: Alfred L. Kroeber’s 1926 expedition. Altamira Press, Walnut Creek. CHAVEZ, S. J., 1992. The conventionalized rules in Pucara pottery technology and iconography: Indications for so- cio-political developments in the Northern Lake Titicaca Basin. Ph.D. dissertation, Michigan State University, Lansing, Michigan. LOSA AVILA Y PALOMARES, G., DE, 1983 [1780]. De los árboles, frutos, plantas, aves y otras cosas medicina- les tiene este reyno. Sociedad Geográfica de La Paz, La Paz. CLARK, N., 1993. The Estuquiña textile tradition: Cultural patterning in late prehistoric fabrics, Moquegua, far Southern Peru. Tesis doctoral, Departamento de Antro- pología, Washington University. St. Louis, Missouri. O’BRIEN, T. G., 1997. A skeletal biological examination of the human mortuary remains from Iwawi, Bolivia. Abstracts of the 62 nd Annual Meeting, Society for American Archaeology, April 2-6, 1997, Nashville, Tennessee, p. 166. Society for American Archaeology, Washington D. C. COBO, B., 1956 [1653]. Historia del Nuevo Mundo. Biblio- teca de autores españoles desde la formación del lengua- je hasta nuestros días, T. 91-92. Ediciones Atlas, Madrid. 140 TIERRAS COMESTIBLES DE LA CUENCIA DEL TITICACA: GEOFAGIA EN… SANTORO, C., 1995. Late prehistoric regional interaction and social change in a coastal valley of Northern Chile. Ph. D. Dissertation, De partment of Anthropology, University of Pittsburgh, Pittsburgh. PAZ SORIA, J. L., 2000. Transición Formativo–Tiwanaku en el sitio de Corralpata, Bolivia. Tesis de Licenciatura, Carrera de Arqueología, Facultad de Ciencias Sociales, Universidad Mayor de San Andrés, La Paz. PETERSEN, G., 1970. Minería y metalurgia en el antiguo Perú. Arqueológicas 12, Lima. TOMOEDA, H., 1993. Los ritos contemporáneos de camélidos y la ceremonia de la Citúa. En El mundo ceremonial andino, L. Millones y Y. Onuki (Eds.), vol. 37: 289-306. Senri Ethnological Studies Nacional Museum of Ethnology, Osaka. RAVINES, R., 1978. Textilería. En Tecnología andina, R. Ravinés (Comp.), pp. 253-268. Instituto de Estudios Pe- ruanos, Lima. VALDIZAN, H. y A. MALDONADO, 1922. La medicina popular peruana. Contribución al folklore médico del Perú (3 tomos). Imprenta Torres Aguirre, Lima. RIVERA, C., 1994. Ch’iji Jawira: Evidencia sobre la pro- ducción de cerámica en Tiwanaku. Tesis de Licenciatura, Carrera de Arqueología, Facultad de Ciencias Sociales, Universidad Mayor de San Andrés, La Paz. WEISS, P., 1943 Los comedores peruanos de tierra; datos históricos, sociales y geográficos; nombres de las tierras comestibles; interpretación fisiológica de la geofagia y la pica. Perú Indígena 5 (12):12-21. ROWE, J. H., 1946. Inca culture at the time of the Spanish conquest. En The Andean Civilizations, J. Steward (Ed.), pp. 183-330. Bureau of American Ethnology, Bulletin 143 (2), Washington D. C. 141 141
https://openalex.org/W2898114739	https://chemrxiv.org/engage/api-gateway/chemrxiv/assets/orp/resource/item/60c73f0fee301c1c93c787f6/original/an-amphiphilic-dna-platform-for-the-design-of-crystalline-frameworks-with-programmable-structure-and-functionality.pdf	English	null	Amphiphilic-DNA Platform for the Design of Crystalline Frameworks with Programmable Structure and Functionality	Journal of the American Chemical Society	2,018	cc-by	10,663	Ryan A. Brady,† Nicholas J. Brooks,‡ Vito Foderà,¶ Pietro Cicuta,∗,† and Lorenzo Di Michele∗,† †Biological and Soft Systems, Cavendish Laboratory, University of Cambridge, Cambridge CB3 0HE, UK ‡Department of Chemistry, Imperial College London, London SW7 2AZ, UK ¶Department of Pharmacy, University of Copenhagen, Universitetsparken 2 2100 Copenhagen, Denmark †Biological and Soft Systems, Cavendish Laboratory, University of Cambridge, Cambridge CB3 0HE, UK ‡Department of Chemistry, Imperial College London, London SW7 2AZ, UK ¶Department of Pharmacy, University of Copenhagen, Universitetsparken 2 2100 Copenhagen, Denmark E-mail: pc245@cam.ac.uk; ld389@cam.ac.uk E-mail: pc245@cam.ac.uk; ld389@cam.ac.uk Abstract allow us to ﬁne-tune the lattice parameter and thus control the partitioning of macromolecules within the frameworks, embed responsive mo- tifs that can induce isothermal disassembly, and include chemical moieties to capture target pro- teins speciﬁcally and reversibly. The reliable preparation of functional, ordered, nanostructured frameworks would be a game changer for many emerging technologies, from energy storage to nanomedicine. Underpinned by the excellent molecular recognition of nu- cleic acids, along with their facile synthesis and breadth of available functionalizations, DNA Nanotechnology is widely acknowledged as a prime route for the rational design of nanostruc- tured materials. Yet, the preparation of crys- talline DNA frameworks with programmable structure and functionality remains a challenge. Here we demonstrate the potential of sim- ple amphiphilic DNA motifs, dubbed C-stars, as a versatile platform for the design of pro- grammable DNA crystals. In contrast to all- DNA materials, in which structure depends on the precise molecular details of individual build- ing blocks, the self-assembly of C-stars is con- trolled uniquely by their topology and symme- try. Exploiting this robust self-assembly prin- ciple we design a range of topologically iden- tical, but structurally and chemically distinct C-stars that following a one-pot reaction self- assemble into highly porous, functional, crys- talline frameworks. Simple design variations Introduction a, C-stars are composed of 4 core-forming oligonucleotides (blue) and 4 cholesterol-functionalized strands (orange). b, Slow cooling of C-star sam- ples leads to the formation of single crystals. Scale bar 30 µm. c, Topology-preserving mod- iﬁcations include; (i) controlling arm length, (ii) inclusion of functional groups, here nitrilo- triacetic acid (NTA), (iii) inclusion of DNA strand-displacement motifs. Here we demonstrate how a platform based on newly introduced amphiphilic DNA nanos- tructures, dubbed C-stars,32 can overcome the aforementioned limitations, oﬀering a one-pot route for the formation of highly porous, bulk crystalline mesophases able to robustly support changes in structure and functionality. Rather than depending on the ﬁne geometrical details of the building blocks, the crystal structure of C-star frameworks is prescribed solely by the topology and symmetry of the amphiphilic DNA motifs.32 We exploit this unique feature to produce a range of topology-preserving C- star designs forming a variety of frameworks with distinct structure and functionalities that nevertheless share the same crystal geometry. We can precisely and continuously tune lattice parameter and framework mesh size, which al- lows us to control the partitioning of a range of Introduction gles have been shown to form diﬀracting single crystals with a remarkable ∼4Å resolution, and overall dimensions exceeding 100 µm.20 How- ever, the compact geometry and rigidity of these building blocks, and the required bond directionality, places substantial limits on the accessible rage of free volume, lattice param- eters, and the possibility of embedding bulky responsive motifs.20,28 Approaches based on DNA “origami’ and single-stranded (ss) DNA “bricks”, can produce 3D architectures of arbi- trary shape, which are however very compact, limiting diﬀusion of solutes and leaving little space to incorporate active elements or molecu- lar cargo.17–19,29,30 An exception is represented by open origami frameworks18 and by the re- cently introduced origami-based tenesegrity tri- angles that form a scaled-up version of the analogous nanoscale lattices,20,28 enabling the incorporation of nanoparticles.31 In all cases, the complexity of origami requires cumbersome sample preparation protocols leading to low yields and high costs. Furthermore, as for wire- frame DNA lattices,29 these approaches have not yet been shown to produce periodic arrays over macroscopic lengthscales. Here we demonstrate how a platform based on newly introduced amphiphilic DNA nanos- tructures, dubbed C-stars,32 can overcome the aforementioned limitations, oﬀering a one-pot route for the formation of highly porous, bulk crystalline mesophases able to robustly support changes in structure and functionality. Rather than depending on the ﬁne geometrical details of the building blocks, the crystal structure of C-star frameworks is prescribed solely by the topology and symmetry of the amphiphilic DNA motifs.32 We exploit this unique feature to produce a range of topology-preserving C- star designs forming a variety of frameworks with distinct structure and functionalities that nevertheless share the same crystal geometry. We can precisely and continuously tune lattice parameter and framework mesh size, which al- lows us to control the partitioning of a range of : NTA c a chol b l 14bp 7-37bp i iii ii Figure 1: Topology preserving modiﬁ- cations in amphiphilic C-stars. a, C-stars are composed of 4 core-forming oligonucleotides (blue) and 4 cholesterol-functionalized strands (orange). b, Slow cooling of C-star sam- ples leads to the formation of single crystals. Scale bar 30 µm. c, Topology-preserving mod- iﬁcations include; (i) controlling arm length, (ii) inclusion of functional groups, here nitrilo- triacetic acid (NTA), (iii) inclusion of DNA strand-displacement motifs. a chol b : NTA c l 14bp 7-37bp i iii ii a c ii Figure 1: Topology preserving modiﬁ- cations in amphiphilic C-stars. Introduction The production of functional crystalline frame- works is arguably the ultimate goal of DNA nanotechnology, and surely one of its most ap- plicable outcomes.1,2 Methodologies have been developed to create a variety of DNA-based ar- rays with high spatial resolution in 1D3–6 and 2D,6–16 and a number of routes exists to ex- tend geometrical control to the third dimen- sion.6,15–20 None of the available approaches, however, has been able provide a general route for the preparation of 3D DNA frameworks that combine high porosity, embedded func- tionality, robustness to design changes, and the ability to retain local order over large length- scales. Frameworks with these characteristics would enable the production of programmable materials in which functional macromolecules, quantum dots, ﬂuorophores, or non-structural DNA motifs are precisely arranged in space, 1 macromolecules. Building blocks tagged with a ligand form frameworks that can speciﬁcally and reversibly entrap a target protein, while the use of C-stars modiﬁed with a responsive DNA motif leads to triggered disassembly of the frameworks. Furthermore, the resilience of the self-assembly mechanism of C-stars makes crys- tallization robust to low-ionic strength condi- tions and to the use of unpuriﬁed components. revolutionizing areas of advanced technology in- cluding energy storage,21,22 photonics,23 sens- ing,24–26 and molecular sieving.27 g, a d o ecu a s ev g Building units in the form of tensegrity trian- gles have been shown to form diﬀracting single crystals with a remarkable ∼4Å resolution, and overall dimensions exceeding 100 µm.20 How- ever, the compact geometry and rigidity of these building blocks, and the required bond directionality, places substantial limits on the accessible rage of free volume, lattice param- eters, and the possibility of embedding bulky responsive motifs.20,28 Approaches based on DNA “origami’ and single-stranded (ss) DNA “bricks”, can produce 3D architectures of arbi- trary shape, which are however very compact, limiting diﬀusion of solutes and leaving little space to incorporate active elements or molecu- lar cargo.17–19,29,30 An exception is represented by open origami frameworks18 and by the re- cently introduced origami-based tenesegrity tri- angles that form a scaled-up version of the analogous nanoscale lattices,20,28 enabling the incorporation of nanoparticles.31 In all cases, the complexity of origami requires cumbersome sample preparation protocols leading to low yields and high costs. Furthermore, as for wire- frame DNA lattices,29 these approaches have not yet been shown to produce periodic arrays over macroscopic lengthscales. Topology-preserving modiﬁcations of C-star motifs C-stars are simple amphiphilic motifs based on DNA nanostars,33–35 composed of 4 core- forming oligonucleotides and 4 cholesterol- functionalized strands (Fig. 1a).32 Slow cooling of a stoichiometric mixture of these compo- nents leads to the self-assembly of macroscopic single crystals in a straightforward one-pot re- 2 action (Fig. 1b).32 In contrast to approaches reliant on Watson-Crick and/or stacking inter- actions alone,20,28,29,31 where a successful self- assembly requires the ﬁne tuning of the building block geometry with atomic resolution, in C- star frameworks long-range order emerges from the frustrated phase separation between the hy- drophobic cholesterol and the hydrophilic DNA. This mechanism is controlled uniquely by the topology and symmetry of the ﬂexible nanos- tar motif,32 which makes C-star self-assembly potentially robust against substantial design changes that preserve these features. To challenge this hypothesis we designed a range of structurally and chemically distinct C-star sub-units which share the same 4-arm topology.32 The investigated design variations are presented schematically in Fig. 1c, and include: (i) a range of C-stars with diﬀerent arm length designed to program the lattice parameter of the frameworks; (ii) a C-star modiﬁed with a chemical group to speciﬁcally capture and reversibly release target proteins; (iii) a C-star modiﬁed with a responsive DNA strand-displacement motif which can be trig- gered to induce isothermal dissolution of the frameworks. action (Fig. 1b).32 In contrast to approaches reliant on Watson-Crick and/or stacking inter- actions alone,20,28,29,31 where a successful self- assembly requires the ﬁne tuning of the building block geometry with atomic resolution, in C- star frameworks long-range order emerges from the frustrated phase separation between the hy- drophobic cholesterol and the hydrophilic DNA. This mechanism is controlled uniquely by the topology and symmetry of the ﬂexible nanos- tar motif,32 which makes C-star self-assembly potentially robust against substantial design changes that preserve these features. electrophoretic migration distance Rf decreases linearly with l in the range 24-51 bp, deviating for l = 21 bp. A consistent trend is observed in the mean hydrodynamic diameter ⟨Dh⟩mea- sured by DLS, increasing with l. For C-star samples with l = 21-42 bp we ob- serve the formation of equilibrium single crys- tals that, irrespective of arm length, feature a rhombic dodecahedral geometry consistent with an underlying cubic symmetry (Fig. 2b, left).36 The relationship between the C-star arm length and the microscopic structure of the self-assembled aggregates was investigated by Small Angle X-ray Scattering (SAXS). Topology-preserving modiﬁcations of C-star motifs Diﬀrac- tion proﬁles obtained from “powder” samples, featuring dense packings of individual crystals demonstrate that all C-star samples exhibiting polyhedral aggregates adopt a body centred- cubic (BCC) unit cell (Fig. 2b, centre and right). Consistent with a crystal geometry in- dependent on arm length, the measured lattice parameter a increases linearly with l, matching very closely the predicted value at (Fig. 2c). We estimate at through a simple geometrical model, where C-star arms are modelled as rigid rods of length equal to dbpl (dbp = 3.4 Å/bp) and the cholesterol-rich cores are approximated to spheres of radius rmici = 30.3 Å, in very good agreement with the previously estimated value for l = 28 C-stars32 To challenge this hypothesis we designed a range of structurally and chemically distinct C-star sub-units which share the same 4-arm topology.32 The investigated design variations are presented schematically in Fig. 1c, and include: (i) a range of C-stars with diﬀerent arm length designed to program the lattice parameter of the frameworks; (ii) a C-star modiﬁed with a chemical group to speciﬁcally capture and reversibly release target proteins; (iii) a C-star modiﬁed with a responsive DNA strand-displacement motif which can be trig- gered to induce isothermal dissolution of the frameworks. Tuning lattice parameter The observed range of viable arm lengths translates into lattice parameters spanning from 18.4 nm to 30.6 nm, reaching a maximum size well above what is achievable 3 l = 24bp a = 201 Å l = 28bp a = 232 Å l = 31bp a = 242 Å l = 35bp a = 267 Å l = 39bp a = 286 Å l = 42bp a = 306 Å l = 51bp Scattering vector, q (Å-1) 0.02 0.06 0.1 log(scattering intensity) Increasing arm length, l l = 21bp a = 184 Å b a dbpl a rmici e d l (bp) 16 20 12 0.58 0.66 30 40 50 20 <Dh> (nm) Rf 8 0.5 a 30 40 50 20 200 250 300 350 400 a (Å) c l (bp) Figure 2: Controlling lattice parameter in C-star crystals. a, Structural characterization of individual nanostars lacking cholesterol functionalization. Increasing l leads to lower migration distance Rf and larger mean hydrodynamic diameter ⟨Dh⟩. b, (left) Bright ﬁeld images show rhombic-dodecahedral single crystals for l = 21-42 bp, and spherical aggregates for l = 51 bp. Scale bars are 10 µm. SAXS powder diﬀraction patterns reveal that C-stars with arm length l = 21-42 bp form BCC crystalline phases. (Centre) 2D diﬀraction patterns. (Right) Radially-averaged proﬁles. Red lines mark best ﬁt to Bragg peaks of a BCC lattice. C-stars with l = 51 bp (bottom) form an amorphous phase. c, Measured lattice parameter increases with l, and closely follows the expected value from geometrical arguments (dashed line). Blue circles indicate conventional C-stars, the green diamond the responsive design (Fig. 6). d, Plausible arrangement of C-stars in a BCC unit cell.32 e, Abstracted view of unit cell. Here, solid blue lines represent DNA duplexes and red spheres represent micelles. For ease of visualization, only 2 nanostars are shown. l (bp) 16 20 12 0.58 0.66 30 40 50 20 <Dh> (nm) Rf 8 0.5 a b a Increasing arm length, l 30 40 50 20 200 250 300 350 400 a (Å) c l (bp) c dbpl a rmici e a d d e Scattering vector, q (Å-1) 0.06 0.1 Figure 2: Controlling lattice parameter in C-star crystals. a, Structural characterization of individual nanostars lacking cholesterol functionalization. Increasing l leads to lower migration distance Rf and larger mean hydrodynamic diameter ⟨Dh⟩. Tuning lattice parameter at = 4 √ 5(dbpl + rmici). (1) While retaining the same 4-arm topology shown in Fig. 1a, we vary nanostar arm length l be- tween 21 and 51 base-pairs (bp). l is controlled by changing the length of the core-forming strands while keeping that of the choles- terolized strand ﬁxed, as shown in Fig. 1c(i). Correct folding of individual nanostar motifs was conﬁrmed by Agarose Gel Electrophoresis (AGE) and Dynamic Light Scattering (DLS), using soluble building blocks in which the cholesterol-modiﬁed strands were replaced by non-functionalized ones of identical sequence. AGE produces single sharp bands for arm lengths of 24-51 bp, while nanostructures with l = 21 give rise to a more smeared band, indi- cating that such small junctions are not indi- vidually stable in running buﬀer (see Fig. S1 in SI). Fig. 2a demonstrates how the relative (1) The prefactor in Eq. 1 is derived from the hypothesized arrangement of C-stars in the unit cell, where each BCC site is occupied by a hydrophobic core featuring 12 choles- terol molecules, linked by C-Stars as shown in Fig. 2d32 and abstracted in Fig. 2e. A more detailed discussion of the derivation of Eq. 1 can be found in the SI (see Fig. S2). Despite being individually unstable, C-stars with l = 21 bp form a stable crystalline phase, demonstrat- ing how collective interactions further stabilize the DNA motifs. The observed range of viable arm lengths translates into lattice parameters spanning from 18.4 nm to 30.6 nm, reaching a maximum size well above what is achievable The prefactor in Eq. 1 is derived from the hypothesized arrangement of C-stars in the unit cell, where each BCC site is occupied by a hydrophobic core featuring 12 choles- terol molecules, linked by C-Stars as shown in Fig. 2d32 and abstracted in Fig. 2e. A more detailed discussion of the derivation of Eq. 1 can be found in the SI (see Fig. S2). Despite being individually unstable, C-stars with l = 21 bp form a stable crystalline phase, demonstrat- ing how collective interactions further stabilize the DNA motifs. Tuning lattice parameter b, (left) Bright ﬁeld images show rhombic-dodecahedral single crystals for l = 21-42 bp, and spherical aggregates for l = 51 bp. Scale bars are 10 µm. SAXS powder diﬀraction patterns reveal that C-stars with arm length l = 21-42 bp form BCC crystalline phases. (Centre) 2D diﬀraction patterns. (Right) Radially-averaged proﬁles. Red lines mark best ﬁt to Bragg peaks of a BCC lattice. C-stars with l = 51 bp (bottom) form an amorphous phase. c, Measured lattice parameter increases with l, and closely follows the expected value from geometrical arguments (dashed line). Blue circles indicate conventional C-stars, the green diamond the responsive design (Fig. 6). d, Plausible arrangement of C-stars in a BCC unit cell.32 e, Abstracted view of unit cell. Here, solid blue lines represent DNA duplexes and red spheres represent micelles. For ease of visualization, only 2 nanostars are shown. with simple all DNA motifs.20,28 Relative peak heights in Fig. 2b gradually change as arm length is increased, implying a change in the electron density distribution within the BCC unit cell. This is to be expected as an increase in l results in a greater fraction of the total material being located within the nanostar arms rather than elsewhere. speciﬁcity of hydrophobic interactions that, along with the free swivelling motion of the cholesterol moieties, relax any constraints on the rotational orientation of individual C-stars. Despite limiting diﬀraction resolution, this in- herent ﬂexibility and rotational degeneracy is the primary cause underlying the insensitivity of C-star self-assembly to ﬁne features, which ultimately enables the design versatility dis- cussed in this paper. Relative peak heights in Fig. 2b gradually change as arm length is increased, implying a change in the electron density distribution within the BCC unit cell. This is to be expected as an increase in l results in a greater fraction of the total material being located within the nanostar arms rather than elsewhere. The diﬀraction resolution of C-star frameworks reaches 4 nm, and is limited by the intrinsic ﬂexibility of the nanostar motifs and the non- Samples with l = 51 bp display no crystallinity, 4 forming instead an amorphous phase (Fig. 2b). Tuning molecular partitioning The conservation of crystal geometry for C- stars with widely diﬀerent arm length of- fers a powerful route for the preparation of nanoporous materials with programmable porosity, which we characterize by deter- mining the degree of penetration of various (macro)molecular probes within the crystals. We chose a variety of ﬂuorescent probes to cover a broad range of molecular weight, hy- drodynamic size, hydrophobicity, and chemical nature, including sodium ﬂuorescein (FAM), ﬂuorescein-modiﬁed dextrans (3kDa DXT- FAM, 10kDa DXT-FAM), rhodamine B, re- combinant green ﬂuorescent protein (rGFP), and ﬂuorescently labelled streptavidin (SAv- A647). Crystals were soaked in solutions con- taining the (macro)molecules, and their pene- tration was determined by extracting the ratio ξ = Iinternal/Iexternal between the ﬂuorescence intensity measured inside and outside the crys- tals from confocal micrographs. The conservation of crystal geometry for C- stars with widely diﬀerent arm length of- fers a powerful route for the preparation of nanoporous materials with programmable porosity, which we characterize by deter- mining the degree of penetration of various (macro)molecular probes within the crystals. We chose a variety of ﬂuorescent probes to cover a broad range of molecular weight, hy- drodynamic size, hydrophobicity, and chemical nature, including sodium ﬂuorescein (FAM), ﬂuorescein-modiﬁed dextrans (3kDa DXT- FAM, 10kDa DXT-FAM), rhodamine B, re- combinant green ﬂuorescent protein (rGFP), and ﬂuorescently labelled streptavidin (SAv- A647). Crystals were soaked in solutions con- taining the (macro)molecules, and their pene- tration was determined by extracting the ratio ξ = Iinternal/Iexternal between the ﬂuorescence intensity measured inside and outside the crys- tals from confocal micrographs. Tuning lattice parameter Blue lines mark best ﬁt to Bragg peaks of a secondary BCC phase with lattice parameter a2. Scale bars 10 µm. Tuning lattice parameter We ascribe this behavior to the increased ﬂex- ibility of the longer DNA arms, which may be too pliant to sustain a rigid network.34 phase with lattice parameter identical to the one found in higher ionic-strength conditions, when reducing sodium concentration we ob- serve the emergence of a secondary BCC phase with slightly expanded lattice parameter. We hypothesize that the secondary phase may ap- pear due to conformational changes of the nanostar junction following reduced electro- static screening.35,37–41 log(scattering intensity) 0.06 0.1 TE, 100 mM NaCl a = 232 Å a2 = 242 Å TE, 200 mM NaCl a = 232 Å a2 = 241 Å PBS a = 232 Å a2 = 244 Å Scattering vector, q (Å-1) Figure 3: Self-assembly at low ionic strength. (left) Bright ﬁeld images show rhombic-dodecahedral single crystals formed by l = 28 bp C-stars in low ionic strength buﬀers. (centre) 2D diﬀraction patterns. (right) Radially-averaged proﬁles. Red lines mark the best ﬁt to the Bragg peaks of a BCC lattice with identical lattice parameter as for the frameworks formed by l = 28 bp C-stars in 300 mM NaCl (2b). Blue lines mark best ﬁt to Bragg peaks of a secondary BCC phase with lattice parameter a2. Scale bars 10 µm. log(scattering intensity) 0.06 0.1 TE, 100 mM NaCl a = 232 Å a2 = 242 Å TE, 200 mM NaCl a = 232 Å a2 = 241 Å PBS a = 232 Å a2 = 244 Å Scattering vector, q (Å-1) log(scattering intensity) log(scattering intensit The robustness of C-star self-assembly also re- laxes the strict need for using highly puri- ﬁed DNA oligonucleotides, which is standard in most DNA nanotechnology preparations. All but the cholesterol functionalized oligonu- cleotides in our experiments are puriﬁed (by the manufacturer) via conventional desalting, signiﬁcantly reducing costs compared to puriﬁ- cation steps based on High-Performance Liquid Chromatography or Polyacrylamide Gel Elec- trophoresis. log(scattering i Scattering vector, q (Å-1) Figure 3: Self-assembly at low ionic strength. (left) Bright ﬁeld images show rhombic-dodecahedral single crystals formed by l = 28 bp C-stars in low ionic strength buﬀers. (centre) 2D diﬀraction patterns. (right) Radially-averaged proﬁles. Red lines mark the best ﬁt to the Bragg peaks of a BCC lattice with identical lattice parameter as for the frameworks formed by l = 28 bp C-stars in 300 mM NaCl (2b). Self-assembly at low ionic strength. FAM FAM+SAv-A647 3kDa DXT-FAM 10kDa DXT-FAM SAv-A647 rGFP Arm length (bp) 35 30 40 25 ξ = Internal/IExternal 0.2 0.4 0.6 0.8 b 28 35 42 24 ξFAM/ξ FAM+SAv-A647 1 1.1 Arm length (bp) 35 30 40 25 d 28 35 42 24 c a FAM FAM+SAv-A647 3kDa DXT-FAM 10kDa DXT-FAM SAv-A647 rGFP Arm length (bp) 35 30 40 25 ξ = Internal/IExternal 0.2 0.4 0.6 0.8 a ξFAM/ξ FAM+SAv-A647 1 1.1 Arm length (bp) 35 30 40 25 c a d Figure 4: Controllable porosity and macromolecular partitioning in C-star networks. The following ﬂuorescent probes were tested: sodium ﬂuorescein (FAM), ﬂuorescein-labelled 3kDa dextran (3kDa DXT-FAM), ﬂuorescein-labelled 10kDa dextran (10kDa DXT-FAM), recombinant GFP (rGFP), Alexa647-labelled streptavidin (SAv-A647). a, Probe penetration was assessed by confocal microscopy, by measuring the ratio ξ between the mean ﬂuorescence levels inside and outside individual crystals soaked in solutions containing the probes. The grey band marks the level of background signal. b, Confocal micrographs of representative samples. c, Ratio between the ξ-values measured for FAM in pristine crystals and crystals previously incubated with SAv- A647. d, Confocal micrographs of crystals soaked in rhodamine B. All scale bars are 10 µm. pore size, we added FAM to each of the samples previously exposed to SAv. As shown in Fig. 4a (bi-color symbols), and further highlighted in Fig. 4c, C-stars with l = 35 and 42 bp show a decrease in the ξ measured for FAM in crystals with SAv in comparison to crystals not soaked in streptavidin - a clear proof that the bulky protein is excluding a fraction of the available free volume to ﬂuorescein. For samples with l = 24 and 28 bp, no change in ξ is observed, conﬁrming that streptavidin does not penetrate these frameworks. Note that SAv displays an aﬃnity for the surface of the crystals and coats them even in conditions in which the mesh size is too small to allow penetration (Fig. 4b). The aﬃnity is likely due to hydrophobic patches on the proteins. As expected, larger molecular weight leads to lower penetration for a ﬁxed l. Self-assembly at low ionic strength. Due to the low packing density of C-star frame- works, self-assembly does not require the high cation concentration or divalent ions needed for compact DNA crystals and origami.20,28,31 When not stated otherwise, all experiments were performed with a sodium concentration of 300 mM and no divalent ions. In Fig. 3 we show that crystallization is robust even at lower monovalent salt concentration, with well deﬁned single crystals observed in 200 mM, 100 mM NaCl, and in physiological phosphate buﬀered saline (PBS). In all cases, SAXS demonstrates unchanged crystal symmetry and scattering resolution compared with samples prepared in 300 mM NaCl. Besides the BCC Due to the low packing density of C-star frame- works, self-assembly does not require the high cation concentration or divalent ions needed for compact DNA crystals and origami.20,28,31 Macromolecule partitioning is clearly depen- dant on l, as shown by Fig. 4a,b, with all of the hydrophilic probes trialled, namely FAM, DXT FAM and rGFP, following the trend of longer arm length leading to increased penetration. 5 FAM FAM+SAv-A647 3kDa DXT-FAM 10kDa DXT-FAM SAv-A647 rGFP Arm length (bp) 35 30 40 25 ξ = Internal/IExternal 0.2 0.4 0.6 0.8 b 28 35 42 24 ξFAM/ξ FAM+SAv-A647 1 1.1 Arm length (bp) 35 30 40 25 d 28 35 42 24 c a Figure 4: Controllable porosity and macromolecular partitioning in C-star networks. The following ﬂuorescent probes were tested: sodium ﬂuorescein (FAM), ﬂuorescein-labelled 3kDa dextran (3kDa DXT-FAM), ﬂuorescein-labelled 10kDa dextran (10kDa DXT-FAM), recombinant GFP (rGFP), Alexa647-labelled streptavidin (SAv-A647). a, Probe penetration was assessed by confocal microscopy, by measuring the ratio ξ between the mean ﬂuorescence levels inside and outside individual crystals soaked in solutions containing the probes. The grey band marks the level of background signal. b, Confocal micrographs of representative samples. c, Ratio between the ξ-values measured for FAM in pristine crystals and crystals previously incubated with SAv- A647. d, Confocal micrographs of crystals soaked in rhodamine B. All scale bars are 10 µm. Speciﬁc and reversible entrapment of a target protein p p of a target protein Although non-speciﬁc entrapment of functional proteins has been successfully demonstrated in all-DNA 3D crystals,43 site speciﬁc positioning has been achieved only in 2D DNA architec- tures.44–46 Owing to their controllable porosity and resilience to changes in building-block de- sign, C-star frameworks oﬀer an attractive plat- form for site-selective entrapment of functional macromolecules, including proteins, in 3D. Fol- lowing the strategy applied for 2D nanostruc- tures,44–46 to obtain protein-binding capability in 3D frameworks we designed a C-star variant with l = 28, in which one arm was modiﬁed to host a nitrilotriacetic acid (NTA) molecule (see Fig. 1c(ii)). When charged with Ni2+ ions, NTA can speciﬁcally bind recombinant proteins featuring poly-histidine residues (His-tags). As shown in Fig. 5a, NTA-functionalized C- stars form macroscopic single crystals visually similar to their non-functional counterparts. Confocal microscopy demonstrates that N- terminal poly-histidine tagged rGFP (rGFPH) strongly partitions within crystals previously washed with a NiCl2 solution. Control exper- iments show that samples lacking either NTA functionalization, NiCl2 or protein exhibit no ﬂuorescence signal under identical imaging con- ditions. Additionally, no ﬂuorescence signal was detected when performing the experiment us- ing a protein lacking the His-tag (rGFP), fully demonstrating the speciﬁcity of the partition- ing. Fig. 5b shows that protein entrapment can be controllably reversed by incubating the crystals loaded with rGFPH with the chelating agent ethylenediaminetetraacetic acid (EDTA), which strips the nickel from the NTA. The pro- tein can again be captured upon removal of EDTA and addition of new NiCl2. Diﬀraction patterns obtained from non-treated NTA C-star frameworks (Fig. 5c) and from those treated with Ni and loaded with rGFPH (Fig. 5d) show the same BCC symmetry and identical lattice parameter as the inert C-star design with the same arm length, demonstrating that neither Although non-speciﬁc entrapment of functional proteins has been successfully demonstrated in all-DNA 3D crystals,43 site speciﬁc positioning has been achieved only in 2D DNA architec- tures.44–46 Owing to their controllable porosity and resilience to changes in building-block de- sign, C-star frameworks oﬀer an attractive plat- form for site-selective entrapment of functional macromolecules, including proteins, in 3D. Fol- lowing the strategy applied for 2D nanostruc- tures,44–46 to obtain protein-binding capability in 3D frameworks we designed a C-star variant with l = 28, in which one arm was modiﬁed to host a nitrilotriacetic acid (NTA) molecule (see Fig. 1c(ii)). Self-assembly at low ionic strength. In- sets and scale bars as for a. c, SAXS diﬀraction patterns of NTA functionalized C-star frame- works. Modiﬁed networks adopt the same BCC lattice as inert C-stars. d, After treating with rGFPH the structure of NTA C-star aggregates remains the same. For c and d, red lines in- dicate the best ﬁt to Bragg peaks of a BCC lattice. 0 20 40 a IFluo 10 30 - NTA - Ni2+ - rGFP - NTA - Ni2+ + rGFPH - NTA + Ni2+ + rGFPH + NTA - Ni2+ - rGFP + NTA - Ni2+ + rGFPH + NTA + Ni2+ + rGFPH + NTA + Ni2+ + rGFP a Self-assembly at low ionic strength. The pene- tration coeﬃcient of ﬂuorescein can be used to estimate the accessible free volume of the framework as discussed in Ref.32 For l = 42 bp the fraction of free volume reaches 85%, a value well above what can be reached with alter- native nanoscale DNA building blocks28 and that compares favourably even against ultra- high porosity metal-organic frameworks.42 g p y g Large SAv molecules are completely excluded from frameworks with l = 24 and 28 bp, for which ξ values are not signiﬁcantly higher than the measured background, indicating no load- ing into crystals. A sharp penetration transi- tion is then observed upon increasing l to 35 and 42 bp, which produce similar values of ξ. This indicates that the pores in the l = 24 and 28 bp crystals are too small to accommodate the rather bulky protein, whereas the larger pores of the l = 35 and 42 bp crystals can. As an additional check to show that SAv was completely excluded from crystals with smaller Rhodamine B, a small hydrophobic molecule, was observed to strongly partition within the crystals regardless of arm length, likely due to an aﬃnity for the cholesterol-rich cores, as is clearly visible in Fig. 4d. 6 c d log(scattering intensity) Scattering vector, q (Å-1) 0.04 0.08 0.12 0.04 0.08 0.12 0 20 40 a IFluo 10 30 - NTA - Ni2+ - rGFP - NTA - Ni2+ + rGFPH - NTA + Ni2+ + rGFPH + NTA - Ni2+ - rGFP + NTA - Ni2+ + rGFPH + NTA + Ni2+ + rGFPH 0 10 20 30 40 b IFluo + EDTA - Ni2+ - rGFPH - EDTA + Ni2+ + rGFPH + EDTA - Ni2+ - rGFPH - EDTA + Ni2+ + rGFPH + NTA + Ni2+ + rGFP Figure 5: Speciﬁc and reversible binding of a target protein. a, Binding of N-terminal 6× histidine tagged rGFP (rGFPH) and rGFP lacking a histidine tag (rGFP) to inert and ni- trilotriacetic acid (NTA) functionalized crystals was assessed by measuring the mean ﬂuores- cence intensity within crystals, IFluo, from con- focal micrographs. Insets show combined bright ﬁeld (top half) and ﬂuorescent (bottom half) micrographs of representative crystals. Scale bars 10 µm. b, Reversibility of rGFPH bind- ing to NTA functionalized crystals upon addi- tion and removal of a competing chelating agent demonstrated through confocal microscopy. Speciﬁc and reversible entrapment of a target protein When charged with Ni2+ ions, NTA can speciﬁcally bind recombinant proteins featuring poly-histidine residues (His-tags). c d log(scattering intensity) Scattering vector, q (Å-1) 0.04 0.08 0.12 0.04 0.08 0.12 0 10 20 30 40 b IFluo + EDTA - Ni2+ - rGFPH - EDTA + Ni2+ + rGFPH + EDTA - Ni2+ - rGFPH - EDTA + Ni2+ + rGFPH b c d log(scattering intensity) Scattering vector, q (Å-1) 0.04 0.08 0.12 0.04 0.08 0.12 g p y ( g ) As shown in Fig. 5a, NTA-functionalized C- stars form macroscopic single crystals visually similar to their non-functional counterparts. Confocal microscopy demonstrates that N- terminal poly-histidine tagged rGFP (rGFPH) strongly partitions within crystals previously washed with a NiCl2 solution. Control exper- iments show that samples lacking either NTA functionalization, NiCl2 or protein exhibit no ﬂuorescence signal under identical imaging con- ditions. Additionally, no ﬂuorescence signal was detected when performing the experiment us- ing a protein lacking the His-tag (rGFP), fully demonstrating the speciﬁcity of the partition- ing. Fig. 5b shows that protein entrapment can be controllably reversed by incubating the crystals loaded with rGFPH with the chelating agent ethylenediaminetetraacetic acid (EDTA), which strips the nickel from the NTA. The pro- tein can again be captured upon removal of EDTA and addition of new NiCl2. Diﬀraction patterns obtained from non-treated NTA C-star frameworks (Fig. 5c) and from those treated with Ni and loaded with rGFPH (Fig. 5d) show the same BCC symmetry and identical lattice parameter as the inert C-star design with the same arm length, demonstrating that neither including the NTA moiety nor trapping the protein aﬀect the microstructure of the frame- Figure 5: Speciﬁc and reversible binding of a target protein. a, Binding of N-terminal 6× histidine tagged rGFP (rGFPH) and rGFP lacking a histidine tag (rGFP) to inert and ni- trilotriacetic acid (NTA) functionalized crystals was assessed by measuring the mean ﬂuores- cence intensity within crystals, IFluo, from con- focal micrographs. Insets show combined bright ﬁeld (top half) and ﬂuorescent (bottom half) micrographs of representative crystals. Scale bars 10 µm. b, Reversibility of rGFPH bind- ing to NTA functionalized crystals upon addi- tion and removal of a competing chelating agent demonstrated through confocal microscopy. In- sets and scale bars as for a. c, SAXS diﬀraction patterns of NTA functionalized C-star frame- works. Modiﬁed networks adopt the same BCC lattice as inert C-stars. Isothermal dissociation of C-star crystals by strand displacement To further demonstrate the robustness of C- star self-assembly to topology-preserving design changes, we produced a C-star variant featur- ing a responsive motif. Relying on toehold- mediated strand displacement,47 we can in- duce the isothermal melting of these modi- ﬁed C-star frameworks upon exposure to a trigger oligonucleotide.48,49 The potential of strand displacement reactions as a means of controlling structural rearrangements in ma- terials has been demonstrated in arrays of DNA-functionalized gold nanoparticles,50 col- loids,48 and liposomes.51 However, previous attempts to program a triggered response in all-DNA crystals have been limited to the graft- ing/displacement of ﬂuorescent DNA oligos, producing color changes but no structural re- sponse.52 Trigger-induced isothermal melting of the frameworks is demonstrated by direct visu- alization of the aggregates, which rapidly dis- solve upon exposure to the trigger (time series in Fig. 6b). The same process, is tracked by SAXS, where we observe progressive loss of crystalline order, demonstrated by the broad- ening ﬁrst, and then disappearance of the BCC Bragg peaks (Fig. 6c). Speciﬁc and reversible entrapment of a target protein d, After treating with rGFPH the structure of NTA C-star aggregates remains the same. For c and d, red lines in- dicate the best ﬁt to Bragg peaks of a BCC lattice. Figure 5: Speciﬁc and reversible binding of a target protein. a, Binding of N-terminal 6× histidine tagged rGFP (rGFPH) and rGFP lacking a histidine tag (rGFP) to inert and ni- trilotriacetic acid (NTA) functionalized crystals was assessed by measuring the mean ﬂuores- cence intensity within crystals, IFluo, from con- focal micrographs. Insets show combined bright ﬁeld (top half) and ﬂuorescent (bottom half) micrographs of representative crystals. Scale bars 10 µm. b, Reversibility of rGFPH bind- ing to NTA functionalized crystals upon addi- tion and removal of a competing chelating agent demonstrated through confocal microscopy. In- sets and scale bars as for a. c, SAXS diﬀraction patterns of NTA functionalized C-star frame- works. Modiﬁed networks adopt the same BCC lattice as inert C-stars. d, After treating with rGFPH the structure of NTA C-star aggregates remains the same. For c and d, red lines in- dicate the best ﬁt to Bragg peaks of a BCC lattice. 7 works. is a 6-base ssDNA dangle one-quarter of the way along the arm. Remarkably, even with these substantial changes, the system retains the same BCC crystalline symmetry of conven- tional C-stars, as demonstrated by SAXS (black trace, Fig. 6c). The measured lattice parame- ter, reaching a = 34.2 nm, further extends the range demonstrated with inert C-stars, while being equally well predicted by Eq. 1 (green di- amond in Fig. 2c), conﬁrming that the crystal structure is fully conserved. Isothermal dissociation of C-star crystals by strand displacement Conclusions Figure 6a shows a schematic of the imple- mented strand displacement mechanism, lead- ing to dissolution of the C-star network. This C-star variant is designed such that the core strands (blue in Fig. 6a) are not bound di- rectly to the cholesterol-functionalized strands (orange in Fig. 6a). Instead, a bridge strand (green in Fig. 6a) connects the nanostar core to the cholesterolized strands, while presenting a dangling 6 nucleotide toe-hold. Upon the addi- tion of the trigger strand (red in Fig. 6a), which is fully complementary to the bridge strand, the core and cholesterolized strands are displaced leaving cholesterol-DNA micelles, free nanos- tars, and a double stranded waste product com- posed of the fully hybridized trigger and bridge strands (bottom-left panel, Fig. 6a). The cor- rect functionality of the triggered disassembly mechanism was demonstrated in freely diﬀus- ing nanostars using DLS and a ﬂuorescence quenching assay (see SI text and Fig. S3). The incorporation of the strand displacement motif leads to two key changes in the struc- ture of C-stars, compared to the conventional design. Namely, two nicks are present in the arm-forming duplex rather than one, and there In summary, we introduced a novel approach for the preparation of 3D crystalline frame- works with pre-designed structural features and embedded responsiveness, programmable through straightforward design variations. The ﬂexibility of our approach derives from the self- assembly mechanism of amphiphilic C-stars, in which network geometry depends on the general topology and symmetry of DNA mo- tifs, rather than on high-resolution molecular details. This feature enables unprecedented design freedom, that we exploit to produce a set of geometrically identical frameworks with ﬁnely controlled lattice parameters spanning a range of nearly 16 nm and reaching a maximum of 34.2 nm. By exploiting the resulting high porosity, the controllable mesh size, and the inherent amphiphilic properties of our mate- rial, we are able to ﬁne-tune the partitioning of macromolecules within the frameworks based on size and hydrophobicity. Furthermore, the resilience of the self-assembly mechanism, and the signiﬁcant free volume available within C- star networks, allowed us to include a respon- The incorporation of the strand displacement motif leads to two key changes in the struc- ture of C-stars, compared to the conventional design. Conclusions Namely, two nicks are present in the arm-forming duplex rather than one, and there 8 47s 71s 91s c a log(scattering intensity) Scattering vector, q (Å-1) 0.02 0.04 0.06 Post trigger addition (min) 60 0 15 45 b Trigger Bridge Toe-hold Figure 6: Embedded responsiveness enables isothermal melting of C-star crystals. a, Schematic of the mechanism underlying isothermal melting of C-star crystals. Addition of a trigger strand (red) causes the chol-DNA micelles to dissociate from the nanostar core through toe-hold mediated strand displacement. b, Series of bright-ﬁeld images showing rapid melting of C-star aggregates upon exposure to the trigger strand. Scale bar 10 µm. c, Despite the spherical macroscopic appearance, SAXS patterns reveal that aggregates formed by responsive C-Stars are indeed crystalline (top). The radially-averaged diﬀraction proﬁle (black solid line) is compatible with a BCC symmetry. Red lines indicate the best ﬁt to the Bragg peaks of a BCC lattice. Adding the trigger strand to the SAXS sample induces progressive disruption of crystalline order (coloured solid lines). The slower melting rate compared to that measured in microscopy experiments (b) is a consequence of the slow passive diﬀusion of the trigger strand through the large sample used for SAXS (see Methods section). log(scattering intensity) Post trigger addition (min) 60 0 15 45 c log(scattering intensity) Scattering vector, q (Å-1) 0.02 0.04 0.06 47s 71s 91s b b a log(scattering intensity) log(scattering in Scattering vector, q (Å-1) 0.02 0.04 0.06 Scattering vector, q (Å-1) 0.02 0.04 0.06 Figure 6: Embedded responsiveness enables isothermal melting of C-star crystals. a, Schematic of the mechanism underlying isothermal melting of C-star crystals. Addition of a trigger strand (red) causes the chol-DNA micelles to dissociate from the nanostar core through toe-hold mediated strand displacement. b, Series of bright-ﬁeld images showing rapid melting of C-star aggregates upon exposure to the trigger strand. Scale bar 10 µm. c, Despite the spherical macroscopic appearance, SAXS patterns reveal that aggregates formed by responsive C-Stars are indeed crystalline (top). The radially-averaged diﬀraction proﬁle (black solid line) is compatible with a BCC symmetry. Red lines indicate the best ﬁt to the Bragg peaks of a BCC lattice. Adding the trigger strand to the SAXS sample induces progressive disruption of crystalline order (coloured solid lines). Methods Agarose gels were prepared at 1.5 wt% agarose in Tris-Borate-EDTA buﬀer (Sigma Aldrich), and precast with DNA stain (SYBR Safe, Ther- moFisher). Annealed nanostars were mixed with loading dye (Thermo Scientiﬁc) and a vol- ume equivalent to 7.5 µg of DNA was loaded into each well. A potential of 75 V (3 V cm−1) was applied for 120 minutes. Gels were imaged using a GelDoc-It imaging system equipped with a UV lamp for illumination and CCD camera for image acquisition. Conclusions The slower melting rate compared to that measured in microscopy experiments (b) is a consequence of the slow passive diﬀusion of the trigger strand through the large sample used for SAXS (see Methods section). ed responsiveness enables isothermal melting of C-star crystals. complexes designed to amplify signals,56,57 per- form computation,58,59 and respond to molec- ular and environmental cues,60–65 opening a new route for producing highly concentrated, well-ordered, sensing/signal-processing archi- tectures with potential applications in point-of- care diagnostics, nanomedicine, or bottom-up synthetic biology. Finally, C-star self-assembly occurs in a one-pot reaction, and does not re- quire the use of highly puriﬁed oligonucleotides, substantially reducing costs and simplifying preparation protocols compared to other avail- able DNA-tile architectures. sive DNA motif leading to isothermal dissolu- tion of the frameworks, as well as a chemical modiﬁcation enabling speciﬁc entrapment and triggered release of functional proteins. The structural responsiveness and programmability of the frameworks, combined with their speciﬁc and non-speciﬁc aﬃnity for various cargoes, hints at the possible application of the material to smart nanomedical vectors. Compared to standard formulations based on lipids,53,54 car- riers prepared from C-stars could be more easily functionalized with ligands or DNA aptamers to achieve selective targeting of diseased cells,53 and programmed to dissolve and release their cargo only in the presence of speciﬁc molecular cues, e.g. disease-related microRNAs.55 Criti- cal for this and other biomedical applications is also the resilience of C-star frameworks to low and physiological ionic strength. More complex C-Star variants could also carry nucleic acid Acknowledgement LDM, PC and NJB acknowledge support from the EPSRC Programme Grant CAPITALS number EP/J017566/1. LDM acknowledges support from the Leverhulme Trust and the Isaac New- ton Trust through an Early Career Fellow- 9 provided by the supplier. ship (ECF-2015-494) and from the Royal So- ciety through a University Research Fellow- ship (UF160152). RAB acknowledges sup- port from the EPSRC CDT in Nanoscience and Nanotechnology (NanoDTC), grant num- ber EP/L015978/1. We acknowledge Diamond Light Source for provision of synchrotron beam- time (SM17271 and SM19371) and we would like to thank A. Smith and T. Snow for assis- tance in operating beamline I22. We thank T. Knowles and B. Mognetti for insightful discus- sions on this project. Free nanostar characterization Folding of nanostars was assessed using agarose gel electrophoresis (AGE) and dynamic light scattering (DLS). For these experiments the cholesterol-modiﬁed strand was replaced with a non-functionalized oligonucleotide of identical sequence to prevent aggregation. Mixtures of required oligonucleotides were prepared in Eppendorf tubes to give a ﬁnal nanostar concentration of 10 µM. Prepared mixtures were cooled from 95◦C to 20◦C at −0.05◦C min−1 using a TechneTM TC-512 ther- mocycler to enable nanostructure hybridiza- tion. Annealed samples were stored at 4◦C and used for electrophoresis and light scattering studies within a week. Supporting Information Available: The following ﬁles are available free of charge. Supporting Information Available: The following ﬁles are available free of charge. Supplementary methods, ﬁgures and DNA se- quences. This material is available free of charge via the Supplementary methods, ﬁgures and DNA se- quences. This material is available free of charge via the Internet at http://pubs.acs.org/. C-Star design and oligonulceotide preparation C-Star structures were designed using NU- PACK.66 Sequences are shown in Tables S1 and S2 of the SI. The nitrilotriacetic acid (NTA) functionalized strand was purchased from Eurogentec, and puriﬁed by the supplier using high-performance liquid chromatogra- phy (HPLC). All other oligonucleotides were purchased from Integrated DNA Technologies (IDT). Non-functionalized strands were pu- riﬁed by the supplier using standard desalt- ing, while cholesterol-functionalized strands were puriﬁed by HPLC. Cy3 and Black Hole Quencher R⃝-2 functionalized strands used for characterizing responsive C-stars (see SI) were puriﬁed by HPLC. As received dehydrated DNA was reconstituted in TE buﬀer (10 mM Tris, 1 mM EDTA, pH 8.0, Sigma Aldrich). All buﬀers were syringe-ﬁltered through 0.22 µm pore size polyethersulfone ﬁlters (Millex) prior to use. The concentration of reconstituted DNA strands was determined by measuring absorbance at 260 nm using a ThermoScien- tiﬁc Nanodrop 2000 UV-Vis spectrophotome- ter. Extinction coeﬃcients for all strands were DLS measurements were performed on a Malvern Zetasizer NanoZSP, equipped with a 633nm He-Ne laser with a maximum power of 10mW. The scattering angle was ﬁxed at 173◦. An ultra low volume quartz cuvette (ZEN2112, Malvern) was ﬁlled with 50 µL of sample syringe-ﬁltered through 0.22 µm polyvinyli- dene ﬂuoride ﬁlters (Millex), and sealed with a polypropylene cap to prevent evaporation. Size distributions shown in Fig. 1 are averaged over 3 runs, each of which consisted of 13-16 indi- vidual measurements. A Peltier heating block was used for temperature control. Preparation of C-star crystals Samples with C-star concentration of 5 µM were prepared by mixing all required strands in 10 of slice thickness 0.5 µm of 20-40 ﬁelds of view, each containing between 2-20 crystals. Pene- tration was quantiﬁed by the ratio of mean ﬂu- orescence intensity inside to outside the crys- tals, ξ, which was determined through manual masking of objects at their equitorial plane us- ing the BioFormats plugin for ImageJ.67 The background level for ξ (grey band in Fig. 4a) was determined using solute-impermeable silica beads as reference (diameter 20 µm). For experiments on trigger-induced isothermal melting (Fig. 6), samples were initially prepared as for all other C-star variants. A volume of trigger strand equivalent to a 1:1 molar ratio of trigger:bridge strand was added carefully in or- der not to disturb sedimented crystals. Directly after addition, a time series was recorded at 0.8 frames per second in transmission bright-ﬁeld mode. equimolar quantities in TE buﬀer with 300 mM NaCl. Samples were loaded into borosili- cate glass capillaries with internal section of 4 × 0.2 mm2 (CM Scientiﬁc). Capillaries were cleaned by ﬁrst sonicating in 2% Hellmanex III water solution (HellmaAnalytics) at 90◦C for 15 minutes. Surfactant was removed by two rounds of thorough rinsing and sonication in ultra-pure water (Milli-Q). Prior to ﬁlling capillaries, oligonucleotide mixtures prepared in Eppendorf tubes were heated to 95◦C us- ing a heating block to ensure complete denat- uration of any aggregates formed upon initial mixing. After loading into capillaries, a small amount of sample was removed and either side was capped oﬀwith mineral oil before sealing permanently onto a microscope slide with epoxy glue (Araldite). Sealed capillaries were then loaded into a fully programmable, Peltier con- trolled, water bath and heated to 95◦C for 30 min, before cooling to 20◦C at −0.01◦C min−1. of slice thickness 0.5 µm of 20-40 ﬁelds of view, each containing between 2-20 crystals. Pene- tration was quantiﬁed by the ratio of mean ﬂu- orescence intensity inside to outside the crys- tals, ξ, which was determined through manual masking of objects at their equitorial plane us- ing the BioFormats plugin for ImageJ.67 The background level for ξ (grey band in Fig. 4a) was determined using solute-impermeable silica beads as reference (diameter 20 µm). For experiments on trigger-induced isothermal melting (Fig. 6), samples were initially prepared as for all other C-star variants. Protein entrapment assay Entrapment of N-terminal 6× His tagged rGFP (rGFPH, ThermoFisher), and rGFP lacking a His-tag (rGFP, Sigma Aldrich), within NTA functionalized C-star networks was character- ized using a confocal microscopy assay with the same confocal setup as for solute penetration assays. An Ar-ion laser (488 nm) was used for excitation. Crystals prepared and extracted as described previously were ﬁrst washed in or- der to remove EDTA present in the incubation buﬀer. This was achieved by 5 rounds of cen- trifugation, supernatant removal, and addition and mixing with washing buﬀer, WB (10 mM tris, 300 mM NaCl, pH 8.0). For imaging, a small volume (approx. 5 µL) of washed crystals was added to a glass microwell slide. Ni2+ ions were introduced by addition of 10 µL NiCl2 con- taining WB (10 mM NiCl2, WB). After allowing to incubate for 1 hour, excess Ni2+ was removed by 5 rounds of washing. Each washing step in- volved the removal of 10 µL of solution from the well, addition of 10 µL of fresh WB, and thor- ough mixing by pipette aspiration. A 5 minute rest between washing steps was included to al- low any dispersed crystals to sediment. After the ﬁnal wash step, 5 µL of either rGFPH incu- bation buﬀer (7 µM rGFPH, 1 mg ml−1 bovine Preparation of C-star crystals A volume of trigger strand equivalent to a 1:1 molar ratio of trigger:bridge strand was added carefully in or- der not to disturb sedimented crystals. Directly after addition, a time series was recorded at 0.8 frames per second in transmission bright-ﬁeld mode. Small angle X-ray scattering SAXS measurements were performed at the I22 beamline of the Diamond Light Source using a radiation wavelength of λ = 1 Å, and beam dimensions of approximately 300 µm wide × 100 µm high. The accessible q range was be- tween 5×10−3 and 0.56 Å−1. Here the scatter- ing q vector is deﬁned as q = (4π/λ) sin θ ≡ 2π/d, where d is the d-spacing, 2θ is the scat- tering angle. The q-scale was calibrated using silver behenate, which has a well deﬁned layer spacing of 58.38 Å. Imaging and solute-penetration as- says 6), after an initial measurement, a volume of trigger strand approximately corre- sponding to a 1:1 molar ratio of trigger:bridge strand was carefully added at the top of the cap- illary and allowed to passively diﬀuse through- out the sample. Measurements were taken as for previous samples, with identical scan loca- tions for all time points. Between measure- ments, care was taken not to move the capil- lary in order to allow for direct comparison of the same pellet region over time. For experiments of protein binding in NTA functionalized networks, the same washing and rGFPH addition steps as described for the con- focal binding assay were used. Here, fresh buﬀer addition and washing was performed with gel-loading pipette tips (Corning R⃝) to allow for thorough mixing and more eﬃcient buﬀer extraction from narrow x-ray capillaries 20 locations per sample. Background subtrac- tion was performed in two steps for the SAXS patterns shown in Fig. 2, Fig. 5 and Fig. 6. Measured scattering from the buﬀer was ﬁrst subtracted, followed by subtraction of an ar- bitrary logarithmic background to account for variable scattering between glass capillaries. For experiments on trigger-induced isothermal melting (Fig. 6), after an initial measurement, a volume of trigger strand approximately corre- sponding to a 1:1 molar ratio of trigger:bridge strand was carefully added at the top of the cap- illary and allowed to passively diﬀuse through- out the sample. Measurements were taken as for previous samples, with identical scan loca- tions for all time points. Between measure- ments, care was taken not to move the capil- lary in order to allow for direct comparison of the same pellet region over time. For experiments of protein binding in NTA functionalized networks, the same washing and rGFPH addition steps as described for the con- focal binding assay were used. Here, fresh buﬀer addition and washing was performed with gel-loading pipette tips (Corning R⃝) to allow for thorough mixing and more eﬃcient buﬀer extraction from narrow x-ray capillaries. Imaging and solute-penetration as- says Confocal images were obtained using a Le- ica TCS SP5 microscope equipped with a HC PL APO CORR CS 40×/0.85 dry objective (Leica). For experiments on macromolecu- lar partitioning, a small volume (2-5 µl) of C- stars extracted from capillaries prepared as de- scribed above was loaded into a glass microw- ell slide (Hamamatsu) containing 20 µl of ﬂu- orescent macromolecule prepared in TE buﬀer with 300 mM NaCl. Samples were sealed with DNase free tape (Grace Bio-Labs FlexWell SealStrips) to prevent evaporation, and left overnight to equilibrate. For excitation of sodium-ﬂuorescein (FAM, Sigma Aldrich), ﬂuo- rescein dextran conjugates (3kDa DXT-FAM & 10kDa DXT-FAM, ThermoFisher), and recom- binant green ﬂuorescent protein (rGFP, Sigma Aldrich) an Ar-ion laser (488 nm) was used, for excitation of rhodamine B (RhodB, Sig- maAldrich) and Ar-ion laser (514 nm) was used, and a HeNe laser (633 nm) was used to im- age Alexa FluorTM 647 conjugated streptavidin (SAv-A647, ThermoFisher) containing samples. For each sample, we recorded confocal z-stacks 11 serum albumin (BSA), WB), or rGFP incu- bation buﬀer (7 µM rGFP, 1 mg ml−1 bovine serum albumin (BSA), WB) was added. Fol- lowing a 1 hour incubation, excess protein was removed by 5 rounds of washing with WB. For the demonstration of the reversibility of protein binding (Fig. 5b), bound rGFPH was removed by 5 rounds of cleaning with EDTA containing washing buﬀer (1 mM EDTA, WB). Following this, crystals could be recharged with rGFPH by repeating the same washing, nickelation, and rGFPH addition steps detailed above. Be- tween washing steps and during incubation pe- riods, sample chambers were sealed with DNase free tape to prevent evaporation. Fluorescent and bright ﬁeld confocal micrographs taken at the equatorial plane of crystals were obtained throughout the process. The degree of pro- tein binding was estimated by taking the mean ﬂuorescent intensity within the crystals, IFluo, through manual masking of objects using the BioFormats plugin for ImageJ.67 20 locations per sample. Background subtrac- tion was performed in two steps for the SAXS patterns shown in Fig. 2, Fig. 5 and Fig. 6. Measured scattering from the buﬀer was ﬁrst subtracted, followed by subtraction of an ar- bitrary logarithmic background to account for variable scattering between glass capillaries. For experiments on trigger-induced isothermal melting (Fig. References (1) Seeman, N. C. J. Theor. Biol. 1982, 99, 237–247. (2) Seeman, N. C. J. Biomol. Struct. Dyn. 1985, 3, 11–34. (3) Li, H.; Park, S. H.; Reif, J. H.; LaBean, T. H.; Yan, H. J. Am. Chem. Soc 2004, 126, 418–419. C-star crystals were prepared in capillaries as described above. The content of 6 capillar- ies per sample was extracted and concentrated by centrifugation and supernatant removal to a ﬁnal C-star concentration of ∼100 µM. Con- centrated C-Star samples were injected into borosilicate glass X-ray capillaries (diameter 1.6 mm) and left to sediment until a clear pel- let became visible. 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Cell Biol. 2010, 189, 777–782. 15 Graphical TOC Entry l 14bp 7-37bp Cholesterol DNA Amphiphilic DNA motif... ...forms 3D single crystals 30 μm Robust to design changes Defined arm length Controlled lattice parameter Controlled porosity Chemical modification Specific & reversible binding of target protein Embedded functionality Isothermal melting through strand displacment Graphical TOC Entry Graphical TOC Entry l 14bp 7-37bp Cholesterol DNA Amphiphilic DNA motif... ...forms 3D single crystals 30 μm Robust to design changes Defined arm length Controlled lattice parameter Controlled porosity Chemical modification Specific & reversible binding of target protein Embedded functionality Isothermal melting through strand displacment l Robust to design changes Amphiphilic DNA motif... ...forms 3D single crystals 16
https://openalex.org/W4309493448	https://essd.copernicus.org/articles/14/5061/2022/essd-14-5061-2022.pdf	English	null	In situ observations of the Swiss periglacial environment using GNSS instruments	Earth system science data	2,022	cc-by	19,939	Earth Syst. Sci. Data, 14, 5061–5091, 2022 https://doi.org/10.5194/essd-14-5061-2022 © Author(s) 2022. This work is distributed under the Creative Commons Attribution 4.0 License. Earth Syst. Sci. Data, 14, 5061–5091, 2022 https://doi.org/10.5194/essd-14-5061-2022 © Author(s) 2022. This work is distributed under the Creative Commons Attribution 4.0 License. Earth Syst. Sci. Data, 14, 5061–5091, 2022 https://doi.org/10.5194/essd-14-5061-2022 © Author(s) 2022. This work is distributed under the Creative Commons Attribution 4.0 License. Published by Copernicus Publications. A. Cicoira, S. Weber et al.: GNSS observations of the Swiss periglacial environment and steep rock walls) at altitudes ranging from 2304 to 4003 ma.s.l. and spread across the Swiss Alps. The primary data products consist of raw GNSS observables in RINEX format, inclinometers, and weather station data. Additionally, cleaned and aggregated time series of the primary data products are provided, including daily GNSS positions derived through two independent processing tool chains. and steep rock walls) at altitudes ranging from 2304 to 4003 ma.s.l. and spread across the Swiss Alps. The primary data products consist of raw GNSS observables in RINEX format, inclinometers, and weather station data. Additionally, cleaned and aggregated time series of the primary data products are provided, including daily GNSS positions derived through two independent processing tool chains. The observations documented here extend beyond the dataset presented in the paper and are cur- rently continued with the intention of long-term monitoring. An annual update of the dataset, available at https://doi.org/10.1594/PANGAEA.948334 (Beutel et al., 2022), is planned. With its future continuation, the dataset holds potential for advancing fundamental process understanding and for the development of applied methods in support of e.g. natural hazard management. In situ observations of the Swiss periglacial environment using GNSS instruments Weber et al.: GNSS observations of the Swiss periglacial environment A. Cicoira, S. Weber et al.: GNSS observations of the Swiss periglacial environment 1 GNSS monitoring in high mountain environments sensor stations, their technical speciﬁcations, an overview of the local geodetic network, and of the data communication system used to collect the data in near real time. The ob- servation positions are distributed throughout the Swiss Alps and range in elevation from 2304 to 4003 ma.s.l.. The data cover sites with differing geomorphological characteristics including rock glaciers (ice-rich creeping landforms indicat- ing the occurrence of permafrost), permafrost-affected land- slides, and steep rock walls. The ﬁeld sites are presented in Sect. 3 and in more detail in Appendix A. The primary data of this paper are the raw GNSS observables of all GNSS sta- tions, accompanied by two-axis inclinometer measurements as well as weather station data for some of the investigated sites. The published dataset contains the complete raw data at full sampling rates of all instruments (primary dataset; see Sect. 4) as well as a selection of derived data products (sec- ondary dataset; see Sect. 5). The derived data products are down-sampled and cleaned time series of weather station and inclinometer data as well as post-processed GNSS daily posi- tions obtained by double-difference processing of the GNSS data. A short discussion about validity and uncertainty of pri- mary and derived data products is given in Sect. 6. In Ap- pendix B, we brieﬂy describe the different research projects that contributed to the consortium, to the development of the sensors, and to the evolution of the ﬁeld-site monitoring net- work. The history of the consortium is tightly linked to the success of the method, and it can not be illustrated without mentioning its practical applications in the ﬁelds of natu- ral hazard management, e.g. in the Swiss Permafrost Mon- itoring Service (PERMOS). A toolset, originally created for the data describing the Matterhorn Hörnligrat ﬁeld-site data (Weber et al., 2019a), allows us to (re-)create and indepen- dently update (living data process) the data documented in this paper. The code, ﬁrst presented in Weber et al. (2019b), is updated and available at https://git.uibk.ac.at/informatik/ neslab/public/permasense/permasense_datamgr (last access: 21 October 2022, Weber et al., 2022). 1 GNSS monitoring in high mountain environments We expect and en- courage the use of this dataset to further develop process- ing methods, provide atmospheric (Hurter et al., 2012) and ground-based (Henkel et al., 2018) observations as well as for educational purposes, but we also foresee future research sensor stations, their technical speciﬁcations, an overview of the local geodetic network, and of the data communication system used to collect the data in near real time. The ob- servation positions are distributed throughout the Swiss Alps and range in elevation from 2304 to 4003 ma.s.l.. The data cover sites with differing geomorphological characteristics including rock glaciers (ice-rich creeping landforms indicat- ing the occurrence of permafrost), permafrost-affected land- slides, and steep rock walls. The ﬁeld sites are presented in Sect. 3 and in more detail in Appendix A. The primary data of this paper are the raw GNSS observables of all GNSS sta- tions, accompanied by two-axis inclinometer measurements as well as weather station data for some of the investigated sites. The published dataset contains the complete raw data at full sampling rates of all instruments (primary dataset; see Sect. 4) as well as a selection of derived data products (sec- ondary dataset; see Sect. 5). The derived data products are down-sampled and cleaned time series of weather station and inclinometer data as well as post-processed GNSS daily posi- tions obtained by double-difference processing of the GNSS data. A short discussion about validity and uncertainty of pri- mary and derived data products is given in Sect. 6. In Ap- pendix B, we brieﬂy describe the different research projects that contributed to the consortium, to the development of the sensors, and to the evolution of the ﬁeld-site monitoring net- work. The history of the consortium is tightly linked to the success of the method, and it can not be illustrated without mentioning its practical applications in the ﬁelds of natu- ral hazard management, e.g. in the Swiss Permafrost Mon- itoring Service (PERMOS). A toolset, originally created for the data describing the Matterhorn Hörnligrat ﬁeld-site data (Weber et al., 2019a), allows us to (re-)create and indepen- dently update (living data process) the data documented in this paper. The code, ﬁrst presented in Weber et al. (2019b), is updated and available at https://git.uibk.ac.at/informatik/ neslab/public/permasense/permasense_datamgr (last access: 21 October 2022, Weber et al., 2022). In situ observations of the Swiss periglacial environment using GNSS instruments Alessandro Cicoira1,2,3,⋆, Samuel Weber1,4,5,6,7,⋆, Andreas Biri4, Ben Buchli4, Reynald Delaloye2, Reto Da Forno4, Isabelle Gärtner-Roer1, Stephan Gruber8, Tonio Gsell4, Andreas Hasler9, Roman Lim4, Philippe Limpach10,18, Raphael Mayoraz11, Matthias Meyer4, Jeannette Noetzli6,7, Marcia Phillips6,7, Eric Pointner12, Hugo Raetzo13, Cristian Scapozza14, Tazio Strozzi15, Lothar Thiele4, Andreas Vieli1, Daniel Vonder Mühll16, Vanessa Wirz1,19, and Jan Beutel17 1Department of Geography, University of Zurich, Zurich, Switzerland 2Department of Geosciences, University of Fribourg, Fribourg, Switzerland 3School of Architecture, Civil and Environmental Engineering, Swiss Federal Institute of Technology, Lausanne, Switzerland 4Computer Engineering and Networks Laboratory, ETH Zurich, Zurich, Switzerland 5Chair of Landslide Research, Technical University of Munich, Munich, Germany 6WSL Institute for Snow and Avalanche Research SLF, Davos, Switzerland 7Climate Change, Extremes and Natural Hazards in Alpine Regions Research Center CERC, Davos Dorf, Switzerland 8Department of Geography and Environmental Studies, Carleton University, Ottawa, Canada 9SensAlpin GmbH, Davos, Switzerland 10Terradata AG, Zurich, Switzerland 11Ct. Valais, Sion, Switzerland 12Rovina und Partner AG, Visp, Switzerland 13Federal Ofﬁce for the Environment FOEN, Ittigen, Switzerland 14Institute of Earth Sciences, University of Applied Sciences and Arts of Southern Switzerland (SUPSI), Mendrisio, Switzerland 15GAMMA Remote Sensing and Consulting AG, Gümlingen, Switzerland 16Personalized Health and Related Technologies, ETH Zurich, Zurich, Switzerland 17Department of Computer Science, University of Innsbruck, Innsbruck, Austria 18Institute of Geodesy and Photogrammetry, ETH Zurich, Zurich, Switzerland 19Pro Natura Schaffhausen, Schaffhausen, Switzerland ⋆These authors contributed equally to this work. Correspondence: Alessandro Cicoira (alessandro.cicoira@geo.uzh.ch) and Samuel Weber (samuel.weber@slf.ch) Received: 21 May 2021 – Discussion started: 11 June 2021 Revised: 13 July 2022 – Accepted: 19 August 2022 – Published: 18 November 2022 Abstract. Monitoring of the periglacial environment is relevant for many disciplines including glaciology, nat- ural hazard management, geomorphology, and geodesy. Since October 2022, Rock Glacier Velocity (RGV) is a new Essential Climate Variable (ECV) product within the Global Climate Observing System (GCOS). How- ever, geodetic surveys at high elevation remain very challenging due to environmental and logistical reasons. During the past decades, the introduction of low-cost global navigation satellite system (GNSS) technologies has allowed us to increase the accuracy and frequency of the observations. Today, permanent GNSS instruments enable continuous surface displacement observations at millimetre accuracy with a sub-daily resolution. In this paper, we describe decennial time series of GNSS observables as well as accompanying meteorological data. The observations comprise 54 positions located on different periglacial landforms (rock glaciers, landslides, Published by Copernicus Publications. 5062 A. Cicoira, S. 2.1 GNSS sensor instruments Each location is equipped with a station consisting of an inte- grated GNSS sensor, a 12 V solar power system mounted on and housed inside a ﬁbreglass reinforced tubular mast, and anchored to the underlying rock (see left panel in Fig. 1). The sensor systems consist of an active GNSS antenna, a GNSS receiver, a two-axis inclinometer, a data logger, and in many cases, a low-power wireless transmission system (see right panel in Fig. 1). Typically, the sensor is mounted on top of a rock outcrop or a boulder large enough for a sta- ble positioning (see Sect. 3.3 for more details). Power for all instruments is provided by a standard 12 V photovoltaic sys- tem: a solar panel, a charge controller, and a 12 V absorbed glass mat (AGM) sealed lead-acid battery housed inside a Pelicase. In order to guarantee maximum exposure to solar radiation, facilitate operation with extended snow cover in winter, and protect the instrument, the instrument is mounted inside the top of the ﬁbreglass mast. All wires are routed in- side the tubular mast. 2.2 Two-axis inclination sensors Two-axis inclinometers (Murata SCA830) are integrated in the sensor systems that are aligned with a marking on the mast mount. This allows a direct determination of the rota- tion along two axes and subsequently allows us to correct the GNSS measurements for the tilt of the mast, i.e. cor- rect for rotational movements, common in the active layer of permafrost landforms (Wirz et al., 2013; Cicoira et al., 2021). Experience has shown that this correction can yield favourable results and provides the coordinates of the mount- ing point (Wirz et al., 2014a), but in most cases, the exact point of rotation still remains unclear because the geometry of the rotating mass (where the station is mounted) is un- known. 1 GNSS monitoring in high mountain environments We expect and en- courage the use of this dataset to further develop process- ing methods, provide atmospheric (Hurter et al., 2012) and ground-based (Henkel et al., 2018) observations as well as for educational purposes, but we also foresee future research Observations based on permanent in situ global navigation satellite system (GNSS) instruments have been investigated intensively over the past decade in pursuit of a better pro- cess understanding of the periglacial environment and its related mass movements (Wirz et al., 2013; Ravanel and Deline, 2014; Cicoira et al., 2019b). The advantage of this method for assessing surface displacements over traditional ﬁeld surveying and remote-sensing techniques is the level of detail that can be obtained at different temporal scales. Today it is possible to monitor at millimetre accuracy with a temporal resolution of minutes, using double-difference processing techniques (Teunissen and Montenbruck, 2017) and commodity receiver hardware similar to those found in consumer products, e.g. mobile phones or automotive sys- tems (Paziewski et al., 2021). Depending on the level of de- tail required, chieﬂy characterised by the accuracy of coor- dinate tuples, their temporal resolution is achieved by op- erating GNSS receiver pairs continuously and subsequently post-processing the observation data derived. While the cost footprint of the required hardware is several orders of mag- nitude lower than that required for traditional geodetic sur- veying equipment (Wirz et al., 2013), the power and com- munication bandwidth required for operating the sensors re- motely remain high. However, it has been shown that by se- lectively duty cycling receivers and transmitting the GNSS observation data on a low-power wireless sensor network (WSN) (Buchli et al., 2012), a trade-off between energy/data volumes and the ﬁdelity required can be obtained. In this way, near-real-time permanent monitoring in remote loca- tions, over large timescales, and in adverse meteorological conditions become possible. In this paper, we document a dataset of continuous GNSS observations of 54 positions obtained in the periglacial en- vironment of the Swiss Alps over the past 14 years in the framework of the X-Sense Project and the PermaSense con- sortium. PermaSense has been a large interdisciplinary re- search consortium targeted to custom-design low-cost wire- less sensors for the detection and analysis of temporal and spatial variability of high-alpine slope movements (Beutel et al., 2011). In Sect. 2, we provide a description of the GNSS https://doi.org/10.5194/essd-14-5061-2022 Earth Syst. Sci. Data, 14, 5061–5091, 2022 5063 A. 1 GNSS monitoring in high mountain environments Cicoira, S. Weber et al.: GNSS observations of the Swiss periglacial environment applications in the ﬁelds of geomorphology, engineering ge- ology, and natural hazard management. SCA830), an ambient temperature/humidity sensor (Sen- sirion SHT31), and power supervisory circuits are integrated. In order to reduce the necessary power footprint, e.g. at times of reduced solar radiation for energy harvesting, a data-acquisition schedule can be set on each device individu- ally. This sampling schedule has hourly granularity, and time windows when the system is running are typically centred at 12:00:00 UTC to facilitate overlapping time intervals for double-difference GNSS processing. 2 Instrumentation technology and data management The measurement network consists of distributed GNSS sta- tions that are permanently installed on locations of interest, i.e. on the surface of a landform under investigation. The GNSS sensor and the ﬁx installations were designed in the framework of the PermaSense Project; both are depicted in Fig. 1. In addition, a MEMS two-axis inclinometer has been fully integrated into the sensor system. At selected locations, automatic weather stations provide auxiliary ambient data that are valuable for the analysis of the GNSS observations. In the following paragraphs, we provide a brief description of the different sensors (GNSS, inclinometers, weather stations) as well as the data communication and management infras- tructure. All sensor types, including their indicative period of operation, unit system, and key characteristics are synthe- sised in Table 1. In 2010, a proof-of-concept study lead to the development of the ﬁrst generation of these instruments, which were ca- pable of logging data to internal SD-card storage only (Wirz et al., 2013). This ﬁrst prototype was subsequently reﬁned into two wireless systems: one focused on experimentation (Beutel et al., 2011), the second being a fully integrated wire- less GNSS sensor (Buchli et al., 2012). This two-step ap- proach allowed for a fast startup of the project, which was initially independent of infrastructure and network coverage. Over time, most of the sensor locations (initially equipped with data loggers only) have been equipped with wireless data transmission, facilitating sensor operation and near-real- time data retrieval (see Tables A1–A6). The data availability is visualised in Fig. A1. 2.1 GNSS sensor instruments https://doi.org/10.5194/essd-14-5061-2022 A. Cicoira, S. Weber et al.: GNSS observations of the Swiss periglacial environment 5064 Figure 1. Structure and components of the GNSS sensor developed in the PermaSense Project. (Left) Structure of the GNSS station. (Right) Detail of the embedded GNSS antenna, data logger, and wireless transmitter. Figure 1. Structure and components of the GNSS sensor developed in the PermaSense Project. (Left) Structure of the GNSS station. (Right) Detail of the embedded GNSS antenna, data logger, and wireless transmitter. Table 1. Overview list of the sensors used, ordered by sensor type. n.a. denotes not available. Table 1. Overview list of the sensors used, ordered by sensor type. n.a. denotes not available. Sensor type Sensor Unit Interval Accuracy L1/L2-GNSS; Leica GRX1200+, AR10/25 antenna m 30 s n.a. L1-GPS; u-blox LEA-6T, Trimble Bullet III m 5, 30 s n.a. Inclination Murata SCA830-D07 Inclinometer ◦ 120 s ±30 mg Air temperature Vaisala WXT520 ◦C 120 s ±0.3 ◦C Barometric pressure Vaisala WXT520 hPa 120 s ±1 hPa Relative humidity Vaisala WXT520 % RH 120 s ±3–5 % RH Wind speed Vaisala WXT520 kmh−1 120 s ±3 % at 10 ms−1 Wind direction Vaisala WXT520 ◦ 120 s ±3 ◦at 10 ms−1 Precipitation Vaisala WXT520 mm 120 s Resolution 0.01 mm Radiation Kipp & Zonen CNR4 Wm−2 120 s Non-linearity < 1 % plied in the data-conversion procedures as advised by the manufacturer. LAN (WLAN) backbone and a ﬁne distribution using a low- power WSN was sought. The topology of the wireless data- communication backbone in the Matter Valley is illustrated in Fig. A6. Using this approach, it is possible to reach back into areas in the back of side valleys that are interesting from the monitoring perspective, but not covered with regards to connectivity. All locations serviced by the WLAN backbone have Internet Protocol (IP) connectivity facilitating the use of standard sensing equipment. For ease of maintenance, a local WLAN hotspot is also available at these sites for all kinds of activities that require an internet connection. In selected lo- cations, standard 3G/4G modems were further implemented as a backup to the network communication system. plied in the data-conversion procedures as advised by the manufacturer. 2.3 Auxiliary weather instruments A GNSS sensor has been installed on stable terrain at se- lect positions in order to provide a stable reference for the double-difference GNSS post-processing. These additional positions ensure a shorter baseline than what would be ob- tained by using the national reference network. Moreover, these reference stations act as the sink of the local WSN and host the instrumentation for the data collection from all GNSS measurement points. More information is provided in Sect. 3.2. A Vaisala WXT520 compact all-in-one weather station was installed at select locations to obtain a more detailed record of local weather data. The measured variables include am- bient air temperature, air pressure, relative humidity, wind (speed and direction), and liquid precipitation. At two lo- cations, a four-component net radiometer (Kipp & Zonen CNR4) was installed without capabilities for ventilation and heating. The WXT520 is capable of heating the rain and wind sensor; but for practical reasons, this feature is only en- abled when enough power is available, which typically corre- sponds to good weather periods and turned off especially in prolonged bad-weather periods. Both instruments have been vendor-calibrated and the respective calibration data are ap- The GNSS sensor instruments are based on commodity L1-GNSS receivers with the extended capability to output the raw satellite observables (u-blox LEA-6T) and an ac- tive antenna (Trimble Bullet III) (Wirz et al., 2013; Buchli et al., 2012). Furthermore, a two-axis inclinometer (Murata https://doi.org/10.5194/essd-14-5061-2022 Earth Syst. Sci. Data, 14, 5061–5091, 2022 3.1.2 Steep rock walls Three ﬁeld sites are focussing on steep bedrock. The Matter- horn Hörnligrat (MH), the Randa Grossgufer (RA), and the Sattelspitz (SA) north-west ridge are all located in the Matter Valley. All sites are particularly relevant from the perspective of natural hazard management. The Hörnligrat, despite its re- moteness, has an important social and economic component due to all the tourist activities that take place on the Matter- horn. The Grossgufer is the remaining lip of the large and 3.1.1 Rock glaciers Eleven rock glaciers across the Swiss Alps have been instru- mented with permanent GNSS sensors. A cluster of ﬁve rock glaciers is concentrated in the right orographic side of the Matter Valley: Dirru (DI), Distelhorn (DIS), Gugla-Bielzug (BH) (positions 10 and 13), Ritigraben (RIT), Steintälli (ST). In the neighbouring Saas Valley, above the village of Saas- Baalen, the rock glaciers Gruben (GRU) and Jaegi (JAE) host two more GNSS stations. These valleys are rich in rock glaciers, many of which move relatively fast, with average displacement rates of up to several metres per year (Delaloye et al., 2013; Strozzi et al., 2020). In order to consistently manage and document the ﬁeld site and all the sensors with homogeneous metadata, a set of rules has been deﬁned: – An individual protocol sheet is used for each interven- tion (ﬁeld work day) where all noteworthy items are recorded (installation, maintenance, removal). Some rock glaciers are being monitored under the auspices of the PERMOS (Noetzli et al., 2019, kinematics data are available online http://www.permos.ch, last access: 21 Oc- tober 2022). They include Ritigraben (RIT1) and Gruben (GRU1) in the Valais, the Stabbio di Largario (LAR1 and LAR2) in Val Blenio, and a second large cluster in the upper Engadine, consisting of the rock glaciers Murtèl-Corvatsch (COR1), Muragl (MUA1), and Schafberg (SCH1). Engadine and the Matter Valley are the two driest valleys in Switzer- land and are characterised by high mountain peaks. For these reasons, they host the majority of rock glaciers in the country (Haeberli and Vonder Mühll, 1996). Many of the sites, espe- cially the PERMOS locations, are also monitored by terres- trial geodetic surveys. The relative data can be obtained from the responsible institutions and from the PERMOS ofﬁce. – Sensor interventions on site take place at different times for each position. To simplify things, the whole day of an intervention is typically assumed to be invalid data. – All sensor devices are mapped to a distinct position ID. The mapping contains start and ﬁnish time stamps, the device ID of the sensor, sensor type, and calibration data. – All data from a speciﬁc data source (sensor type) are kept in an individual data structure. Queries are typi- cally made per data type and position ID. – Detailed circumstances (orientation angles, status of the station) are recorded using auxiliary data formats: text ﬁles, Excel ﬁles or photographs. 2.4 Near-real-time data communication The locations monitored in the scope of the research and the data presented in this paper are located in high-alpine areas, where cellular network coverage is limited or unavailable. Some ﬁeld sites have been instrumented following a long- term monitoring strategy, where retrieval of the data once or twice a year is sufﬁcient for the continuation of the time series and their analysis. However, many of the GNSS sta- tions, especially in the Mattertal, are particularly relevant from a natural hazard perspective, and timely data commu- nication is essential for their applicability to management strategies. For this reason, large effort was put into the de- sign and implementation of a data-communication system in near real time. A unique solution comprising of a wireless Since WLAN equipment serving the communication backbone is energy-consuming, the actual sensors are inte- grated with a custom low-power WSN operating on ISM- band radio transceivers and a specialised communication protocol for data gathering (Beutel et al., 2009). Using a hier- https://doi.org/10.5194/essd-14-5061-2022 Earth Syst. Sci. Data, 14, 5061–5091, 2022 5065 A. Cicoira, S. Weber et al.: GNSS observations of the Swiss periglacial environment are located in the periglacial environment or in its proximity. We classify the instrumented landforms into three types: (i) rock glaciers, (ii) landslides, and (iii) steep rock walls. In this section, we present an overview of the landform types and instrumented ﬁeld sites. A more detailed description of the investigated landforms, accompanied by some scientiﬁc ref- erences which can be used as a starting point for additional information, is given in Appendix A. All the landforms and their types are illustrated in Fig. 2 alongside the number of the GNSS sensors installed (details can be investigated in the Supplement of the paper). An example of a small-scale local geodetic network and the wireless communication geometry are provided in Sect. 3.2. archical system of commodity components for the backbone and a highly optimised and custom network for the ﬁne distri- bution to spatially separated sensing locations, it is possible to operate on a more reduced resource footprint, over long periods of time, and minimising the number of interventions. https://doi.org/10.5194/essd-14-5061-2022 2.5 Data-management infrastructure All data collected are organised and stored so that repro- ducible research and reuse of the data in different contexts and in future projects are possible. The data infrastructure must also allow for ﬂexibility with respect to extensions (e.g. integrating new sensor types), support of different sampling rates, metadata integration and life-cycle management. The data back-end is implemented using a data-streaming mid- dleware, where a dedicated processing structure called a vir- tual sensor is responsible for processing a speciﬁc data type, e.g. one virtual sensor for temperature measurements and an- other virtual sensor for GNSS positions. Complete process- ing chains can be implemented by concatenating virtual sen- sors, either within the same instance of the Global Sensor Network (GSN; Aberer et al., 2006) or also across multiple instances of the GSN. More details about data processing and storing are given in Weber et al. (2019a). 3.1.3 Landslides Five permafrost-affected landslides have been instrumented in the Matter Valley with the goal of monitoring unstable and potentially dangerous slopes: (i) the south-west ﬂank of the Breithorn (BH), (ii) one of the culminating peaks of the Mischabel ridge, (iii) the west ﬂank of the Gugla sum- mit, locally called Längenschnee (LS), (iv) the Grabengufer (GG) landslide above the village of Randa, and (v) the Wisse Schijen (WYS) on the eastern ﬂank of the Weisshorn, di- rectly facing it on the other side of the valley. All these sites are well-known by the local population and the cantonal au- thorities for a long-lasting history of displacement and in some cases for hazard phenomena. The Längenschnee and the Grabengufer landslides are particularly interesting from a historical and geomorphological perspective, respectively. More details can be found in Appendices A and B. Based on the aforementioned factors, a set of local refer- ence positions has been deﬁned and equipped with continu- ously operating GNSS receivers as well as high-bandwidth data transmission (see Table 2) forming a local geodetic ref- erence network. In cases where only few or single observa- tion points have been deployed, stations from the permanent GNSS network in Switzerland (AGNES) serve as a refer- ence. Periodic checks against the national GNSS network have shown that all but two (Längenschnee/RL01 and Hörn- ligrat/HOGR) reference locations are stable and do not ex- hibit local movements (see Fig. A2). The station RL01 was abandoned after the discovery that the ridge where it is lo- cated is actually moving at a rate of a few centimetres per year, contrary to the assumptions made at an initial site sur- vey. The reference station HOGR represents a special case since it is located on a moving buttress on the Matterhorn Hörnligrat Ridge. In this case however, due to the extreme nature of the environment in which the station is installed, no stable terrain at the same elevation is available. Therefore, the 3 Field sites The dataset described can be divided into ﬁve main geo- graphical areas. Moving from west to east, they are (i) the Matterhorn Hörnli Ridge, (ii) the main trunk of the Matter Valley, (iii) the upper Saas Valley in the Canton of Valais, (iv) Stabbio di Largario in Ticino, and (v) the upper Engadin Valley in Canton Graubünden. All of the ﬁeld sites monitored https://doi.org/10.5194/essd-14-5061-2022 Earth Syst. Sci. Data, 14, 5061–5091, 2022 A. Cicoira, S. Weber et al.: GNSS observations of the Swiss periglacial environment A. Cicoira, S. Weber et al.: GNSS observations of the Swiss periglacial environment 5066 Figure 2. Overview map of the ﬁeld sites. The ﬁeld sites are divided into ﬁve major geographical and geomorphological areas, for which a panel provides a more detailed sight of the sensor network. Further details can be investigated in the Supplement of the paper. Satellite imagery from the Swiss Federal Ofﬁce of Topography, swisstopo. Figure 2. Overview map of the ﬁeld sites. The ﬁeld sites are divided into ﬁve major geographical and geomorphological areas, for which a panel provides a more detailed sight of the sensor network. Further details can be investigated in the Supplement of the paper. Satellite imagery from the Swiss Federal Ofﬁce of Topography, swisstopo. famous Randa rockslide which took place in 1991. The Sat- telspitz north-west ridge is a currently accelerating buttress, potentially endangering some constructions and activities in the Täsch-Alp. (Wirz et al., 2013; Bu et al., 2021). For the chosen approach using single-frequency GNSS receivers, a short baseline on the order of hundreds of metres to a few kilometres maxi- mum, is favourable. Furthermore, the non-moving reference position and the rover receiver should be situated in the same altitude regime, exhibit similar shading by topographic ob- stacles (sky view), and need to be operated in overlapping time windows in order to generate the best results. https://doi.org/10.5194/essd-14-5061-2022 3.2 Local geodetic network The most accurate position data can be computed using dif- ferential GNSS processing techniques (see Sect. 5) when cor- rection data from a GNSS receiver on a non-moving position are available in close proximity to the rover (short baseline) https://doi.org/10.5194/essd-14-5061-2022 Earth Syst. Sci. Data, 14, 5061–5091, 2022 A. Cicoira, S. Weber et al.: GNSS observations of the Swiss periglacial environment 5067 Table 2. Local geodetic network: reference stations: RD01 – Reference Dirru 01, RL01 – Reference Längenschnee 01, RG01 – Reference Grabengufer 01, RAND – Reference Randa, HOGR – Hörnligrat Reference. Reference Area Topographic Feature Sensor Period RD01 Next to Dirru rock glacier Large bedrock feature u-blox LEA-6T, Trimble Bullet III March 2011–ongoing RL01 Between Längenschnee and Breithorn Fractured ridge u-blox LEA-6T, Trimble Bullet III August 2011–May 2013 RG01 Above Grabengufer rock glacier Bedrock on ridge u-blox LEA-6T, Trimble Bullet III September 2011–ongoing RAND Top of Grossgufer Bedrock above rockfall Leica GRX1200+, AR25 antenna May 2011–ongoing HOGR Hörnligrat ridge Rock buttress on ridge Leica GRX1200+, AR10 antenna December 2010–ongoing Table 2. Local geodetic network: reference stations: RD01 – Reference Dirru 01, RL01 – Reference Länge Grabengufer 01, RAND – Reference Randa, HOGR – Hörnligrat Reference. ii. From a geomorphological perspective, the chosen point must be representative of the investigated process. As explained in Wirz et al. (2014b) and later in Cicoira et al. (2021), the velocity measured at the surface of a periglacial landform is typically the result of three super-imposed signals. On bedrock, the representatives of the selected point is directly connected with the struc- tural geology of the site, making the selection less prone to personal interpretation. In order to minimise the noise due to stochastic movement within the active layer and at the terrain surface, it is favourable to choose large boulders well embedded in the active layer (with two metaphors: a stable boat in water or a tooth with a deep root; see Fig. 3). The additional two-axis incli- nation measurements allow us to retrieve critical infor- mation about the three-dimensional nature of the roto- translation of the surveyed point (not known a priori) and with the use of proper post-processing analysis, re- duce the relative error associated in the measurements (Wirz et al., 2014a). reference position is maintained, but the observations have to be corrected for the movement of the reference position be- fore further analysis, as explained in more detail in Weber et al. 3.3 Field site selection and prerequisites for in situ GNSS sensor installation by a rockfall (see Fig. A7b) or a snow avalanche. In this case, the posi- tion has to be replaced. A location as close as possible and with similar characteristics has to be chosen in or- der to minimise the inﬂuence of spatial variability and guarantee (as much as possible in this unwanted condi- tion) data homogeneity and the continuation of the time series. Within the PermaSense project, the installation of an in situ GNSS sensor on an observation point had to com- ply with minimum technical and geomorphological require- ments, which can be met fairly easy, depending on the ﬁeld site. i. From a technical perspective, good visibility to the hori- zon in direction of the Equator (southern sky when working in the Northern Hemisphere) is required both for good and persistent satellite visibility as well as for maximum solar radiation for power generation (see Fig. 3). Where different positions are equivalent with regard to the other criteria, the technical feasibility of the measurements can be used as a discriminant for a decision. In case of abundant snow cover or large peri- ods without sunlight, a tall mast and conservative power set-ups are critical. 3.2 Local geodetic network (2019a). 3.3 Field site selection and prerequisites for in situ GNSS sensor installation 3.3 Field site selection and prerequisites for in situ GNSS sensor installation The variety of the ﬁeld sites presented in this paper is the re- sult of the historical development of the PermaSense project (a short summary is given in Appendix B). The multitude of measuring points and ﬁeld sites presented embodies the variety of applications that the project has served over time. Natural hazard management applications are often very well constrained in the selection of the site and the single loca- tion of the measurement positions, as these are the relevant ones from the hazard perspective. On the other hand, long- term climatic monitoring requires more thorough thinking and preliminary investigations to select the best-suited ﬁeld sites ﬁrst and then the single measurement points. It is impor- tant to note that the measurements provide a detailed descrip- tion of the kinematic behaviour of surface boulder, which are only a proxy of deep-seated geomorphological processes. The results have to be evaluated with care and expert knowl- edge; see e.g. Cicoira et al. (2021) for the components of rock glacier velocities. In this paragraph, we provide a brief overview of three guiding criteria (technical and geomorpho- logical) that all the GNSS positions have to respect. iii. The longevity of the measuring point must be antici- pated prior to installing the GNSS station. In extreme cases with blocks sliding on the surface or strongly ro- tating above the surface, it may be required to re-level the GNSS instrument back to vertical after some time (see Fig. A7a). Finally, it may happen that a station is severely damaged or destroyed, e.g. by a rockfall (see Fig. A7b) or a snow avalanche. In this case, the posi- tion has to be replaced. A location as close as possible and with similar characteristics has to be chosen in or- der to minimise the inﬂuence of spatial variability and guarantee (as much as possible in this unwanted condi- tion) data homogeneity and the continuation of the time series. iii. The longevity of the measuring point must be antici- pated prior to installing the GNSS station. In extreme cases with blocks sliding on the surface or strongly ro- tating above the surface, it may be required to re-level the GNSS instrument back to vertical after some time (see Fig. A7a). Finally, it may happen that a station is severely damaged or destroyed, e.g. 4.2 Inclinometer data Almost all L1-GPS sensors are equipped with an integrated two-axis inclinometer based on a MEMS component (Mu- rata SCA830-D07). An example of the inclinometer data for station BH13 is given in Fig. 4. These are sampled at 2 min intervals during periods at which the GNSS sensor is run- ning. The schedule for the inclinometer data acquisition is the same as for the GNSS sensor. Data are provided in CSV ﬁles. The data can be used to constrain the rotational move- ment of the sensor around a centre of rotation, which in prin- ciple is not known. Wirz et al. (2014b) provided a framework to use this information to correct the position measurement on the ground for the component of the rotation between the anchor and the sensor (given by the mast height). For some applications (e.g. Leinauer et al., 2022), the inclinometer data have been proven to be a valuable proxy of more expensive position/displacement measurements. The sensor labels are given due to historical reasons and follow a geographical and chronological order. Each sensor is given a four-character alphanumeric label, with two or three letters indicating the ﬁeld site (as illus- trated in Sect. 3) and the number identifying a given sensor on a ﬁeld site. The labelling of the data ﬁles consists of this four-character alphanumeric label preﬁx- ing a string for the sensor type, and a string indicat- ing the time period of the measurements included in the ﬁle: e.g. BH10_gps_inclinometer_2018.csv con- tains GPS inclinometer data for the year 2018 for the position Breithorn 10. More information on the structure of the data repository is provided in Sect. 7 and Table 3. 4 Primary data products The mast height indi- cates the height of the antenna above the rock instrumented. Additional to this basic information, the ﬁeld “online data” allow us to identify all the sensors that are currently con- nected with the online data-management infrastructure via the near-real-time WSN (see Sect. 2.4 for more details). Un- der the ﬁeld “location”, the coordinates of the station at the time of deployment are indicated in the EPSG:2056 – Swiss CH1903+ / LV95 coordinate system. The current coordinates are available in the dataset. Finally, two ﬁelds are used to il- lustrate the sensors installed at each station. We distinguish between kinematics for all GNSS stations, including optional inclinometers, and automatic weather stations. A more de- tailed overview of the data availability for all sensors is given in Fig. A1, where all operating periods, data gaps, and dis- continued sensors are visualised. allows for more ﬂexible analysis, especially for the explo- ration of real-time kinematic applications. As explained in Sect. 2.1, the GNSS rovers employed (Wirz et al., 2013; Buchli et al., 2012) are not continuously sampling data at the given rate. In order to reduce energy consumption, the data generation duration can be conﬁgured based on a user- deﬁned daily schedule. The schedule is deﬁned using hourly granularity with a minimum duration of 1 h per day. 4 Primary data products In this section, we present the primary data products pro- vided within the scope of this paper. The primary data prod- ucts are the raw ﬁles as provided by the sensors. They com- prise GNSS observables in the form of compressed RINEX ﬁles, as well as inclinometer and weather station data (CSV ﬁles). Technical speciﬁcations about the sensors are given https://doi.org/10.5194/essd-14-5061-2022 Earth Syst. Sci. Data, 14, 5061–5091, 2022 5068 A. Cicoira, S. Weber et al.: GNSS observations of the Swiss periglacial environment A. Cicoira, S. Weber et al.: GNSS observations of the Swiss periglacial environment Figure 3. Two GNSS stations mounted atop large boulders on the Breithorn landslide 2982 ma.s.l., Herbriggen (a) and on the Largario Rock Glacier 2355 ma.s.l., Val di Blenio (b) illustrate how a ﬁeld deployment including all sensor setup and anchoring appears. Figure 3. Two GNSS stations mounted atop large boulders on the Breithorn landslide 2982 ma.s.l., Herbriggen (a) and on the Largario Rock Glacier 2355 ma.s.l., Val di Blenio (b) illustrate how a ﬁeld deployment including all sensor setup and anchoring appears. Glacier 2355 ma.s.l., Val di Blenio (b) illustrate how a ﬁeld deployment including all sensor setup and anchoring appears. Figure 4. A decade of air temperature and precipitation from weather station DH13, as well as displacement, inclination, and azimuth for Gugla Bielzug Rock Glacier (position BH13) and displacements for reference station RD01. Figure 4. A decade of air temperature and precipitation from weather station DH13, as well as displacement, inclination, and azimuth for Gugla Bielzug Rock Glacier (position BH13) and displacements for reference station RD01. Earth Syst. Sci. Data, 14, 5061–5091, 2022 https://doi.org/10.5194/essd-14-5061-2022 5069 A. Cicoira, S. Weber et al.: GNSS observations of the Swiss periglacial environment in Sect. 2 and in Table 1. More comprehensive information about the data availability, operation window of all sensors, their instrumentation, and additional plots are given in the appendix. Tables A1–A6 provide a detailed list of all GNSS and automatic weather stations, along with some basic infor- mation about them. The label of the station is indicated on the very left. Thereafter, we indicate the period of operation, from the date of deployment to the end date of the operations, or an indication if it is currently ongoing. Next, the name of the reference station (as in Table 2) used for the double- difference GNSS processing is given. 4.3 Weather station data The Dirruhorn and Matterhorn sites are instrumented with automatic weather stations. The data comprise ambient air temperature, air pressure, relative humidity, wind (speed and direction), and precipitation as well as four-component net radiation. The time series for the Dirruhorn ﬁeld site is given in Fig. 4. The raw weather station data sampled at 2 min in- tervals are provided in the form of CSV ﬁles. 4.1 GNSS raw observation data national coordinate system by using the online REFRAME conversion service (REST API) by swisstopo. The geode- tic datum of all daily position data is EPSG:2056 – Swiss CH1903+ / LV95 coordinate system with the reference frame Bessel (ellipsoidal). After post-processing, the derived data are uploaded again to the PermaSense database for conve- nient online data access. A custom-developed set of wrapper scripts to be used with RTKLIB is described in Appendix D. The processing using the Bernese GNSS software is a similar process and follows the same steps presented in Fig. 5. However, further details on this tool are omitted due to its license-only availability. Because the two processing tools use different algorithms and parameter sets, the output data can show differences. Most noticeably, due to different parameter-threshold settings, it may happen that a daily com- puted position is available for a given tool, but not for the other. Given the long-term nature of the observations, these often spurious missing values can be safely interpolated in a further processing step if deemed necessary by the applica- tion. 5.1 GNSS processing The main data-transformation step performed within this paper is the transformation from the raw primary GNSS data to calculate daily static GNSS positions using double- difference GNSS post-processing. Double-difference GNSS processing (Teunissen and Montenbruck, 2017) is based on data obtained in a common observation interval from a sta- tion pair. The workﬂow is illustrated in Fig. 5. Positions for the so-called “rover” station can be calculated with high pre- cision under the assumption that the location of the “refer- ence” station is quasi-stationary and that observations from both stations are subject to similar perturbations. Care should be taken that the baseline distance between any station pair is short, the ﬁeld of view to the satellites (horizon) is similar, and a station pair is located in the same altitude regime. The main quality indicators of the input data (GNSS observables) are the number of visible satellites, the signal-to-noise ratio, and the observation duration. For the derived data products, the ratio of ﬁxed ambiguities as well as the standard devia- tions per coordinate axis are key quality indicators. Double- difference processing achieves best accuracy when utilising the precision ﬁnal GNSS data products from the International GNSS Service (IGS), although other GNSS data products can be used as well, e.g. if near-real-time solutions are re- quired for practical applications in landslide-monitoring pro- cedures. 4.1 GNSS raw observation data In order to favour direct usage of the data presented in this pa- per, we provide data products derived from post-processing of the raw sampled data presented above. This process con- sists of three main steps: data transformations, cleaning, and aggregation. The only transformation needed for the pre- sented dataset is from the raw GNSS observables to daily positions; more details are discussed in the next subsection. In the second step, artefacts that have been identiﬁed man- ually or are known a priori from the metadata (e.g. due to ﬁeld interventions) are corrected in the data-cleaning step. Available cleaning operations are to delete, offset, or replace single or multiple data points. Finally, for all data, aggregates are computed. For inclinometer and weather data, the aggre- gates are given in hourly values. Different aggregation func- Raw GNSS observables are the key primary data product of this paper. The raw observables are provided in the form of industry standard daily RINEX 2.11 ﬁles for each station pre- sented. They include carrier-phase measurements, pseudo- range values as well as Doppler shift and raw signal strength. Furthermore, the two positions, RAND and HOGR, equipped with a reference grade L1-/L2-GNSS receiver, and used as reference stations, contain both GPS and GLONASS obser- vation data. For both receiver types employed, signals are tracked at an interval of 30 s with the exception of the L1- GPS reference stations that were changed from 30 to 5 s mid- way through the project. By using a higher sampling rate, a very large amount of data is obtained, but the approach https://doi.org/10.5194/essd-14-5061-2022 Earth Syst. Sci. Data, 14, 5061–5091, 2022 A. Cicoira, S. Weber et al.: GNSS observations of the Swiss periglacial environment 5070 tions are used. In most cases, the arithmetic mean is applied. For the cumulative precipitation, we used sum. Peak rain in- tensity is obtained with the maximum. From the GNSS data, the daily positions are calculated with the GNSS processing routine. The aggregation function in the toolchain can further provide aggregation over longer time periods, e.g. to weekly, monthly, or yearly values. In the following discussion, we provide a brief explanation of the methods used, which rep- resent operational best practices emerged from the context of the PermaSense project. A more detailed description of the processing steps and the related algorithm (also provided in the paper) is given in Appendices C and D. 4.1 GNSS raw observation data The GNSS processing ﬂow employed uses two dif- ferent post-processing toolchains, namely the Bernese GNSS Software (Dach et al., 2015) and the open-source RTK- LIB toolchain (http://www.rtklib.com, last access: 31 De- cember 2021). A. Cicoira, S. Weber et al.: GNSS observations of the Swiss periglacial environment Figure 5. Typical GNSS post-processing workﬂow using the Bernese GNSS software or RTKLIB. Figure 6. Data cleaning example: Inclinometer west-component time series data for location BH13 on the Gugla-Bielzug rock glacier has severe outliers and jumps in the raw data that can be removed, compensated for, and aggregated to smooth time series using the PermaSense Data Manager. Figure 5. Typical GNSS post-processing workﬂow using the Bernese GNSS software or RTKLIB. Figure 5. Typical GNSS post-processing workﬂow using the Bernese GNSS software or RTKLIB. Figure 6. Data cleaning example: Inclinometer west-component time series data for location BH13 on the Gugla-Bielzug rock glacier has severe outliers and jumps in the raw data that can be removed, compensated for, and aggregated to smooth time series using the PermaSense Data Manager. Figure 6. Data cleaning example: Inclinometer west-component time series data for location BH13 on the Gugla-Bielzug rock glacier has severe outliers and jumps in the raw data that can be removed, compensated for, and aggregated to smooth time series using the PermaSense Data Manager. 5.2.2 Inclinometer data All GNSS-derived daily positions are converted to rel- ative coordinates starting with a zero value at the on- set of the measurement period and have undergone a rudimentary data-cleaning step: outliers and jumps per- taining to device changes on ﬁeld service days have been removed. All cleaned daily ﬁles showing displace- ments from the beginning are available in the folder gnss_derived_data_products. Such cleaned time series easily enable us to visualise and compare the displace- ment rates measured at the several locations and to distin- guish different patterns linked to landforms (see Fig. 7). Similarly, the raw data from inclinometer and weather sta- tion sensors are available at sampling intervals of 120 s. Based on the example of the Gugla-Bielzug rock glacier described earlier (see Sect. 5.2 and Fig. 6), all time-series data from inclinometer and weather stations are cleaned, offset-compensated, and aggregated to hourly data prod- ucts. All of these data ﬁles are available in the folder timeseries_derived_data_products. 5.2 Data cleaning and aggregation Weber et al.: GNSS observations of the Swiss periglacial environment https://doi.org/10.5194/essd-14-5061-2022 5.2 Data cleaning and aggregation A generic toolchain to access, compile, clean, aggregate, and validate both primary as well as derived data from the online PermaSense database has been developed by Weber et al. (2019b) (DOI: http://doi.org/10.5281/zenodo.2542715) and an update is available at https://git.uibk.ac.at/informatik/ neslab/public/permasense/permasense_datamgr (last access: 21 October 2022, Weber et al., 2022). This tool allows us to query the database based on location and sensor type/data type, compile raw CSV ﬁles, apply metadata, data cleaning and correction functions, aggregate time-series data to pre- selectable aggregation intervals, and produce intuitive vali- dation plots. The data cleaning and correction step allows us to delete outliers based on time intervals or value thresholds as well as perform simple mathematical corrections, e.g. to apply a constant factor or an offset for a given interval. Us- ing this latter feature allows us to correct artefacts introduced during site maintenance, e.g. from changing sensor equip- ment or from re-levelling a GNSS mast that is required when the tilt angle changes very much. An example for such a cor- rection is shown in Fig. 6 for the north component of the in- clinometer data of station BH13 that is located on the Gugla- Bielzug rock glacier. Additionally, outliers from days with sensor-device changes are removed using the del operation, and offsets due to differences between two sensor devices are corrected using the offset operation. The ﬁltered original data (with a number of artefacts) are plotted in orange, the cleaned data are shown in blue, and the aggregated data over 1400 min (daily values) are shown in red in Fig. 6. A quick tutorial for this tool is given in Appendix C. The post-processing with RTKLIB starts from the daily observation ﬁles, compiled using the online database as one ﬁle per day per position. To constitute a baseline pair between a rover position and a reference station, in most cases we use a sensor from the local geodetic network. Differently, we use reference positions from AGNES for determining the coordi- nates of the reference stations, and in cases where the base- line distance is too high for the calculation of accurate posi- tions. All rover–reference pairs are indicated in Tables A1– A6. Thereafter, coordinates are calculated. In a ﬁnal step, the daily coordinates are re-projected from the WGS84 to Swiss https://doi.org/10.5194/essd-14-5061-2022 Earth Syst. Sci. Data, 14, 5061–5091, 2022 5071 A. Cicoira, S. 5.3 Standardised analysis plots For each position, a number of standardised graphs are generated (see Figs. A3, A4 and A5 ). These plots are available for all derived data products in the folder https://doi.org/10.5194/essd-14-5061-2022 Earth Syst. Sci. Data, 14, 5061–5091, 2022 5072 A. Cicoira, S. Weber et al.: GNSS observations of the Swiss periglacial environment A. Cicoira, S. Weber et al.: GNSS observations of the Swiss periglacial environment 5072 A. Cicoira, S. Weber et al.: GNSS observations of the Swiss periglacial environment Figure 7. Time series of all GNSS positions presented in this paper, classiﬁed by landform type and range of displacement rates. The outlier in the Landslides Mattertal (LS05, Summer 2020) was due to a superﬁcial roto-translatory movement of the boulder on which the sensor was placed, as conﬁrmed by a 24◦rotation visible in the inclinometer data. Figure 7. Time series of all GNSS positions presented in this paper, classiﬁed by landform type and range of displacement rates. The outlier in the Landslides Mattertal (LS05, Summer 2020) was due to a superﬁcial roto-translatory movement of the boulder on which the sensor was placed, as conﬁrmed by a 24◦rotation visible in the inclinometer data. Figure 7. Time series of all GNSS positions presented in this paper, classiﬁed by landform type and range of displacement rates. The outlier in the Landslides Mattertal (LS05, Summer 2020) was due to a superﬁcial roto-translatory movement of the boulder on which the sensor was placed, as conﬁrmed by a 24◦rotation visible in the inclinometer data. timeseries_sanity_plots of the data repository and also as a supplement to this paper. hindering direct comparison to other sensors, typically used for validation. The processing of the derived data products is completely transparent: the complete processing workﬂow from raw sen- sor data to the derived data products applying cleaning and aggregation is described in Sect. 5. Standardised visualisa- tions for every data channel (an example is given in Fig. 6) are provided at https://doi.org/10.1594/PANGAEA.948334 (Beutel et al., 2022) and allow to check the data cleaning and aggregation on a per-channel basis. Note that data offset were corrected only when ﬁeld site interventions included in the meta data provided an objective reason. No arbitrary stylistic correction is performed. https://doi.org/10.5194/essd-14-5061-2022 6 Validation and uncertainty of data products The dataset presented in this manuscript comprises primary (raw) and derived data products for a series of sensors. In Table 1 we provide an overview of the sensor types, their sampling intervals and their nominal accuracy. For most of the sensor systems used, the work presented here represents a novel application domain, with custom developed sensor systems applied in a challenging operating regime. In many cases, the extreme environmental conditions and demanding logistics make so that our measurements are the only ones performed in the area over such extended periods of time, The processing accuracy of the GNSS observables (input data), to daily coordinates (primary data product) heavily de- Earth Syst. Sci. Data, 14, 5061–5091, 2022 https://doi.org/10.5194/essd-14-5061-2022 5073 A. Cicoira, S. Weber et al.: GNSS observations of the Swiss periglacial environment 5073 Table 3. Structure, description, formats, and sizes of the dataset components. File Data description Format # Data points # Files Size gnss_data_raw.zip Raw daily position data csv 231’136 885 61.3 MB gnss_data_raw_observables.zip GNSS raw observations RINEX 2.11 199’551’511 126’395 38.1 GB timeseries_data_raw.zip Raw primary sensor data csv 60’622’944 472 10.4 GB gnss_derived_data_products.zip Daily position data csv 229’669∗ 774 23 MB timeseries_derived_data_products.zip Sensor data after cleaning/aggregation csv 3’290’599 416 161.3 MB timeseries_sanity_plots.zip Standard plots for all data png – 694 174.1 MB dirruhorn_nodepositions.xlsx General metadata ﬁle xlsx – 1 98 kB matterhorn_nodepositions.xlsx General metadata ﬁle xlsx – 1 58 kB permos_nodepositions.xlsx General metadata ﬁle xlsx – 1 37 kB README.md – md – 1 4 kB Total – – 263’925’859 129’636 48.9 GB ∗In part the data have been processed by two different toolchains, RTKLIB and Bernese. Table 3. Structure, description, formats, and sizes of the dataset components. this as noise. This noise can be considered irrelevant, because as the feature under investigation usually exhibit a yearly dis- placement 2–3 orders of magnitude higher than the seasonal, instrumental noise itself (displacement in metres, noise in millimetres). Nevertheless, short-term outliers in the obser- vations should be interpreted carefully, as they can be an am- pliﬁed result of noise in the reference station (e.g. spikes in RAND and HOGR in Fig. A2). A last caveat should be given on the interpretation of the data, which despite hourly to daily resolution, should be interpreted with care at such shorter timescales, critically distinguishing between noise and sig- nals in the data. pends on the quantity and quality of the input data. 6 Validation and uncertainty of data products This in turn depends on the satellite availability, the meteorological and atmospheric conditions, on the duration of the data ac- quisition intervals that in turn depend on the available power as well as the ground kinematics observed. In effect, the ac- curacy of the primary GNSS data products varies over time and space (between different positions). For this reason, no average/typical values are given for the whole dataset. In- stead, quality indicators are available for each daily position ﬁx calculated. For the GNSS data the standard deviation for each axis (E, N, vertical) as well as the ratio of ﬁxed am- biguities is given, the ratio is a percentage that speciﬁes how well the GNSS solver was able to resolve integer ambiguities for ﬁxing a solution – a commonly used quality indicator in GNSS post-processing. For the rtklib solution a static param- eter set is chosen for each reference position, i.e. all baseline pairs to the same reference are processed in the same way. The details are in the respective documentation of the tools used. https://doi.org/10.5194/essd-14-5061-2022 Earth Syst. Sci. Data, 14, 5061–5091, 2022 8 Conclusions and outlook This paper documents the data resulting from a multiple ﬁeld-site monitoring effort using in situ GNSS sensors on dif- ferent landforms in the Swiss Alps. This dataset constitutes one of the largest and highest ﬁdelity datasets document- ing mass movement by means of permanent ground mea- surement points, a method that has been developed and put into practice by the authors. The data are obtained mainly at mass movement sites in the periglacial ﬁeld with a few place- ments at lower altitudes at or beyond the fringe of permafrost. Most of these sites are subject to further investigations using a multitude of methods, e.g. terrestrial surveys, seismome- ters, time-lapse photography, UAV surveys, InSAR. A basic overview of these ﬁeld sites, methods employed, and data available are documented. As such, this dataset provides an important step for future work and development of novel methods, further process understanding, and help for miti- gating natural hazards as well as adaptation strategies. Since October 2022, Rock Glacier Velocity (RGV) is a new Essen- tial Climate Variable (ECV) product within the Global Cli- mate Observing System (GCOS) (WMO, 2022). The Gugla-Bielzug rock glacier (BH 10/13) is situated to- wards the north above the village of Herbriggen be- tween the peaks of the Gugla and Breithorn. The frontal part of the rock glacier opens up into a very steep gully with frequent and high-volume discharge of loose ma- terial. The Gugla-Bielzug rock glacier and Bielzug de- bris ﬂow are serious hazard sources in the area. Signiﬁ- cant efforts have been undertaken to (i) study the details and (ii) implement protective measures for the safety of the valley habitat and infrastructure. Speciﬁcally, a debris catchment, several damming structures, and an early warning system using geophones have been im- plemented (Kummert et al., 2018a; Kummert and De- laloye, 2018; Kummert et al., 2018b; Wirz et al., 2014b; Guillemot et al., 2021; Oggier et al., 2016). The Stabbio di Largario rock glacier (LAR) is located in the Adula/Rheinwaldhorn massif and therefore adds to the monitoring concept in a different weather zone (Scapozza et al., 2014). 7 Code and data availability western ﬂank of the Distelhorn, above the municipality of Grächen in the Matter Valley. The two instrumented GNSS positions are located on two fronts. The upper one (at 2500 ma.s.l.) shows a bulgy morphology, while the lower one (at 2420 ma.s.l.) is more distinct. Ac- cess to the rock glacier is facilitated by the presence of nearby ski slopes. – https://git.uibk.ac.at/informatik/neslab/public/ permasense/rtklib_processing (last access: 26 Oc- tober 2022) (DOI: https://doi.org/10.5281/zenodo. 7251288, Beutel, 2022). The Gruben rock glacier (GRU) and Jaegi rock glacier (JAE) are located in the adjacent Saas Valley, near Saas Grund, (Switzerland). They are among the ﬁrst rock glaciers investigated in Switzerland (Haeberli et al., 1979; Haeberli, 1996, 1985; Haeberli and Schmid, 1988). The Jaegi rock glacier consists of two over- riding fronts at around 2550 ma.s.l. The rock glacier has shown signs of destabilisation since the 1950s (Ghirlanda et al., 2016). Currently, the upper lobe – between 2670 and 2550 ma.s.l. – has to be considered destabilised, with large displacement rates and geomor- phological signs of degradation. The GNSS position is located close to this fast-moving front. The dataset (structure is described in Table 3) and its an- nual update are available from the following website: – Dataset https://doi.org/10.1594/PANGAEA.948334 (Beutel et al., 2022) 7 Code and data availability The dataset published with this paper contains data from 1 March 2011 until 31 December 2021. An overview of the structure, ﬁle types, and size of the datasets, for both the raw primary data and derived data products, is given in Table 3. Furthermore, the dataset also contains the key metadata ﬁles for the ﬁeld sites. Annual updates of this dataset are planned (living data process). Using the toolset described in Appendix C and using the online repository at http://data.permasense.ch, last access: 26 October 2022 (see Weber et al., 2019a for details), the data user can also create custom updates of the dataset independently. Furthermore, a set of wrapper scripts for GNSS post-processing using the open-source RTKLIB toolchain (http://www.rtklib.com, last access: 26 October 2022) are described in Appendix D. This toolchain allows us to compute the differential GNSS daily positions from both the RINEX ﬁles contained in this dataset as well as the online data from the PermaSense database. Furthermore, the accuracy of the daily coordinate ﬁxes de- pends also on the physical behaviour observed (the kinematic of the measured landforms). If the observation point moves signiﬁcantly during one observation interval (epoch) an aver- age position is computed. For static (non-moving positions) this effect can be easily neglected. For fast motion, or stick- slip events on the contrary, this effect can negatively impact the accuracy of the measurements that basically results in a time offset for the observed displacement. If required a de- tailed analysis using ﬁner grained temporal scales for the GNSS post-processing can be applied to the input data. On longer temporal scales, a seasonal period in noise can be ob- served for most stations and especially notable for the refer- ence station. In Figs. 4 and B2, we show two examples of the seasonal variability in the position of a reference station. This pattern could be explained by the physical behaviour of the boulder on which the sensor is mounted. However, because no validation measurements are present, we have to consider The toolset (code) for preparing, processing, validating, and updating the data contained in this publication is avail- able from the following providers and links: Earth Syst. Sci. Data, 14, 5061–5091, 2022 https://doi.org/10.5194/essd-14-5061-2022 A. Cicoira, S. Weber et al.: GNSS observations of the Swiss periglacial environment 5074 – https://git.uibk.ac.at/informatik/neslab/public/ permasense/permasense_datamgr (last access: 26 Oc- tober 2022) (DOI: https://doi.org/10.5281/zenodo. 7251255, Weber et al., 2022). 8 Conclusions and outlook This rock glacier is known as an important source of debris, reworked by debris ﬂow descending from the Soi Valley, and having caused sev- eral damages at the village of Dangio-Torre in the past, destroying the chocolate plant located at the conﬂu- ence of Soi Valley into the Blenio Valley on 28–29 Au- gust 1908. Appendix A: Detailed description of the ﬁeld sites A1 Rock glaciers It hosts the longest time series of permafrost temperatures in an alpine rock glacier, dating back to 1987 (Vonder Mühll and Haeberli, 1990; Hoelzle et al., 2002). Dur- ing the same period, vertical inclinometer proﬁles were also measured manually, providing a unique dataset. Recent detailed process-oriented studies are based on the dataset presented (Cicoira et al., 2019a, 2021). The Sattelspitz buttress (SA) is a minor buttress on the west ridge above Täsch (Switzerland). Due to slope instabil- ities and hazard potential for the fresh water supply of the village of Täsch, a kinematic monitoring has been set up at this site. A ﬁxed GNSS sensor has been in- stalled on top of a rock tower that is currently showing the largest displacement rates on the rock face. The data have not been analysed in a scientiﬁc publication. The Sattelspitz buttress (SA) is a minor buttress on the west ridge above Täsch (Switzerland). Due to slope instabil- ities and hazard potential for the fresh water supply of the village of Täsch, a kinematic monitoring has been set up at this site. A ﬁxed GNSS sensor has been in- stalled on top of a rock tower that is currently showing the largest displacement rates on the rock face. The data have not been analysed in a scientiﬁc publication. The Sattelspitz buttress (SA) is a minor buttress on the west ridge above Täsch (Switzerland). Due to slope instabil- ities and hazard potential for the fresh water supply of the village of Täsch, a kinematic monitoring has been set up at this site. A ﬁxed GNSS sensor has been in- stalled on top of a rock tower that is currently showing the largest displacement rates on the rock face. The data have not been analysed in a scientiﬁc publication. The Ritigraben rock glacier (RI) is a well-studied landform in the vicinity of the ski slopes of Grächen (Switzer- land). In addition to the kinematics data, borehole tem- peratures and inclinometers data are available (Kenner et al., 2017, 2018; Cicoira et al., 2019a). The Schafberg rock glacier (SCH) is located above the mu- nicipality of Pontresina (Switzerland). The rock glacier delivers debris to steep slopes prone to snow avalanches and debris ﬂows that endanger the infrastructure and the village in the valley bottom. Therefore, it has been the object of investigations for this and other projects related to natural hazards. A1 Rock glaciers The Dirru Rock Glacier (DI) is a well-studied landform for this peculiar kinematic behaviour (Delaloye et al., 2010). Continuous kinematic measurements have been available since 2011 for three positions in the upper part and the ﬁrst steep ﬂank of the rock glacier. In year 2018, two more positions were instrumented on the fast- moving tongue, with the goal of creating a spatially re- solved network for the analysis of the spatial variability of rock glacier velocities at a daily scale (Cicoira et al., 2019b, 2021). The Muragl rock glacier (MUR) is located in the homonym valley in the municipality of Samedan (Switzerland). The rock glacier has already been investigated 20 years ago in some of the ﬁrst detailed photogrammetric stud- ies from aerial and satellite imagery in the periglacial environment (Kääb et al., 1997, 1998). On site, one ﬁxed GNSS sensor is located a few hundred metres up from the PERMOS borehole (Cicoira et al., 2019a). The Distelhorn rock glacier (DIS) is the northernmost rock glacier of the Matter Valley cluster. It is located on the The Distelhorn rock glacier (DIS) is the northernmost rock glacier of the Matter Valley cluster. It is located on the The Distelhorn rock glacier (DIS) is the northernmost rock glacier of the Matter Valley cluster. It is located on the Earth Syst. Sci. Data, 14, 5061–5091, 2022 https://doi.org/10.5194/essd-14-5061-2022 A. Cicoira, S. Weber et al.: GNSS observations of the Swiss periglacial environment A. Cicoira, S. Weber et al.: GNSS observations of the Swiss periglacial environment 5075 The Randa Grossgufer (RA) is a 30 × 106 m3 rockslide site on the orographic left side of the Matter Valley, just opposite from the Dirru rock glacier. It has been thor- oughly investigated by numerous researchers (Willen- berg et al., 2008b, a; V. Gischig et al., 2011; Fäh et al., 2012-12; S. Gischig et al., 2011; Moore et al., 2011; Burjánek et al., 2010; Eberhardt et al., 2004). Moni- toring activity has increased in the past years with one GNSS reference and 3× GNSS sensor rovers situated at the lip of the detachment. The Murtèl-Corvatsch rock glacier (COR) is one of the ﬁrst rock glaciers to be studied intensively worldwide. Its displacement rates are 1–2 orders of magnitude smaller than most other rock glaciers presented in this dataset. A3 Landslides It is not directly connected to the valley bottom and is characterised by gentler slope gradients in com- parison to the other rock glaciers in the valley. How- ever, it carries two exemplary steep frontal zones above which the two GNSS sensors are positioned. The creep rates and the seasonal variability of this rock glacier are less pronounced and are more related to the general pat- terns observed in the Alps, rather than its steeper neigh- bours in the Matter Valley (Noetzli et al., 2019). The Steintälli rock glacier (ST) is located a few hundred metres higher in the same catchment as the Dirru rock glacier. It is not directly connected to the valley bottom and is characterised by gentler slope gradients in com- parison to the other rock glaciers in the valley. How- ever, it carries two exemplary steep frontal zones above which the two GNSS sensors are positioned. The creep rates and the seasonal variability of this rock glacier are less pronounced and are more related to the general pat- terns observed in the Alps, rather than its steeper neigh- bours in the Matter Valley (Noetzli et al., 2019). A1 Rock glaciers The kinematic measure- ments are complemented by borehole temperatures and past inclinometer proﬁle measurements (Arenson et al., 2002), available through PERMOS. Detailed studies about rock glacier dynamics have investigated this well- monitored site (Cicoira et al., 2019a; Kenner et al., 2020). 1Geologisches Gutachten, Dr. R. Urs Winterhalter, Zurich, 1959, personal communication, Rosie Allmendinger, 2018, Her- briggen, various news reports from 1959. A3 Landslides The Breithorn landslide (BH) is a landslide situated on the south-west slope of the Breithorn summit (3176 m a.s.l.). It is located in close proximity, a few hundred metres north, of the Gugla-Bielzug rock glacier, which holds the same label. Positions 3, 7, 9, 12, and 68 are/were located on the landslide. Positions 10 and 13 are located on the rock glacier (see below). The Längenschnee (LS) and Gugla (GU) landslides can be found one ridge line further south in the valley. This area features a large, relict landslide that surged at least once in 1959, when the village of Herbriggen was evacuated for a number of days in mid-winter1. The lower parts of the slopes leading down from the Gugla are known as Längenschnee, a sediment and debris-rich area charac- terised by sharp steepening drop, leading into the valley and towering above the village of Herbriggen. Here, the downslope movement of the whole area poses a signif- icant and real risk for the habitat in the valley. Impor- tant countermeasures apart from observation and mon- itoring have been undertaken recently, e.g. a protective dam is currently being constructed and a large boulder (Grosse Stei) in the area of Längenschnee has been ﬁxed by anchoring and concrete under-ﬁlling. This boulder has been monitored since 2018 using a GNSS sensor (position LS11). The Längenschnee (LS) and Gugla (GU) landslides can be found one ridge line further south in the valley. This area features a large, relict landslide that surged at least once in 1959, when the village of Herbriggen was evacuated for a number of days in mid-winter1. The lower parts of the slopes leading down from the Gugla are known as Längenschnee, a sediment and debris-rich area charac- terised by sharp steepening drop, leading into the valley and towering above the village of Herbriggen. Here, the downslope movement of the whole area poses a signif- icant and real risk for the habitat in the valley. Impor- tant countermeasures apart from observation and mon- itoring have been undertaken recently, e.g. a protective dam is currently being constructed and a large boulder (Grosse Stei) in the area of Längenschnee has been ﬁxed by anchoring and concrete under-ﬁlling. This boulder has been monitored since 2018 using a GNSS sensor (position LS11). The Steintälli rock glacier (ST) is located a few hundred metres higher in the same catchment as the Dirru rock glacier. A2 Steep rock walls The Matterhorn Hörnli ridge (MH) is the east ridge of the Matterhorn. The measurements are mainly clustered in and around a prominent high-alpine rockfall (at an el- evation of about 3500 ma.s.l.) that took place in year 2003 (Hasler et al., 2008, 2011, 2012). The highest po- sition at this site and in the entire dataset is located at the Solvey Hut at 4003 ma.s.l. Apart from the ther- mal and kinematic measurements documented in Weber et al. (2019a), recent years have also seen experimenta- tion with seismic sensors (Weber et al., 2018b, c). The Matterhorn Hörnli ridge (MH) is the east ridge of the Matterhorn. The measurements are mainly clustered in and around a prominent high-alpine rockfall (at an el- evation of about 3500 ma.s.l.) that took place in year 2003 (Hasler et al., 2008, 2011, 2012). The highest po- sition at this site and in the entire dataset is located at the Solvey Hut at 4003 ma.s.l. Apart from the ther- mal and kinematic measurements documented in Weber et al. (2019a), recent years have also seen experimenta- tion with seismic sensors (Weber et al., 2018b, c). The Grabengufer landslide (GG) is a complex landform comprising of a landslide (sagging) originating at ap- prox. 2800 ma.s.l. as well as a very fast-moving rock The Grabengufer landslide (GG) is a complex landform comprising of a landslide (sagging) originating at ap- prox. 2800 ma.s.l. as well as a very fast-moving rock Earth Syst. Sci. Data, 14, 5061–5091, 2022 https://doi.org/10.5194/essd-14-5061-2022 A. Cicoira, S. Weber et al.: GNSS observations of the Swiss periglacial environment A. Cicoira, S. Weber et al.: GNSS observations of the Swiss periglacial environment 5076 glacier releasing its debris into a hazard debris ﬂow ending just north of the village of Randa (Delaloye et al., 2013). It has seen mitigation measures for many decades with the earliest on record dating back to 1945, when a protective wall was constructed to fence off a lateral part of the moving rock glacier in the area of the “Grüne Garten” above the village of Randa2. The Wisse Schijen landslide (WYS) is situated on the orographic left side of the Matter Valley at around 3100 m a.s.l., to the west of Randa. A large landslide is affecting the top of the permafrost slope equipped with avalanche protection structures (eight rows of snow nets). 2Protocol of the Swiss Federal Council, 15 February 1945. Earth Syst. Sci. Data, 14, 5061–5091, 2022 A2 Steep rock walls These nets retain snow in the Wisse Schijen avalanche release area and protect numerous rows of steel snow bridges below. A GNSS sensor is situated in the centre of the landslide to monitor slope displace- ments parallel to borehole inclinometer measurements. https://doi.org/10.5194/essd-14-5061-2022 Earth Syst. Sci. Data, 14, 5061–5091, 2022 5077 A. Cicoira, S. Weber et al.: GNSS observations of the Swiss periglacial environment A. Cicoira, S. Weber et al.: GNSS observations of the Swiss periglacial environment A. Cicoira, S. Weber et al.: GNSS observations of the Swiss periglacial environment Data availability for all primary data products. The time periods when data are available are indicated in green. imary data products. The time periods when data are available are indicated in green. Data availability for all primary data products. The time periods when data are available are indicated in green. Earth Syst. Sci. Data, 14, 5061–5091, 2022 https://doi.org/10.5194/essd-14-5061-2022 A. Cicoira, S. Weber et al.: GNSS observations of the Swiss periglacial environment 5078 78 A. Cicoira, S. Weber et al.: GNSS observations of the Swiss periglacial environment gure A2. Time series (weekly mean) the three components of all reference stations used. A detailed description of each station is given in ble 2. For a discussion of the spike in HOGR that also appears in the nearby RAND, see Sect. 6. Figure A2. Time series (weekly mean) the three components of all reference stations used. A detailed description of each station is given in Table 2. For a discussion of the spike in HOGR that also appears in the nearby RAND, see Sect. 6. Figure A2. Time series (weekly mean) the three components of all reference stations used. A detailed description of each station is given in Table 2. For a discussion of the spike in HOGR that also appears in the nearby RAND, see Sect. 6. Earth Syst. Sci. Data, 14, 5061–5091, 2022 https://doi.org/10.5194/essd-14-5061-2022 A. Cicoira, S. Weber et al.: GNSS observations of the Swiss periglacial environment 5079 Figure A3. Station BH13 on the Breithorn/Bielzug rock glacier above Herbriggen; VS exhibits a steady acceleration downslope. Figure A3. Station BH13 on the Breithorn/Bielzug rock glacier above Herbriggen; VS exhibits a steady acce BH13 on the Breithorn/Bielzug rock glacier above Herbriggen; VS exhibits a steady acceleration downslope. Figure A4. Station BH13 on the Breithorn/Bielzug rock glacier above Herbriggen; VS exhibits a steady acceleration downslope. Figure A4. Station BH13 on the Breithorn/Bielzug rock glacier above Herbriggen; VS exhibits a steady acceleration downslope. Earth Syst. Sci. Data, 14, 5061–5091, 2022 https://doi.org/10.5194/essd-14-5061-2022 a Location coordinates are given for the ﬁrst day of deployment. b Sensor was destroyed in an avalanche ending the time series. c Sensor location detached in a rockfall ending the time series. a Location coordinates are given for the ﬁrst day of deployment. b Sensor was destroyed in an avalanche ending the time series. c Sensor loc detached in a rockfall ending the time series A. Cicoira, S. Weber et al.: GNSS observations of the Swiss periglacial environment 5080 Figure A5. Station BH13 on the Breithorn/Bielzug rock glacier above Herbriggen; VS exhibits a steady acceleration downslope. Figure A5. Station BH13 on the Breithorn/Bielzug rock glacier above Herbriggen; VS exhibits a steady acceleration downslope. Figure A6. Wireless LAN (WLAN) backbone and ﬁne distribution of wireless connectivity to all sensor locations in the Matter Valley. The basis for such connection is a WLAN access point located at the cable car station of the Klein Matterhorn 3883 ma.s.l. about 6.5 km away, where the network is attached to a local Internet service provider using glass ﬁbre. In a previous publication, Weber et al. (2019a) provided a more comprehensive and detailed description of the technology of the WSN. Figure A6. Wireless LAN (WLAN) backbone and ﬁne distribution of wireless connectivity to all sensor locations in the Matter Valley. The basis for such connection is a WLAN access point located at the cable car station of the Klein Matterhorn 3883 ma.s.l. about 6.5 km away, where the network is attached to a local Internet service provider using glass ﬁbre. In a previous publication, Weber et al. (2019a) provided a more comprehensive and detailed description of the technology of the WSN. Figure A7. In very active movement zones ﬁnding a single block to ﬁx the measurement instrument can be challenging, both w.r.t. longevity and the representative motion of the investigated landform. The two locations shown in this ﬁgure are very active zones in steep parts of two rock glaciers above Herbriggen (Switzerland): Dirru rock glacier (a) and Breithorn/Bielzug rock glacier (b). Photos from PermaSense archive. Figure A7. In very active movement zones ﬁnding a single block to ﬁx the measurement instrument can be challenging, both w.r.t. longevity and the representative motion of the investigated landform. The two locations shown in this ﬁgure are very active zones in steep parts of two rock glaciers above Herbriggen (Switzerland): Dirru rock glacier (a) and Breithorn/Bielzug rock glacier (b). Photos from PermaSense archive. Earth Syst. Sci. Data, 14, 5061–5091, 2022 https://doi.org/10.5194/essd-14-5061-2022 5081 a Location coordinates are given for the ﬁrst day of deployment. b Data from the permanent GNSS network in Switzerland (AGNES) are used here. A. Cicoira, S. Weber et al.: GNSS observations of the Swiss periglacial environment ira, S. Weber et al.: GNSS observations of the Swiss periglacial environment 1. Per position overview of the sensors in the Matter Valley ﬁeld site (see Fig. 2): Part 1. General Locationa Kinematics Weather Station Period of operation (yyyy-mm-dd) Reference Mast height [m] Online data East North Altitude L1/L2-GNSS L1-GPS Inclination Radiation Weather data BH03 2015-10-07–ongoing RD01 1.0 X 2629378 1109934 2754 X X BH07 2011-05-18–ongoing RD01 1.0 X 2629787 1110178 2982 X X BH09 2011-05-18–ongoing RD01 1.5 X 2630158 1110218 3159 X X BH10 2011-08-17–2018-01-08b RD01 1.5 X 2629255 1109755 2662 X X BH12 2012-02-24–ongoing RD01 1.0 X 2629596 1110114 2872 X X BH13 2012-02-24–ongoing RD01 1.0 X 2629332 1109779 2701 X X DI02 2011-05-02–ongoing RD01 1.5 X 2629569 1107710 2770 X X DI03 2017-06-09–ongoing RD01 1.5 X 2629354 1107798 2676 X X DI04 2017-06-09–ongoing RD01 1.0 X 2629241 1107861 2599 X X DI07 2010-12-16–ongoing RD01 1.5 X 2629355 1107810 2673 X X DI55 2011-03-09–ongoing RD01 1.0 X 2629457 1107876 2694 X DI57 2011-03-01–2014-09-11 RD01 1.5 2629354 1107816 2673 X DIS1 2012-07-19–ongoing RD01 1.0 2632748 1115588 2426 X X DIS2 2012-07-19–ongoing RD01 1.0 2632911 1115403 2501 X X GG01 2011-09-29–ongoing RG01 1.0 X 2628937 1104474 2906 X X GG02 2011-09-29–2020-05-17c RG01 1.0 X 2628872 1104513 2894 X X GG52 2011-10-03–2016-12-30 RG01 1.0 2628872 1104514 2894 X GG66 2012-10-26–2016-12-30 RG01 1.3 X 2628817 1104430 2905 X GG67 2012-11-20–2016-12-30 RG01 1.3 X 2628782 1104448 2889 X GU02 2011-05-18–2013-05-24 RD01 1.0 2629715 1109034 2969 X X GU03 2011-05-18–2013-05-24 RD01 1.5 2629761 1108988 2996 X X GU04 2011-08-17–2013-05-24 RD01 1.5 2629946 1108966 3130 X X LS01 2011-05-18–2013-08-19 RD01 1.5 2629465 1109281 2804 X X LS04 2011-05-18–2013-08-19 RD01 1.5 2629455 1109179 2802 X X LS05 2012-02-24–ongoing RD01 1.0 X 2629059 1109378 2611 X X LS06 2018-04-17–ongoing RD01 1.0 X 2628942 1109255 2524 X X LS11 2014-11-21–2020-10-03c RD01 1.0 X 2629018 1109451 2588 X X LS12 2014-11-21–ongoing RD01 1.0 X 2629053 1109268 2604 X X a Location coordinates are given for the ﬁrst day of deployment. b Sensor was destroyed in an avalanche ending the time series. c Sensor location detached in a rockfall ending the time series. Table A1. Per position overview of the sensors in the Matter Valley ﬁeld site (see Fig. 2): Part 1. Earth Syst. Sci. Data, 14, 5061–5091, 2022 https://doi.org/10.5194/essd-14-5061-2022 5082 A. Cicoira, S. Weber et al.: GNSS observations of the Swiss periglacial environment Table A2. A. Cicoira, S. Weber et al.: GNSS observations of the Swiss periglacial environment Per position overview of the sensors in the Matter Valley ﬁeld site (see Fig. 2): Part 2. General Locationa Kinematics Weather Station Period of operation (yyyy-mm-dd) Reference Mast height [m] Online data East North Altitude L1/L2-GNSS L1-GPS Inclination Radiation Weather data RAND 2011-05-28–ongoing ZERMb X 2625632 1107181 2415 X RA01 2015-10-08–ongoing RAND 1.0 X 2625797 1107096 2326 X X RA02 2015-10-08–ongoing RAND 1.0 X 2625772 1107086 2336 X X RA03 2016-05-27–ongoing RAND 1.0 X 2625736 1107044 2325 X X RD01 2011-03-01–ongoing RAND 0.5 X 2629577 1108071 2706 X RG01 2011-09-29–ongoing RAND 0.5 X 2628984 1104371 2974 X RIT1 2012-07-19–ongoing RD01 1.0 2631650 1113771 2605 X X RL01 2011-08-17–2013-05-24 RD01 1.0 2629491 1109569 2873 X X SA01 2018-08-06–ongoing RAND 1.0 X 2628397 1099584 3079 X X SATT 2018-08-29–ongoing RAND X 2628357 1099535 3127 X X ST02 2011-05-18–ongoing RD01 1.0 2630157 1108556 2997 X X ST05 2011-05-18–ongoing RD01 1.5 2630237 1108650 3029 X X WYS1 2014-11-20–ongoing RAND 1.0 X 2624011 1105068 3056 X X DH13 2011-03-08–ongoing X 2629563 1108035 2690 X DH42 2011-08-17–ongoing X 2628985 1104370 2827 X DH68 2013-08-20–ongoing X 2629598 1110119 2870 X DH69 2018-07-10–2020-06-03 X 2629385 1107919 2644 X DH73 2018-07-10–2020-06-03 X 2629385 1107919 2644 X a Location coordinates are given for the ﬁrst day of deployment. b Data from the permanent GNSS network in Switzerland (AGNES) are used here. Earth Syst. Sci. Data, 14, 5061–5091, 2022 https://doi.org/10.5194/essd-14-5061-2022 5083 A. Cicoira, S. Weber et al.: GNSS observations of the Swiss periglacial environment A3. Per position overview of the sensors in the Matterhorn ﬁeld site. General Locationa Kinematics Weather Station Period of operation (yyyy-mm-dd) Reference Mast height [m] Online data East North Altitude L1/L2-GNSS L1-GPS Inclination Radiation Weather data HOGR 2011-02-03–ongoing ZERMb X 2618012 1092200 3463 X MH33 2014-08-16–ongoing HOGR X 2617961 1092175 3487 X X MH34 2014-08-14–ongoing HOGR X 2618001 1092197 3463 X X MH35 2015-06-02–ongoing HOGR X 2617961 1092175 3487 X X MH40 2015-06-03–ongoing HOGR X 2617957 1092175 3489 X MH43 2018-08-15–2020-07-08 HOGR X 2617957 1092175 3489 X MH15 2015-06-02–ongoing X 2618019 1092200 3402 X MH25 2010-12-17–ongoing X 2618019 1092200 3402 X MH51 2019-06-26–ongoing X 2617392 1091918 4003 X a Location coordinates are given for the ﬁrst day of deployment. b Data from the permanent GNSS network in Switzerland (AGNES) are used here. Table A3. Per position overview of the sensors in the Matterhorn ﬁeld site. Table A4. Per position overview of the sensors in the Saas Tal ﬁeld site. e A4. Per position overview of the sensors in the Saas Tal ﬁeld site. General Locationa Kinematics Weather Station Period of operation (yyyy-mm-dd) Reference Mast height [m] Online data East North Altitude L1/L2-GNSS L1-GPS Inclination Radiation Weather data GRU1 2012-07-25–ongoing RD01 0.5 2640436 1113468 2823 X X JAE1 2012-07-26–ongoing RD01 0.5 2639856 1111235 2585 X X a Location coordinates are given for the ﬁrst day of deployment. Earth Syst. Sci. Data, 14, 5061–5091, 2022 https://doi.org/10.5194/essd-14-5061-2022 5084 Appendix B: Project context and history The most notable event was in 2018 when the debris discharged by the Ritigraben Rock Glacier obstructed the river and caused se- vere ﬂooding all the way into the central sewage treatment plant of the valley (Kenner et al., 2017, 2018). X-Sense, the initial project, conducted from 2010 to 2013, focused on a study area on the orographic right side of the Matter Valley above the municipalities of Randa and Her- briggen (Switzerland). This area is dominantly situated in permafrost, exhibits several features (Wirz et al., 2013) (see Fig. 2), and has a rich history w.r.t. mass movement-related natural hazards. Speciﬁcally, the earliest known records for hazard mitigation efforts date back to 1945 (subsidies by the Swiss federal government for rock-wall protection measures near the Grabengufer) and February 1959 (evacuation of the village of Herbriggen due to an excessive landslide sponta- neously developing on the Längenschnee/Gugla area). y p g g g ) Apart from obtaining sensor data and working on geosci- entiﬁc process studies, this project also focussed on devel- oping and proving the utility of low-powered wireless GNSS sensors in the scope of the application described. As men- tioned earlier in Sect. 2, a new set of sensors was developed based on commodity L1-GPS receivers and ubiquitous wire- less data access based on previous work on the Matterhorn (Talzi et al., 2007; Hasler et al., 2008, 2012; Weber et al., 2017) and Jungfraujoch (Hasler et al., 2011; Girard et al., 2012). The main challenge, besides designing a robust and long-lived sensing system suitable for year-round operation in a high-alpine setting, lies in the fact that the GNSS sen- sors employed are characterised by (i) large data volumes and (ii) a signiﬁcant power consumption compared to many other in situ sensors used in this domain. This is due to the fact that the GNSS receiver needs to be operated continuously over large periods of time (typically hours) and without us- ing any low-power operating modes and also using an active antenna in order to obtain sufﬁcient observation data from the satellite constellation w.r.t. both quality and quantity. For the detection of very small displacements, such as in compact bedrock or the ability to react to changing displacement dy- namics quickly, e.g. in natural hazard scenarios, a 24/7 oper- ation of the sensors is required. A. Cicoira, S. Weber et al.: GNSS observations of the Swiss periglacial environment 5085 decision-making by the Swiss cantonal and federal author- ities. A selection of the most noteworthy events and mea- sures are described in the following: In spring 2013, exces- sive discharge from the Gugla-Bielzug rock glacier caused severe debris ﬂow in the Bielzug torrent, causing a partial evacuation of the village of Herbriggen. Subsequently, a new catchment with dam as well as geophone-based monitoring was projected and erected. In order to protect hikers crossing the Grabengufer, a hanging bridge spanning the upper part of the discharge gully was constructed in 2010. Due to the rapid evolution of the Grabengufer rock glacier and the landslide above it, the bridge was hit by discharged debris multiple times, subsequently closed and dismantled. In 2017, a new bridge with a span of 494 m was erected further downslope in the gully. In 2018, a large boulder on the order of 2000 m3 was blasted in a 2-month effort to protect the village of Randa below (see Figs. B1 and B2). This freestanding boulder was located at the front of the landslide feeding into the Graben- gufer rock glacier and was gradually revealed due to continu- ous erosion happening because of the excessive slope move- ments in the area. Figure B2 shows the long-term evolution of position GG02. The seasonal accelerations throughout the time series and the anomalous behaviour (exponential accel- eration) during 2020 are notable; this lead to the detachment of the boulder from the slope and a consequent rockfall. The process is visible in both the displacement and inclinome- ter data. Another relevant case interests the area of Längen- schnee, where a large rock boulder (2524 ma.s.l.) endanger- ing the municipality of Herbriggen was stabilised with an- chors and concrete under-ﬁlling in 2014. Here, the monitor- ing of slope movement using 3× GPS on unstable masses and 1× GNSS sensor on the stabilised rock serve as inte- gral parts of the protection measures for the village of Her- briggen. Due to the recent evolution of the landslide, the vil- lage has received a new hazard zonation in 2018, and four large protective dams have recently been erected on the upper limit of the village. In the Ritigraben area, the rock glacier has repeatedly led to severe debris ﬂow with impact on the road, railway track, and Matter Vispa river below. A. Cicoira, S. Weber et al.: GNSS observations of the Swiss periglacial environment The most notable event was in 2018 when the debris discharged by the Ritigraben Rock Glacier obstructed the river and caused se- vere ﬂooding all the way into the central sewage treatment plant of the valley (Kenner et al., 2017, 2018). The method devised in this initial project (Buchli et al., 2012; Wirz et al 2013) has proven to be very successful Appendix B: Project context and history A. Cicoira, S. Weber et al.: GNSS observations of the Swiss periglacial environment Table A5. Per position overview of the sensors in the Val Blenio ﬁeld site. Table A5. Per position overview of the sensors in the Val Blenio ﬁeld site. General Locationa Kinematics Weather Station Period of operation (yyyy-mm-dd) Reference Mast height [m] Online data East North Altitude L1/L2-GNSS L1-GPS Inclination Radiation Weather data LAR1 2014-09-26–ongoing SANBb 1.0 2718881 1148509 2355 X X LAR2 2014-09-28–ongoing SANBb 1.0 2718731 1148483 2304 X X a Location coordinates are given for the ﬁrst day of deployment. b Data from the permanent GNSS network in Switzerland (AGNES) are used here. Table A6. Per position overview of the sensors in the Engadine ﬁeld site. A6. Per position overview of the sensors in the Engadine ﬁeld site. General Locationa Kinematics Weather Station Period of operation (yyyy-mm-dd) Reference Mast height [m] Online data East North Altitude L1/L2-GNSS L1-GPS Inclination Radiation Weather data COR1 2015-12-17–ongoing SAMEb 0.8 2783147 1144727 2669 X X MUA1 2012-08-04–ongoing SAMEb 1.0 2791144 1153620 2609 X X SCH1 2012-08-04–ongoing SAMEb 1.0 2791062 1152725 2809 X X a Location coordinates are given for the ﬁrst day of deployment. b Data from the permanent GNSS network in Switzerland (AGNES) are used here. Earth Syst. Sci. Data, 14, 5061–5091, 2022 https://doi.org/10.5194/essd-14-5061-2022 A. Cicoira, S. Weber et al.: GNSS observations of the Swiss periglacial environment https://doi.org/10.5194/essd-14-5061-2022 Appendix B: Project context and history decision making by the Swiss cantonal and federal author ities. A selection of the most noteworthy events and mea- sures are described in the following: In spring 2013, exces- sive discharge from the Gugla-Bielzug rock glacier caused severe debris ﬂow in the Bielzug torrent, causing a partial evacuation of the village of Herbriggen. Subsequently, a new catchment with dam as well as geophone-based monitoring was projected and erected. In order to protect hikers crossing the Grabengufer, a hanging bridge spanning the upper part of the discharge gully was constructed in 2010. Due to the rapid evolution of the Grabengufer rock glacier and the landslide above it, the bridge was hit by discharged debris multiple times, subsequently closed and dismantled. In 2017, a new bridge with a span of 494 m was erected further downslope in the gully. In 2018, a large boulder on the order of 2000 m3 was blasted in a 2-month effort to protect the village of Randa below (see Figs. B1 and B2). This freestanding boulder was located at the front of the landslide feeding into the Graben- gufer rock glacier and was gradually revealed due to continu- ous erosion happening because of the excessive slope move- ments in the area. Figure B2 shows the long-term evolution of position GG02. The seasonal accelerations throughout the time series and the anomalous behaviour (exponential accel- eration) during 2020 are notable; this lead to the detachment of the boulder from the slope and a consequent rockfall. The process is visible in both the displacement and inclinome- ter data. Another relevant case interests the area of Längen- schnee, where a large rock boulder (2524 ma.s.l.) endanger- ing the municipality of Herbriggen was stabilised with an- chors and concrete under-ﬁlling in 2014. Here, the monitor- ing of slope movement using 3× GPS on unstable masses and 1× GNSS sensor on the stabilised rock serve as inte- gral parts of the protection measures for the village of Her- briggen. Due to the recent evolution of the landslide, the vil- lage has received a new hazard zonation in 2018, and four large protective dams have recently been erected on the upper limit of the village. In the Ritigraben area, the rock glacier has repeatedly led to severe debris ﬂow with impact on the road, railway track, and Matter Vispa river below. A. Cicoira, S. Weber et al.: GNSS observations of the Swiss periglacial environment 5086 Figure B1. The Grabengufer rock glacier in winter. Panel (a) shows an overview of the catchment with the Grabenhorn on top, the Graben- gufer landslide, and the Grabengufer rock glacier above the Dorfbächji channel, with exposed eroded debris. Panel (b) shows the zoomed-in area highlighted with the yellow box, the Grabengufer landslide in summer. The different measurement positions, the reference station, and the freestanding block (approx. 2000 m3) blasted in summer of 2018 are labelled. Photos from PermaSense archive. Figure B1. The Grabengufer rock glacier in winter. Panel (a) shows an overview of the catchment with the Grabenhorn on top, the Graben- gufer landslide, and the Grabengufer rock glacier above the Dorfbächji channel, with exposed eroded debris. Panel (b) shows the zoomed-in area highlighted with the yellow box, the Grabengufer landslide in summer. The different measurement positions, the reference station, and the freestanding block (approx. 2000 m3) blasted in summer of 2018 are labelled. Photos from PermaSense archive. Figure B2. A decade of displacement, inclination, and azimuth for the Grabengufer rock glacier (position GG02). In 2020, the boulder where the GNSS station was installed showed an anomalous behaviour and strong acceleration until falling into the gully. The data show this process until the last day. Figure B2. A decade of displacement, inclination, and azimuth for the Grabengufer rock glacier (position GG02). In 2020, the boulder where the GNSS station was installed showed an anomalous behaviour and strong acceleration until falling into the gully. The data show this process until the last day. Appendix C: PermaSense data manager Appendix B: Project context and history The resulting energy and data footprint of the GNSS sensor alone (without data logging and data transmission) is on the order of watts and megabytes per station and per day. Therefore, it signiﬁcantly exceeds typical requirements of geoscientiﬁc data-acquisition systems, e.g. a typical data logger with sensors attached. The method devised in this initial project (Buchli et al., 2012; Wirz et al., 2013) has proven to be very successful and was thus expanded to other locations and applications of monitoring (Kenner et al., 2018; Cicoira et al., 2021) as well as natural hazard mitigation (Kenner et al., 2020) in collab- oration with partners of PERMOS, the Swiss cantonal and federal authorities (Randa Grossgufer, Wisse Schijen, PER- MOS GNSS sites) (Noetzli et al., 2019). The deployment activities of GNSS sensors started in the summer of 2010 on the central orographic right side of the Matter Valley above the village of Herbriggen. From then on, the newly developed GNSS sensors were tested and put to use to survey kinematics across different landforms and haz- ard areas (Wirz et al., 2014b). Further extensions took place at the Steintälli rock glacier, the Gugla/Bielzug rock glacier, the Längenschnee, Breithorn, Gugla landslide areas as well as the Grabengufer above Randa. During the study period, further hazard and mitigation events took place where the data documented by and sup- plementing this paper served as integral components for Earth Syst. Sci. Data, 14, 5061–5091, 2022 A. Cicoira, S. Weber et al.: GNSS observations of the Swiss periglacial environment A. Cicoira, S. Weber et al.: GNSS observations of the Swiss periglacial environment the online PermaSense database at http://data.permasense.ch (last access: 21 October 2022) into a local ﬁle system. Speciﬁcally, the PermaSense data manager allows us to Appendix D: GNSS processing using the open-source RTKLIB The output format of the position data CSV ﬁles produced by RTKLIB is described in Table D1. A set of scripts automates the computation of static double- difference GNSS solutions using the open-source toolchain RTKLIB (http://www.rtklib.com, last access: 21 Octo- ber 2022). The scripts are conﬁgurable for each baseline pair w.r.t. input data, conﬁguration parameter, and the toolchain to be used. The conﬁguration ﬁles and especially paths are set up speciﬁcally for processing and producing the PermaSense GNSS data contained in this dataset, but they can be adapted to other processing needs accordingly. This allows ﬂexibility for individual processing needs for each position, should that be required. The scripts are designed to run on x86 Linux, but porting this to other platforms is straightforward. D1 Prerequisites In order to run these scripts, the following prerequisites must be installed: – query data from the PermaSense GSN server and save them locally as CSV ﬁles – RTKLIB processing scripts https://git.uibk.ac.at/ informatik/neslab/public/permasense/rtklib_processing (last access: 21 October 2022, Beutel, 2022) – load the locally store CSV ﬁles – ﬁlter according to reference values if available – RTKL (last a – RTKLIB can be obtained from http://www.rtklib.com/ (last access: 21 October 2022) or alternatively https:// github.com/rtklibexplorer/RTKLIB (last access: 21 Oc- tober 2022) – ﬁlter according to reference values if available – clean data manually if needed – generate aggregates using an arithmetic mean (excep- tions for weather data) – RINEX ﬁle compression tools https://terras.gsi.go.jp/ja/ crx2rnx.html (last access: 21 October 2022) – RINEX ﬁle compression tools https://terras.gsi.go.jp/ja/ crx2rnx.html (last access: 21 October 2022) – generate per-year CSV ﬁles for each position and data type Some of the double-difference baselines are conﬁgured us- ing reference positions from the Permanent GNSS network in Switzerland (AGNES) and therefore these observation data must be obtained directly from swisstopo as they are not con- tained in this dataset. – generate standard plots for all positions as an intuitive sanity check – query images from the PermaSense database server, convert to JPEG and save them locally. D2 Processing sequence and conﬁguration setup A README.md with this software package explains its us- age. In short, a suitable Python environment is required. Us- ing anaconda, you can install the requirements by executing the following command: A single processing job always computes a single daily posi- tion for a given baseline pair. There are two kinds of conﬁgu- ration ﬁles that are required for each processing job: (i) a pa- rameter ﬁle that speciﬁes a baseline pair, data down-/upload location, starting dates, default directories, and (ii) tools to use for processing as well as a RTKLIB tool-conﬁguration ﬁle. The script package ﬁrst collects the necessary data from either the online server at http://data.permasense.ch (last ac- cess: 21 October 2022) from the IGS service or from a lo- cal ﬁle repository from disk and copies these ﬁles together with the necessary conﬁguration ﬁles into a local tempo- rary directory. If required, RINEX ﬁles are produced using the conv2bin tool from the RTKLIB toolchain. Thereafter, the post-processing tool rnx2rtkp is called, producing an output ﬁle with the coordinate data (see Table D1). Lastly, the coordinates are transformed from WGS84 to the Swiss national coordinate system by using the online REFRAME conversion service (REST API) by swisstopo. conda env create -f condaEnvironment.yaml conda activate permasense_datamgr conda env create -f condaEnvironment.yaml onda activate permasense_datamgr Individual positions can be enabled/disabled in the main Python ﬁle manage_GSNdata.py, the metadata for ﬁlter- ing and cleaning is contained in the folder ./metadata. Finally the tool is run by python manage_GSNdata.py By default, data are generated in the directory ./data. https://doi.org/10.5194/essd-14-5061-2022 Appendix C: PermaSense data manager (last access: 21 October 2022, Weber et al., 2022). It con- tains both a Python toolbox for downloading and processing primary as well as secondary data. The toolbox contains routines for the compilation, cleaning, aggregation, and vali- dation of both primary as well as derived data products from Codes for the management and processing of data associ- ated with this paper are available at https://git.uibk.ac.at/ informatik/neslab/public/permasense/permasense_datamgr https://doi.org/10.5194/essd-14-5061-2022 Earth Syst. Sci. Data, 14, 5061–5091, 2022 5087 D3 Processing wrapper script syntax The main processing wrapper script is responsible for down- loading all required data, creating a local temporary com- puted space, executing the post-processing tool rnx2rtkp, uploading the resulting data to the PermaSense database, and archiving input and output data as well as cleaning up the temporary ﬁle space. Earth Syst. Sci. Data, 14, 5061–5091, 2022 5088 5088 Table D1. GNSS computed daily position data CSV ﬁle format. Value Unit Description time [–] Timestamp of the position data position [–] Inventory position number from the database label [–] Station label processing_time ms Processing time in Unix time in milliseconds device_type [–] Sensor type version [–] Processing framework version reference_label [–] Reference station label e m Easting n m Northing h m Altitude sd_e m Standard deviation easting sd_n m Standard deviation northing sd_h m Standard deviation altitude ratio_of_fixed_ambiguities [–] Quality metric from post-processing Acknowledgements. We are extremely grateful for the extraor- dinary local support that we received during our research activi- ties in the Matter and Saas valleys, speciﬁcally from the munici- palities of Zermatt (Romy Biner-Hauser), St. Niklaus (Gaby Fux), Herbriggen, Randa, Taesch (Klaus Tscherrig), and Saas Grund; the whole team of Air Zermatt (Gerold Biner); Kurt Lauber, Stephanie Mayor, Martin Lehner, Edith Lehner, and the Hörnlihütte team; Europahütte (Marcel Brantschen); Kinnhütte (Victor Imbo- den); Alpin Center Zermatt; Zermatter Bergbahnen (Kurt Guntli); Sprengtechnik-GFS (Willy Gitz and Angelo Gruber); Hotel Bahn- hof, Zermatt (Fabi Lauber); and the local mountain guides (Her- mann Biner, Robert Andenmatten, Willy Taugwalder, Urs Lerjen, Benedikt Perren, Bruno Jelk, Hannes Walser, Simon Anthamat- ten, Yann Dupertuis, and Anjan Truffer). Without this strong pos- itive welcome, this work would not have been possible. We do not know what we would have done without our “home base” at Ho- tel Bergfreund in Herbriggen (CH). A big thank you to all gener- ations of the Rosi and Rudi Allmendinger families for their gen- erous support. Furthermore, we are thankful for technical support and consultancy from the Art of Technology (Rolf Schmid), Zurich, CH, as well as Swisstopo (Elmar Brockmann), Wabern, CH, and to the many friends and helpers who were involved in supporting the ﬁeld work: Lucas Girard, Stephanie Gubler, Christoph Walser, Robert Kenner, Johann Müller, Jeff Moore, and Valentin Gischig. Finally, we acknowledge the two anonymous referees as well as Ken Mankoff and Inga Beck for handling the peer-review process and substantially improving the manuscript. References Cicoira, A., Marcer, M., Gärtner-Roer, I., Bodin, X., Arenson, L. 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Bu, J., Yu, K., Qian, N., Zuo, X., and Chang, J.: Per- formance Assessment of Positioning Based on Multi- Frequency Multi-GNSS Observations: Signal Quality, PPP and Baseline Solution, IEEE Access, 9, 5845–5861, https://doi.org/10.1109/ACCESS.2020.3048352, 2021. Girard, L., Beutel, J., Gruber, S., Hunziker, J., Lim, R., and Weber, S.: A custom acoustic emission monitoring system for harsh en- vironments: Application to freezing-induced damage in alpine rock walls, Geosci. Instrum. Method. Data Syst., 1, 155–167, https://doi.org/10.5194/gi-1-155-2012, 2012. Buchli, B., Sutton, F., and Beutel, J.: GPS-equipped Wireless Sen- sor Network Node for High-accuracy Positioning Applications, Lecture Notes on Computer Science 7158. Proc. of 9th Euro- pean Conference on Wireless Sensor Networks (EWSN 2012), Trento, Italy, 15–17 February 2012, https://link.springer.com/ chapter/10.1007/978-3-642-28169-3_12 (last access: 31 Octo- ber 2022), 2012. Gischig, S., Moore, J. R., Evans, K. F., Amann, F., and Loew, S.: Thermomechanical forcing of deep rock slope deformation: 2. The Randa rock slope instability, J. Geophys. Res.-Earth, 116, F04011, https://doi.org/10.1029/2011JF002007, 2011. Gischig, V., Amann, F., Moore, J., Loew, S., Eisenbeiss, H., and Stempfhuber, W.: Composite rock slope kinematics at the current Randa instability, Switzerland, based on remote sensing and numerical modeling, Eng. Geol., 118, 37–53, https://doi.org/10.1016/j.enggeo.2010.11.006, 2011. Burjánek, J., Gassner-Stamm, G., Poggi, V., Moore, J. R., and Fäh, D.: Ambient vibration analysis of an unstable mountain slope, Geophys. J. Int., 180, 820–828, https://doi.org/10.1111/j.1365- 246X.2009.04451.x, 2010. A. Cicoira, S. Weber et al.: GNSS observations of the Swiss periglacial environment 5089 Review statement. This paper was edited by Inga Beck and Kenneth Mankoff and reviewed by two anonymous referees. Review statement. This paper was edited by Inga Beck and Kenneth Mankoff and reviewed by two anonymous referees. through heat conduction on rock glacier dynamics: a nu- merical modelling approach, The Cryosphere, 13, 927–942, https://doi.org/10.5194/tc-13-927-2019, 2019a. Cicoira, A., Beutel, J., Faillettaz, J., and Vieli, A.: Water controls the seasonal rhythm of rock glacier ﬂow, Earth Planet. Sc. Lett., 528, 115844, https://doi.org/10.1016/j.epsl.2019.115844, 2019b. D3 Processing wrapper script syntax compute_solution.sh -p [parameter_file] -d -b -r -c -f -u YYYY MM DD # -d: igs data download # -b: no data download and no conversion for the basestation # -r: no data download and no conversion for the roverstation # -c: no conversion # -f: use IGS final data product # -u: upload to GSN database Supplement. The supplement related to this article is available online at: https://doi.org/10.5194/essd-14-5061-2022-supplement. Supplement. The supplement related to this article is available online at: https://doi.org/10.5194/essd-14-5061-2022-supplement. Author contributions. JB, AC, and SW developed the concept and prepared the manuscript. JB, SG, AH, SW, BB, AB, MM, RL, TG, and RDF developed the sensor technology, the data- management architecture as well as the tools for managing the data. VW, SG, HR, and JB conceived the initial GNSS sensor deploy- ments with the help of LT, TS, AV, and DVM that jointly had instru- mental roles in launching and executing the initial X-Sense project. PL implemented the ﬁrst GNSS post-processing prototype. JB, AV, SW, AC, AH, HR, LT, TS, RD, IGR, RM, JN, MP, EP, CS, and DVM contributed to the scaling and application of the technology to further ﬁeld sites and applications, especially in the domain of long-term monitoring, natural hazard mitigation and early warning. All the authors contributed to the article and approved the submitted manuscript. Financial support. This research has been supported by fund- ing from the Swiss National Science Foundation NCCR-MICS, the ETH Zurich Competence Center Environment and Sustainabil- ity (CCES), the Swiss Federal Ofﬁce of the Environment (FOEN), nano-tera.ch (grant no. 530659), and the Swiss Permafrost Moni- toring Network (PERMOS). Support in the form of equipment has been given by Hilti Schweiz AG, Arc’teryx, Petzl, and Beal. The technical workshops at ETHZ and UniZH as well as Art of Tech- nology, Zurich, contributed to the successful development and im- plementation of various pieces of equipment. Competing interests. The contact author has declared that none of the authors has any competing interests. Disclaimer. Publisher’s note: Copernicus Publications remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. Earth Syst. Sci. Data, 14, 5061–5091, 2022 https://doi.org/10.5194/essd-14-5061-2022 Earth Syst. Sci. Data, 14, 5061–5091, 2022 A. Cicoira, S. 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Permafrost in Switzerland 2014/2015 to 2017/2018, Glacio- logical Report (Permafrost) No. 16-19 of the Cryospheric Commission of the Swiss Academy of Sciences (SCNAT), https://doi.org/10.13093/permos-rep-2019-16-19, 2019. Hurter, F., Geiger, A., Perler, D., and Rothacher, M.: GNSS water vapor monitoring in the Swiss Alps, in: 2012 IEEE International Geoscience and Remote Sensing Sympo- sium, Munich, Germany, 22–27 July 2012, 1972–1975, https://doi.org/10.1109/IGARSS.2012.6351115, 2012. Oggier, N., Graf, C., Delaloye, R., and Burkard, A.: Integral pro- tection concept “Bielzug” – Integrales Schutzkonzept Bielzug, in: Proc. INTERPRAEVENT, Lucerne, Switzerland, 30 May– 2 June 2016, 525–534, https://interpraevent2016.ch/wp-content/ uploads/2019/03/IP16_CP_digital.pdf (last access: 31 Octo- ber 2022), 2016. Kääb, A., Haeberli, W., and Gudmundsson, G. H.: Analysing the creep of mountain permafrost using high precision aerial photogrammetry: 25 years of moni- toring Gruben rock glacier, Swiss Alps, Permafrost Periglac., 8, 409–426, https://doi.org/10.1002/(SICI)1099- 1530(199710/12)8:4<409::AID-PPP267>3.0.CO;2-C, 1997. References Paziewski, J., Fortunato, M., Mazzoni, A., and Odolinski, R.: An analysis of multi-GNSS observations tracked by recent Android smartphones and smartphone-only rel- ative positioning results, Measurement, 175, 109162, https://doi.org/10.1016/j.measurement.2021.109162, 2021. Kääb, A., Gudmundsson, G. H., and Hoelzle, M.: Surface deformation of creeping mountain permafrost. Photogram- metric investigations on Murtel Rock Glacier, Swiss Alps, in: Proc. Seventh International Conference on Permafrost, International Permafrost Association, Yellowknife, North- west Territories, Canada, 23–27 June 1998, 531–537, https: //www.uspermafrost.org/assets/docs/Publications/Proceedings/ Ravanel, L. and Deline, P.: Rockfall hazard in the Mont Blanc mas- sif increased by the current atmospheric warming, in: IAEG 12th Congress, edited by: Lollino, G., Manconi, A., Clague, J., Shan, W., and Chiarle, M., Climate Change and Engineering Geol- https://doi.org/10.5194/essd-14-5061-2022 Earth Syst. Sci. Data, 14, 5061–5091, 2022 A. Cicoira, S. Weber et al.: GNSS observations of the Swiss periglacial environment 5091 Weber, S., Beutel, J., and Meyer, M.: Code for Per- maSense GSN data management, Zenodo [code], https://doi.org/10.5281/zenodo.2542715, 2019b. ogy, Torino, Italy, 425–428, https://hal-sde.archives-ouvertes.fr/ hal-01896005 (last access: 21 October 2022), 2014. ogy, Torino, Italy, 425–428, https://hal-sde.archives-ouvertes.fr/ hal-01896005 (last access: 21 October 2022), 2014. Scapozza, C., Lambiel, C., Bozzini, C., Mari, S., and Conedera, M.: Assessing the rock glacier kinematics on three different timescales: a case study from the south- ern Swiss Alps, Earth Surf. Proc. Land., 39, 2056–2069, https://doi.org/10.1002/esp.3599, 2014. Weber, S., Beutel, J., and Meyer, M.: Code for management and processing of PermaSense data (3.1), Zenodo [code], https://doi.org/10.5281/zenodo.7251255, 2022. Willenberg, H., Evans, K. F., Eberhardt, E., Spillmann, T., and Loew, S.: Internal structure and deformation of an unstable crystalline rock mass above Randa (Switzerland): Part II – Three-dimensional deformation patterns, Eng. Geol., 101, 15– 32, https://doi.org/10.1016/j.enggeo.2008.01.016, 2008a. Strozzi, T., Caduff, R., Jones, N., Barboux, C., Delaloye, R., Bodin, X., Kääb, A., Mätzler, E., and Schrott, L.: Monitoring Rock Glacier Kinematics with Satellite Synthetic Aperture Radar, Re- mote Sens., 12, 559, https://doi.org/10.3390/rs12030559, 2020. Talzi, I., Hasler, A., Gruber, S., and Tschudin, C.: PermaSense: In- vestigating Permafrost with a WSN in the Swiss Alps, in: Pro- ceedings of the 4th Workshop on Embedded Networked Sen- sors, EmNets ’07, ACM, New York, NY, USA, June 2007, 8–12, https://doi.org/10.1145/1278972.1278974, 2007. Willenberg, H., Loew, S., Eberhardt, E., Evans, K. F., Spillmann, T., Heincke, B., Maurer, H., and Green, A. G.: Internal struc- ture and deformation of an unstable crystalline rock mass above Randa (Switzerland): Part I – Internal structure from integrated geological and geophysical investigations, Eng. References Geol., 101, 1–14 , https://doi.org/10.1016/j.enggeo.2008.01.015, 2008b. Teunissen, P. J. and Montenbruck, O. (Eds.): Handbook of Global Navigation Satellite Systems, 1st edn., Springer In- ternational Publishing, Hardcover ISBN 978-3-030-73172-4, eBook ISBN 978-3-319-42928-1, https://doi.org/10.1007/978-3- 319-42928-1, 2017. Wirz, V., Beutel, J., Buchli, B., Gruber, S., and Limpach, P.: Temporal Characteristics of Different Cryosphere-Related Slope Movements in High Mountains, edited by: Margottini, C., Canuti, P., and Sassa, K., Springer, Berlin, Heidelberg, 383– 390, ISBN 978-3-642-31336-3, eBook ISBN 978-3-642-31337- 0, https://doi.org/10.1007/978-3-642-31337-0_49, 2013. Vonder Mühll, D. and Haeberli, W.: Thermal Characteristics of the Permafrost within an Active Rock Glacier (Murtèl/- Corvatsch, Grisons, Swiss Alps), J. Glaciol., 36, 151–158, https://doi.org/10.3189/S0022143000009382, 1990. Wirz, V., Beutel, J., Gruber, S., Gubler, S., and Purves, R. S.: Estimating velocity from noisy GPS data for investigat- ing the temporal variability of slope movements, Nat. Hazards Earth Syst. Sci., 14, 2503–2520, https://doi.org/10.5194/nhess- 14-2503-2014, 2014a. Weber, S., Beutel, J., Faillettaz, J., Hasler, A., Krautblatter, M., and Vieli, A.: Quantifying irreversible movement in steep, fractured bedrock permafrost on Matterhorn (CH), The Cryosphere, 11, 567–583, https://doi.org/10.5194/tc-11-567-2017, 2017. Wirz, V., Geertsema, M., Gruber, S., and Purves, R. S.: Tempo- ral variability of diverse mountain permafrost slope movements derived from multi-year daily GPS data, Mattertal, Switzer- land, Landslides, 13, 67–83, https://doi.org/10.1007/s10346- 014-0544-3, 2014b. Weber, S., Beutel, J., Gruber, S., Gsell, T., Hasler, A., and Vieli, A.: Rock-temperature, fracture displacement and acoustic/micro- seismic data measured at Matterhorn Hörnligrat, Switzerland, Zenodo [data set], https://doi.org/10.5281/zenodo.1163037, 2018a. World Meteorological Organization (WMO): The 2022 GCOS ECVs Requirements, https://library.wmo.int/doc_num.php? explnum_id=11318, last access: 1 November 2022. Weber, S., Fäh, D., Beutel, J., Faillettaz, J., Gruber, S., and Vieli, A.: Ambient seismic vibrations in steep bedrock permafrost used to infer variations of ice-ﬁll in fractures, Earth Planet. Sc. Lett., 501, 119–127, https://doi.org/10.1016/j.epsl.2018.08.042, 2018b. Weber, S., Faillettaz, J., Meyer, M., Beutel, J., and Vieli, A.: Acous- tic and micro-seismic characterization in steep bedrock per- mafrost on Matterhorn (CH), J. Geophys. Res.-Earth, 123, 1363– 1385, https://doi.org/10.1029/2018JF004615, 2018c. Weber, S., Beutel, J., Da Forno, R., Geiger, A., Gruber, S., Gsell, T., Hasler, A., Keller, M., Lim, R., Limpach, P., Meyer, M., Talzi, I., Thiele, L., Tschudin, C., Vieli, A., Vonder Mühll, D., and Yücel, M.: A decade of detailed observations (2008– 2018) in steep bedrock permafrost at the Matterhorn Hörn- ligrat (Zermatt, CH), Earth Syst. Sci. Data, 11, 1203–1237, https://doi.org/10.5194/essd-11-1203-2019, 2019a (data avail- able at: https://doi.org/10.1594/PANGAEA.897640). Earth Syst. Sci. Data, 14, 5061–5091, 2022 https://doi.org/10.5194/essd-14-5061-2022
https://openalex.org/W2566935689	http://www.scielo.br/pdf/coluna/v15n4/1808-1851-coluna-15-04-00295.pdf	English	null	COMPARATIVE STUDY ON THE TREATMENT OF DISC HERNIATIONS	Coluna/Columna	2,016	cc-by	4,630	Original Article/Artículo Original/Artigo Original Original Article/Artículo Original/Artigo Original ESTUDIO COMPARATIVO DEL TRATAMIENTO DE LAS HERNIAS DISCALES ESTUDO COMPARATIVO DE TRATAMENTO DE HÉRNIAS DE DISCO María Luz Suárez-Huerta1, Elena Iglesia-Diez1, Alfredo Álvarez Castro1, Jesús Betegón Nicolás1, Sonia Sánchez Campos1, Antonio Luis Mostaza Saavedra1, Manuel Fernández-González1 1. Complejo Asistencial Universitario de León, Leon, Spain. RESUMEN Objetivo: Comparar los resultados del tratamiento de pacientes con lumbalgia y radiculalgia secundaria a hernia discal asociada a de- generación del disco, mediante la instrumentación con tornillos pediculares y barra dinámica, liberando la raíz sin discectomía frente a otras técnicas no instrumentadas (microdiscectomía con o sin foraminotomía). Métodos: Se ha realizado un estudio descriptivo retrospectivo de las intervenciones realizadas en pacientes con hernias discales por los servicios de Traumatología y Neurocirugía, recogiendo las siguientes variables: edad, sexo, tipo de técnica, tiempo de intervención, tiempo de evolución, grado de satisfacción y complicaciones. Se constituyeron dos grupos: instrumentación con barras dinámicas y técnicas no instrumentadas, comparando los resultados de cada grupo. Se utilizó el software SPSS v20.0. Resultados: Presentamos 142 intervenciones realizadas entre 2009 y 2012, 86 mediante instrumentación dinámica y 56 por técnicas de descompresión sin instrumentación. No se observaron diferencias estadísticamente significativas entre los grupos respecto a edad, sexo, ni tiempo de evolución hasta la intervención. Se observaron diferencias estadísticamente significativas (p = 0,001) en el tiempo de intervención, siendo menor en la técnica instrumentada. Respecto a las complicaciones de las técnicas no se hallaron diferencias significativas ni en las reintervenciones ni en las infecciones. Conclusiones: No encontramos diferencias significativas entre la utilización de instrumentación con barras dinámicas frente a otras técnicas quirúrgicas no instrumentadas en el tratamiento de las hernias discales de más de 6 meses de evolución, ni respecto a las complicaciones y ni al grado de satisfacción de los pacientes. Descriptores: Desplazamiento del disco intervertebral/terapia; Discectomía; Tornillos pediculares. ABSTRACT Objective: To compare the results of treatment of patients with low back pain and radiculalgia resulting from disc herniation associated with disc degeneration through instrumentation with pedicle screws and dynamic rod, with root release and without diskectomy compared with other non-instrumented techniques (microdiskectomy with or without foraminotomy). Methods: This is a retrospective descriptive study of interventions for patients with herniated discs in the Traumatology and Neurosurgery that used the following variables: age, sex, type of technique, surgical time, time of evolution, degree of satisfaction, and complications. Two groups were formed: instrumentation with dynamic rods and non-instrumented techniques, comparing the results of each group. The software used was the SPSS v20.0. Results: We presented 142 interventions carried out between 2009 and 2012, 86 with dynamic instrumentation and 56 by other decompression techniques without instrumentation. No statistically significant differences were observed between age and sex groups and time elapsed until intervention. We found statistically significant differences (p=0.001) in surgical time, which was lower in the instrumented technique. No significant differences were found in complications between the techniques in both re-operations and in infections. Conclusions: In this study, we found no significant differences between the use of instrumentation with dynamic rods with respect to other non-instrumented surgical techniques in the treatment of herniated discs over 6 months of evolution or the complications and the degree of the patients’ satisfaction. Keyword: Intervertebral disc displacement/therapy; Diskectomy; Pedicle screws. This study was conducted at the Complejo Asistencial Universitario de Leon, Leon, Spain. Correspondence: Avenida de la costa N°30 5°B, Gijón Astúrias, España. CP.3205. msuarez83@hotmail.com METHOD There was no significant difference either in the levels of patient satisfaction between the groups (p=0.825), averaging higher than 3 in both groups, or in terms of responses to the question about whether they would undergo the surgery again (chi square p=0.103). We organized the patients into two groups by technique: one group with dynamic rod instrumentation and release of the compressed root without discectomy and the other with simple discectomy. We compared the results obtained in each group. For the statistical analysis, we used SPSS v20.0 software. The level of significance was established at 95%. y g g y g ( q p ) The only point where differences were encountered was in the use of corticosteroids prior to surgery, higher in the group of patients without instrumentation (p=0.001). The population sample with the lowest satisfaction with the surgery (those who left a poor or average rating, who would not recommend the technique, and who would not undergo the surgery again) was a group of 23 patients (65% of whom were women) with an average age of 44 years ±8, slightly overweight (BMI=25.6), with L4-L5 involvement (52%), and being treated with psychoactive drugs (56%). INTRODUCTION induction and two hours prior to the surgery. The patients who un- derwent surgery in the Neurosurgery Service received antibiotic prophylaxis 90 minutes before the surgery began. The level to be operated on was determined intraoperatively using a fluoroscope. Chronic back pain is one of the greatest problems related to decreased quality of life of the patient. Nonetheless, the surgical approach to disc hernias of more than six months of evolution, as- sociated with degenerative discopathies that do not respond to conservative treatment, continues to be a challenge.1 Release and dynamic stabilization technique Pedicular instrumentation system with dynamic rods: The con- ventional technique without a microscope was used. An incision was made in the skin and then a dissection by planes with an electric scalpel. A foraminotomy or a flavectomy was performed to decom- press the nerve root. The procedure continued with the placement of transpedicle screws assisted by a fluoroscope and the dynamic rod was installed. Discectomies or bone grafts (arthrodesis) were not performed in any of the cases. g We often encounter studies in scientific journals that present poor disc hernia surgery outcomes within the context of degenerative pathology, and even with no degenerative pathology, in cases where they have evolved over time.2 Thus, the concept of postdiscectomy syndrome has emerged to define a clinical profile of the recurrence of lumbar and radicular pain related to periradicular fibrosis, instability of the operated segment, degeneration of the segment, etc.3 Distinct surgical techniques have been developed to treat this problem, among which is included dynamic stabilization of the oper- ated segment.4 The goal of this technique is to stabilize the segment, without fusion in order to reduce mobility and also to reduce and/or prevent postsurgical degeneration.5,6 Other indications, not addressed in this study, are used to prevent the adjacent segment syndrome, only applicable in cases of fusion with or without instrumentation.7 RESULTS One hundred and forty-two patients, who underwent surgery be- tween 2009 and 2012, with an average follow-up time of 44 months (ranging from 16-65 months), were included in the study. Instrumentation with dynamic rods was used in 86 patients and other techniques without instrumentation were used in the other 56 patients (18 patients under- went microdiscectomies with foraminotomy and 38, microdiscectomies without foraminotomy). The distribution by sex was 70 males (49%) and 72 females (51%). The average age was 43.67 years (ranging from 21 to 65 years of age) and the average BMI was 25.25 (from 15.4 to 36.9). The average surgical time was 109 minutes (ranging from 45 to 275 minutes). The average clinical follow-up time prior to surgery was 17.9 months (ranging from 6 to 192 months). The most common locations were L4-L5 right (39 patients, 25.7%), L5-S1 left (39 patients, 25.7%), L4-L5 left (35 patients, 23%), and L5-S1 right (25 patients, 16.4%). Simple discectomy technique Microdiscectomy: An incision is made in the skin, then dissection by planes using an electric scalpel or a cold scalpel and scissors is performed. Once the space is located, it is confirmed using the fluoroscope. From this point on, the procedure continues under the microscope. Once the space where the disc hernia is found, the root is located and the protruding disc and the foraminal disc fragments are removed, and then we move on to the discectomy. The patient receives a vial of extended release corticosteroid and a compound to prevent fibrosis. A foraminotomy is performed in cases where the space is compromised and a laminectomy when there is a disk fragment sequestered inside the channel or in cases of large volume disc hernias. y pp Our objective is to compare the results of treatment in patients with low back pain and radiculalgia secondary to a herniated disc associated with disc degeneration, using instrumentation with pedicle screws and dynamic rods, releasing the root without discectomy, with those of the other technique still considered the gold standard for disc surgery, microdiscectomy with or without foraminotomy. Given that we compared two different techniques, we analyzed whether the impingement of the intervertebral disc, a recognized cause of the onset of fibrosis,8 and whether laminectomy and/or foraminotomy, possible causes for segmental instability,9 would have better clinical outcomes in reducing possible postlaminectomy segmental instability than to not carry out discectomy and than the use of dynamic stabilization. RESUMO Objetivo: Comparar os resultados do tratamento de pacientes com dor lombar e radiculalgia decorrente de hérnia de disco associada à degeneração do disco, por meio de instrumentação com parafusos pediculares e barra dinâmica com liberação da raiz sem discotomia em com- paração com outras técnicas não instrumentadas (microdiscotomia com ou sem foraminotomia). Métodos: Foi realizado um estudo retrospectivo e descritivo de intervenções realizadas em pacientes com hérnia de disco nos serviços de Traumatologia e Neurocirurgia, empregando-se as seguintes variáveis: idade, sexo, tipo de técnica, tempo de cirurgia, tempo de evolução, grau de satisfação e complicações. Foram formados dois grupos: instrumentação com barras dinâmicas e técnicas não instrumentadas, comparando os resultados de cada grupo. O software utilizado foi o SPSS v20.0. Resultados: Apresentamos 142 intervenções realizadas entre 2009 e 2012, 86 por instrumentação dinâmica e 56 por técnicas de descompressão sem instrumentação. Não foram observadas diferenças estatisticamente significativas entre os grupos de idade, sexo e tempo de evolução até a intervenção. Constataram-se diferenças estatisticamente significativas quanto ao tempo cirúrgico (p = 0,001), sendo menor na técnica instrumentada. Com relação às complicações das técnicas, não houve diferença significativa nas re-operações nem nas infecções. Conclusões: Não foram encontradas diferenças significativas entre o uso de instrumentação com barras dinâmicas com respeito a outras técnicas cirúrgicas não instrumentadas no tratamento da hérnia de disco com mais de 6 meses de evolução nem a complicações e ao grau de satisfação dos pacientes. Descritores: Deslocamento do disco intervertebral/terapia; Discotomia; Parafusos pediculares. This study was conducted at the Complejo Asistencial Universitario de Leon, Leon, Spain. Correspondence: Avenida de la costa N°30 5°B, Gijón Astúrias, España. CP.3205. msuarez83@hotmail.com http://dx.doi.org/10.1590/S1808-185120161504146381 Received on 02/24/2015, accepted on 06/03/2016. Coluna/Columna. 2016;15(4):295-8 296 METHOD This was a retrospective review of the surgeries performed in patients with disc herniations by the Traumatolgy and Neurosurgery Services from 2009 to 2012. The variables collected were: age, sex, BMI, type of technique used, surgical time, time of disease progression until surgery, patient satisfaction level (1=poor, 2=average, 3=good, 4=excellent), and the presence of complications (infections, failure of the osteosynthesis materials, reinterventions, follow-up in pain treatment centers). We did not observe any statistically significant differences in the composition of the groups in terms of age, sex, time of disease progression until the intervention, BMI, location of the hernia, or duration of postoperative follow-up. Statistically significant differences were observed (p=0.001) between surgical times, with an average of 84 minutes ±22.58) for the dynamic technique and 150 minutes (±38.76) for the discectomies. (Table 1) The study was approved by Institutional Rewiew Board of the Hospital de León (the number of the research protocol was 1622), the patients signed the informed consent form for participation in the study, and all the data was anonymized. The inclusion criteria were: Patients between 18 and 65 years of age, who suffered from low back pain and radiculalgia secondary to disc herniation associated with disc degeneration at a single level (confirmed by magnetic resonance imaging), and who underwent medical treatment and rehabilitation for at least six months. All the patients were informed about the surgical procedures and signed the informed consent form. We excluded patients with non-lumbar disc hernias or without medical histories, patients with less than six months of disease progression, who had undergone previous surgeries, and who had stenosis, listhesis, segmental instability, infections, tumors, scoliosis, vertebral fractures, or severe systemic illnesses. There were 25 reinterventions (16.4%), 5 infections (3.3%), and 13 referrals to the pain treatment center for follow-up (8.6%). There were no significant differences between the comparison groups either for reinterventions (p=0.344), infections (p=0.447), or for follow-up in the pain treatment center (p=0.706). (Table 2) There was no significant difference either in the levels of patient satisfaction between the groups (p=0.825), averaging higher than 3 in both groups, or in terms of responses to the question about whether they would undergo the surgery again (chi square p=0.103). The only point where differences were encountered was in the use of corticosteroids prior to surgery, higher in the group of patients without instrumentation (p=0.001). Coluna/Columna. 2016;15(4):295-8 DISCUSSION Furthermore, this study should have been conducted with a randomized population, recording the symptoms as a function of time, as was not the case. In their observational studies, Nygaard et al.14, Ng and Sell15 con- cluded that surgery following from eight to twelve months of sciatica produced worse results than surgery performed earlier. However, we must keep in mind that it is difficult to make patients with persistent sciatica wait for 8-12 months of conservative treatment. Furthermore, this study should have been conducted with a randomized population, recording the symptoms as a function of time, as was not the case. However, the general conclusions from the random trials conducted by Weinstein, Osterman, and Butterman do not suggest unsatisfactory outcomes from long-term conservative treatment.16-18 In our review of the articles that analyze the outcomes of treatments for disc herniations, we observed that there are discrepancies among the scientific works in terms of methodology, surgical outcomes, and follow-up. Perhaps the main issues are data collection bias and the great variability among the individual characteristics of the patients presented by the different studies.29 In our study, the fact that the patients were separated into two cohorts, each of which was operated, treated, and analyzed by a different service was a limitation because the results are not entirely comparable. However, the general conclusions from the random trials conducted by Weinstein, Osterman, and Butterman do not suggest unsatisfactory outcomes from long-term conservative treatment.16-18 From all these results, we can conclude that early surgery (a clinical history of sciatica for 6-12 weeks) does not lead to better long-term results. The only benefits are a faster decline of the radiculopathy and an earlier recovery. This, however, can be considered a valuable advantage for the part of the population that is unable to, unwilling to, or cannot wait for the natural course of the disease or for the possibility of a delayed surgery if necessary. Coluna/Columna. 2016;15(4):295-8 Surgical intervention and indication These systems are technically more complex, require longer surgical time, and have more complications.2 Instead, dynamic stabilization devices function together with the movement segment, without replacing any anatomical structure. Semirigid fixation is the most commonly used term to describe these devices, a questionable concept and one we do not share since dynamic systems do not produce fusion. In fact, there is no graft involved, it is only intended to offer stabilization without the tension that conventional rigid fixation produces. Back pain and sciatica following discectomy may be due to segmen- tal instability and to the concept of chronic degenerative instability.22,24 Formerly, fixation systems were static, but today dynamic stabilization systems are beginning to be used. These devices preserve movement and can be classified as prosthetic or dynamic. With prosthetic devices, the disc, the nucleus, and the facet joints are completely substituted by the prosthesis, replacing the anatomic structure and functions of the lumbar movement segment. These systems are technically more complex, require longer surgical time, and have more complications.2 Instead, dynamic stabilization devices function together with the movement segment, without replacing any anatomical structure. Semirigid fixation is the most commonly used term to describe these devices, a questionable concept and one we do not share since dynamic systems do not produce fusion. In fact, there is no graft involved, it is only intended to offer stabilization without the tension that conventional rigid fixation produces. The main complications reported in the dynamic approach group were residual pain and instrumentation failure, while in the other tech- nique group hernia recurrence and dural injuries were predominant. DISCUSSION The devices restrict movement to a certain extent and allow the load to be shared between the device and the movement segment. For the long-term survival of the device, the loads and the movement must be shared with the device, which complements the kinematics of the segment in motion. If this does not happen, the device can end up failing from overuse (breakage or loosening). This must be avoided since the device must endure for an indefinite period of time. They bring the advantages of easy conversion to conventional stabilization or replacement of parts, compatibility with minimally invasive procedures, and restoration of anatomical lordosis.25 Degenerative disc disease and disc hernias are the most com- mon problems in patients with low back pain10 and one of the most common causes of work leave. Nevertheless, the treatment of disc hernias is very controversial and there are a multitude of studies that present contradictory conclusions.11 For example, the results published by Weber10 reported that prolonged conservative treatment has outcomes after four years of follow-up similar to those achieved through early surgery. After this, several observational cohort studies were conducted that presented worse results from conservative treatment as compared to early surgery. They came to the conclusion that, after two months of sci- atica, outcomes from conservative treatment are worse than those from surgical intervention.12,13 All of these results must be viewed with caution because the studies were not based on randomized populations and included patients who did not receive the same analgesic regimens or follow the same recommendations, making the outcomes not totally comparable. p Dynamic stabilization is used to eliminate lumbar pain and stabilize degenerated discs. These systems enable a more physiological transmission of forces between the anterior and posterior compo- nents of the lumbar spine, while maintaining mobility and controlling abnormal movements in the lumbar segment.26,27 These semirigid stabilization systems restore normal spine functions and protect the adjacent segments.27,28 In our review, there was only one case of degeneration of the adjacent disk. In their observational studies, Nygaard et al.14, Ng and Sell15 con- cluded that surgery following from eight to twelve months of sciatica produced worse results than surgery performed earlier. However, we must keep in mind that it is difficult to make patients with persistent sciatica wait for 8-12 months of conservative treatment. Surgical intervention and indication The patients were operated in the prone position under general anesthesia. They received prophylactic antibiotics with the anesthetic Coluna/Columna. 2016;15(4):295-8 Coluna/Columna. 2016;15(4):295-8 COMPARATIVE STUDY ON THE TREATMENT OF DISC HERNIATIONS 297 Table 1. Comparison of variables between the instrumented technique and dis- cectomy. Technique Statistical Significance Instrumentation Discectomy Age 44.3 42.7 0.111 Sex Male 43 27 0.835 Female 43 29 Time since the surgery (months) 18.9 16.9 0.311 BMI 25.7 25.2 0.362 Time of postoperative follow-up (months) 42.9 45.7 0.256 Surgical time (mintues) 84 150 0.002 Table 2. Complications. Technique P value Instrumentation No instrumentation Number of reinterventions 12 13 0.344 Number of infections 2 3 0.447 Number of patients in follow-up in pain treatment centers 8 5 0.706 The general recommendation is to wait for a period of 6 to 12 weeks after the onset of symptoms, except in cases of cauda equina or rapid loss of motor function.13,19 However, taking all the studies into account, perhaps we should rethink this indication because in our study we excluded patients who had undergone surgery after less than 6 months of progression. p g Currently, there are numerous techniques for treating degenerative disc disease with debatable results.15 In the last century, surgical treat- ment for pain in degenerative disc diseases began with discectomies and decompressions. The first lumbar discectomy was performed by Mixter and Barr20 in 1934, and became the most used technique. Today, the microdiscectomy, a less traumatic procedure that permits smaller incisions of the skin and muscle tissue, is being performed. The simple discectomy and the microdiscectomy are considered to be the gold standards for the surgical treatment of lumbar disc hernias.2 However, these techniques have not yielded good results in the treatment of chronic back pain following disc suppression, since low back pain and sciatica persisted in up to 40% of cases, although in only 20% in our sample. g y Back pain and sciatica following discectomy may be due to segmen- tal instability and to the concept of chronic degenerative instability.22,24 Formerly, fixation systems were static, but today dynamic stabilization systems are beginning to be used. These devices preserve movement and can be classified as prosthetic or dynamic. With prosthetic devices, the disc, the nucleus, and the facet joints are completely substituted by the prosthesis, replacing the anatomic structure and functions of the lumbar movement segment. REFERENCES follow up. J Neurosurg. 2000;92(2 Suppl):131-4. 1. Nyström B. Spinal fusion in the treatment of chronic low back pain: rationale for improvement. Open Orthop J. 2012;6:478-81. 1. Nyström B. Spinal fusion in the treatment of chronic low back pain: rationale for improvement. Open Orthop J. 2012;6:478-81. 15. Ng LC, Sell P. Predictive value of the duration of sciatica for lumbar disce prospective cohort study. J Bone Joint Surg Br. 2004;86(4):546-9. improvement. Open Orthop J. 2012;6:478-81. 2. Mirza SK, Deyo RA. Systematic review of randomized trials comparing lumbar fusion surgery to nonoperative care for treatment of chronic back pain. Spine (Phila Pa 1976). 2007;32(7):816-23. 2. Mirza SK, Deyo RA. Systematic review of randomized trials comparing lumbar fusion surgery to nonoperative care for treatment of chronic back pain. Spine (Phila Pa 1976). 2007;32(7):816-23. 16. Jacobs WC, Arts MP, van Tulder MW, Rubinstein SM, van Middelkoop M, Ostelo RW, Verhagen AP, Koes BW, Peul WC. Surgical techniques for sciatica due to herniated disc, a systematic review. Eur Spine J. 2012;21(11):2232-51. 3. Anandjiwala J, Seo JY, Ha KY, Oh IS, Shin DC. Adjacent segment degeneration after instrumented posterolateral lumbar fusion: a prospective cohort study with a minimum five-year follow-up. Eur Spine J. 2011;20(11):1951-60. 17. Osterman H, Seitsalo S, Karppinen J, Malmivaara A. Effectiveness of microdiscectomy for lumbar disc herniation: a randomized controlled trial with 2 years of follow-up. Spine (Phila Pa 1976). 2006;31(21):2409-14. 4. Kaner T, Ozer AF. Dynamic stabilization for challenging lumbar degenerative diseases of the spine: a review of the literature. Adv Orthop. 2013;2013:753470. of the spine: a review of the literature. Adv Orthop. 2013;2013:7 18. Buttermann GR. Treatment of lumbar disc herniation: epidural steroid injection compared with discectomy. A prospective, randomized study. J Bone Joint Surg Am. 2004;86-A(4):670-9. 5. Coe JD, Kitchel SH, Meisel HJ, Wingo CH, Lee SE, Jahng TA. NFlex Dynamic Stabilization System : Two-Year Clinical Outcomes of Multi-Center Study. J Korean Neurosurg Soc. 2012;51(6):343-9. 19. Deyo RA. Back surgery--who needs it? N Engl J Med. 2007;356(22):2239-43. 19. Deyo RA. Back surgery--who needs it? N Engl J Med. 2007 g 6. Sénégas J, Vital JM, Pointillart V, Mangione P. Clinical evaluation of a lumbar interspinous dynamic stabilization device (the Wallis system) with a 13-year mean follow-up. Neurosurg Rev. 2009;32(3):335-41. 20. Mixter WJ, Barr JS. Rupture of the intervertebral disc with involvement of the spinal canal. N Engl J Med. 1934;211:210–5. 21. Striffeler H, Gröger U, Reulen HJ. CONCLUSIONS There are no significant differences between the use of dynamic rods and other non-instrumented surgical techniques in the treatment of disc herniations with more than 6 months of evolution in terms of complications or of the level of patient satisfaction. p p We did find a statistically significant difference between the 298 surgical times required, which was shorter in the patients with dynamic instrumentation. herniations within the context of degenerative disc disease, longer-term studies need to be conducted and compared with other alternative surgical techniques. We also observed a statistically significant difference in the proportion of corticosteroids used prior to surgery, which was lower among the patients with dynamic instrumentation. All the authors declare that there are no potential conflicts of interest regarding this article. Because few long-term studies have been published comparing the different surgical techniques used for the treatment of disc CONTRIBUTION OF THE AUTHORS: Each author made significant individual contributions to the development of the manuscript. They all reviewed and approved the final version of the work. MLSH Data collection, bibliographical research, and writing of the manuscript. EID Data collection and analysis. AAC Data analysis and interpretation. JBN Critical review of the manuscript. SSC Bibliographical research. ALMS Data collection. MFG Design and critical i f th t d CONTRIBUTION OF THE AUTHORS: Each author made significant individual contributions to the development of the manuscript. They all reviewed and approved the final version of the work. MLSH Data collection, bibliographical research, and writing of the manuscript. EID Data collection and analysis. AAC Data analysis and interpretation. JBN Critical review of the manuscript. SSC Bibliographical research. ALMS Data collection. MFG Design and critical review of the study. REFERENCES “Standard” microsurgical lumbar discectomy vs. “conservative” microsurgical discectomy. A preliminary study. Acta Neurochir (Wien). 1991;112(1-2):62-4. p g 7. Sengupta DK, Herkowitz HN. Pedicle screw-based posterior dynamic stabilization: literature review. Adv Orthop. 2012;2012:424268. 8. Ross JS, Robertson JT, Frederickson RC, Petrie JL, Obuchowski N, Modic MT, deTribolet N. Association between peridural scar and recurrent radicular pain after lumbar discectomy: magnetic resonance evaluation. ADCON-L European Study Group. Neurosurgery. 1996;38(4):855-61. 22. Caspar W, Campbell B, Barbier DD, Kretschmmer R, Gotfried Y. The Caspar microsurgical discectomy and comparison with a conventional standard lumbar disc procedure. Neurosurgery. 1991;28(1):78-86. 23. Vaughan PA, Malcolm BW, Maistrelli GL. Results of L4-L5 disc excision alone versus disc excision and fusion. Spine (Phila Pa 1976). 1988;13(6):690-5. p g y 9. Mullin BB, Rea GL, Irsik R, Catton M, Miner ME. The effect of postlaminectomy spinal instability on the outcome of lumbar spinal stenosis patients. J Spinal Disord. 1996;9(2):107-16. 24. Panjabi MM. Clinical spinal instability and low back pain. J Electromyogr Kinesiol. 2003;13(4):371-9. 10. Weber H. Lumbar disc herniation. A controlled, prospective study with ten years of observation. Spine (Phila Pa 1976). 1983;8(2):131-40. 25. Schwarzer AC, Aprill CN, Derby R, Fortin J, Kine G, Bogduk N. The relative contributions of the disc and zygapophyseal joint in chronic low back pain. Spine (Phila Pa 1976). 1994;19(7):801-6. 11. Peul WC, van den Hout WB, Brand R, Thomeer RT, Koes BW; Leiden-The Hague Spine Intervention Prognostic Study Group. Prolonged conservative care versus early surgery in patients with sciatica caused by lumbar disc herniation: two year results of a randomised controlled trial. BMJ. 2008;336(7657):1355-8. 26. Sengupta DK. Dynamic stabilization devices in the treatment of low back pain. Neurol India. 2005;53(4):466-74. 27. Bothmann M, Kast E, Boldt GJ, Oberle J. Dynesys fixation for lumbar spine degeneration. Neurosurg Rev. 2008;31(2):189-96. 12. Hurme M, Alaranta H. Factors predicting the result of surgery for lumbar intervertebral disc herniation. Spine (Phila Pa 1976). 1987;12(9):933-8. 28. Kaner T, Sasani M, Oktenoglu T, Cosar M, Ozer AF. Utilizing dynamic rods with dynamic screws in the surgical treatment of chronic instability: a prospective clinical study. Turk Neurosurg. 2009;19(4):319-26. 13. Rothoerl RD, Woertgen C, Brawanski A. When should conservative treatment for lumbar disc herniation be ceased and surgery considered? Neurosurg Rev. 2002;25(3):162-5. 29. Grieve GP. Movilizacion de la columna vertebral: Manual basico de metodo clinico. Barcelona: Paidotribo; 2001. 14. Nygaard OP, Kloster R, Solberg T. REFERENCES Duration of leg pain as a predictor of outcome after surgery for lumbar disc herniation: a prospective cohort study with 1-year 14. Nygaard OP, Kloster R, Solberg T. Duration of leg pain as a predictor of outcome after surgery for lumbar disc herniation: a prospective cohort study with 1-year Coluna/Columna. 2016;15(4):295-8
https://openalex.org/W1971914405	https://wwwnc.cdc.gov/eid/article/14/12/pdfs/08-0695.pdf	English	null	Enzootic Angiostrongyliasis in Shenzhen, China	Emerging infectious diseases	2,008	cc-by	1,745	LETTERS Address for correspondence: Laura Tomassone, Dipartmento di Produzioni Animali, Ecologia ed Epidemiologia, Università di Torino, Italy, via L da Vinci 44, 10095 Grugliasco, Torino, Italy; email: laura.tomassone@unito.it cantonensis occurs in wild rats and snails in Shenzhen. Persons in Shenzhen eat raw or undercooked freshwater and terrestri- al snails and slugs. This practice pro- vides opportunities for infection with A. cantonensis, particularly given that P. canaliculata has been aquacultured intensively for human consumption. The prevalence of A. cantonensis in wild rats and snails in Shenzhen poses substantial risk for future outbreaks of human eosinophilic meningitis. Moreover, public health ofﬁ cials, epidemiologists, researchers, clini- cal technicians, medical practitioners, parasitologists, and veterinarians, as well as the general public, should be aware of such risks, and integrated strategies should be taken to reduce or eliminate such risks. To examine A. cantonensis infec- tion in intermediate host snails, 302 terrestrial snails (Achatina fulica) were collected from 10 investigation sites across Shenzhen, and 314 fresh- water snails (Pomacea canaliculata) were sampled from 6 investigation sites. We examined the snails for A. cantonensis larvae by using pepsin digestion standardized procedures (3). To survey the prevalence of adult A. cantonensis in deﬁ nitive host rats, we collected 187 Rattus norvegicus rats and 121 R. ﬂ avipectus rats collected from 4 sites where positive snails pos- itive for A. cantonensis were found. These rats were examined for the pres- ence of adult A. cantonensis in their cardiopulmonary systems. Acknowledgment p y y A. cantonensis larvae were found in 96 (15.6%) of 616 examined snails. Of these, P. canaliculata had an aver- age infection rate of 20.7% (65/314), signiﬁ cantly higher (p<0.01) than that of A. fulica (10.3%, 31/302), an indication that P. canaliculata may be the principal intermediate host for A. cantonensis in Shenzhen. A. can- tonensis adults were recovered from the cardiopulmonary systems of 37 (12%) of 308 examined rats. Infec- tion rate for R. norvegicus rats was 16.6% (31/187), signiﬁ cantly higher (p<0.01) than that for R. ﬂ avipec- tus (4.9%, 6/121), an indication that R. norvegicus may be the principal deﬁ nitive host for A. cantonensis in Shenzhen, possibly due to the rat’s preference for eating snails. Infec- tion rates were higher for female rats (25.6% for R. norvegicus and 7.8% for R. ﬂ avipectus) than for male rats (8.9% for R. norvegicus, 2.9% for R. ﬂ avipectus), possibly because female rats eat more snails to supply proteins for reproduction. This report of enzo- otic A. cantonensis infection in wild rats and snails in Shenzhen demon- strates the existence of natural origins of infection with A. cantonensis for humans in this city. We thank Alasdair Nisbet for his as- sistance in improving the manuscript. Project support was provided in part by a grant from Shenzhen Municipal Bu- reau of Science and Technology (grant no. 2007079) to R.-L.Z. and a grant from the Program for Changjiang Scholars and Innovative Research Team in University (grant no. IRT0723) to X.-Q.Z. Shenzhen municipality is situated in the most southern part of main- land People’s Republic of China be- tween the northern latitudes of 22°27′ to 22°52′ and eastern longitudes of 113°46′ to 114°37′; it shares a border with the Hong Kong Special Admin- istrative Region, China, in the south. The climate is subtropical, with an av- erage annual temperature of 23.7 °C. The city is 1,952.84 km2 and has a population of 10 million. Enzootic Angiostrongyliasis in Shenzhen, China To the Editor: Angiostrongylus cantonensis is a zoonotic parasite that causes eosinophilic meningitis in hu- mans after they ingest infective larvae in freshwater and terrestrial snails and slugs, paratenic hosts (such as fresh- water ﬁ sh, shrimps, frogs, and crabs), or contaminated vegetables. With the increase of income and living stan- dards, and the pursuit of exotic and delicate foods, populations around the world have seen angiostrongyliasis become an important foodborne para- sitic zoonosis (1–9). DOI: 10.3201/eid1412.080695 DOI: 10.3201/eid1412.080695 1. Alicata JE. The discovery of Angiostrongy- lus cantonensis as a cause of human eo- sinophilic meningitis. Parasitol Today. 1991;7:151–3. DOI: 10.1016/0169-4758- (91)90285-V Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 14, No. 12, December 2008 Ren-Li Zhang, Mu-Xin Chen, Shi-Tong Gao, Yi-Jie Geng, Da-Na Huang, Jian-Ping Liu, Yuan-Liang Wu, and Xing-Quan Zhu Author afﬁ liations: Shenzhen Center for Disease Control and Prevention, Shen- zhen, People’s Republic of China (R.-L. Zhang, M.-X. Chen, S.-T. Gao, Y.-J. Geng, D.-N. Huang, J.-P. Liu, Y.-L. Wu); and South China Agricultural University, Guangzhou, People’s Republic of China (M.-X. Chen, X.-Q. Zhu) Since 2006, thirty-two sporadic cases of human eosinophilic menin- gitis caused by consumption of un- dercooked aquacultured snails have been documented in Shenzhen (Shen- zhen Center for Disease Control and Prevention, unpub. data). To identify the source of these infections and as- sess the risk for an outbreak of eo- sinophilic meningitis, we conducted a survey to investigate whether A. Knowledge about Avian Inﬂ uenza, European Region Eosinophilic meningitis in a returned traveler from Santo Domingo: case report and review. J Travel Med. 2007;14:407–10. DOI: 10.1111/j.1708-8305.2007.00152.x 6. Tsai HC, Liu YC, Kunin CM, Lai PH, Lee SS, Chen YS, et al. Eosinophilic meningi- tis caused by Angiostrongylus cantonensis associated with eating raw snails: correla- tion of brain magnetic resonance imaging scans with clinical ﬁ ndings. Am J Trop Med Hyg. 2003;68:281–5. ( ) Respondents were asked 7 ques- tions about their knowledge of the risks humans face regarding avian inﬂ uenza (Table). When we looked at these results with the aim of set- ting future public health information campaign objectives, we considered incorrect or “don’t know” responses to indicate public health information campaign failures. Uncertainty regard- ing avian inﬂ uenza risks appeared to involve consumption of eggs and vac- cinated, cooked poultry and whether the virus can be transmitted between humans. However, for all questions asked, more than half of the respon- dents answered correctly except when asked about eating poultry that had been vaccinated against avian inﬂ uen- za. This question also had the highest number of “don’t know” responses. Respondents are most knowledge- able about the preventive measure of culling chickens, perhaps because of the media attention these events at- tract. The large percentage of correct answers for some questions points to successes of previous information campaigns and media coverage, but the 40% of respondents answering in- correctly or “don’t know” to questions about poultry and egg consumption 7. Waugh CA, Shaﬁ r S, Wise M, Robinson RD, Eberhard ML, Lindo JF. Human Angiostrongylus cantonensis, Jamaica. Emerg Infect Dis. 2005;11:1977–8. 8. Lv S, Zhang Y, Steinmann P, Zhou XN. Emerging angiostrongyliasis in Mainland China. Emerg Infect Dis. 2008;14:161–4. DOI: 10.3201/eid1401.061529 pp p p A 2006 Eurobarometer survey asked 29,170 residents of the 27 coun- tries in the European Union, Croatia, and Turkey about their knowledge of avian inﬂ uenza risks (2). Eurobarom- eter surveys are undertaken by the Eu- ropean Commission to monitor the EU public’s social and political opinions. The survey was conducted on a mul- tistage random sampling basis. There- fore, the sample is representative of the whole territory surveyed. Each country’s population was randomly sampled according to rural, metropoli- tan, and urban population densities. A cluster of addresses was selected from each primary sampling unit by using country-dependent resources such as electoral registers. References 1. Alicata JE. The discovery of Angiostrongy- lus cantonensis as a cause of human eo- sinophilic meningitis. Parasitol Today. 1991;7:151–3. DOI: 10.1016/0169-4758- (91)90285-V Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 14, No. 12, December 2008 1955 1955 LETTERS Knowledge about Avian Inﬂ uenza, European Region by face-to-face interviews in respon- dents’ homes. 2. Hochberg NS, Park SY, Blackburn BG, Sejvar JJ, Gaynor K, Chung H, et al. Distribution of eosinophilic meningi- tis cases attributable to Angiostrongylus cantonensis, Hawaii. Emerg Infect Dis. 2007;13:1675–80. Data were collected from March 27 through May 1, 2006. This period is especially interesting when looking at Europeans’ knowledge about avian inﬂ uenza risk because the ﬁ rst Euro- pean cases of avian inﬂ uenza (H5N1) were found in October 2005 in Tur- key; additional cases were found later that month in Romania, Croatia, and the United Kingdom. Therefore, the period would have included media coverage about avian inﬂ uenza as well as any targeted public health efforts to inform residents about avian inﬂ uenza risks. By the end of this survey’s ﬁ eld- work period, 17 of the 29 countries surveyed had reported inﬂ uenza virus (H5N1) in birds, 3 in mammals, and 1 in humans (3). To the Editor: Since the ﬁ rst iden- tiﬁ cations of avian inﬂ uenza (H5N1) in Europe in late 2005 and early 2006, Eurobarometer survey data obtained during April–May 2006 have provided a unique opportunity to examine the knowledge of respondents across the European Union, Croatia, and Turkey about the risks and transmission of avian inﬂ uenza. The H5N1 strain of avian inﬂ uenza virus has caused >240 human deaths in central and Southeast Asia, the Middle East, and Africa (1). Four of these deaths occurred in Tur- key in 2006. Understanding gaps in the public’s knowledge about avian inﬂ uenza risks and transmission pro- vides guidance on which issues future public health information campaigns may wish to focus. From a public health perspective, a more informed general public will be less likely to un- necessarily alter their travel and food consumption behavior and more likely to take appropriate preventive actions. 3. Lindo JF, Waugh C, Hall J, Cunningham- Myrie C, Ashley D, Eberhard ML, et al. Enzootic Angiostrongylus cantonensis in rats and snails after an outbreak of human eosinophilic meningitis, Jamaica. Emerg Infect Dis. 2002;8:324–6. 4. Slom TJ, Cortese MM, Gerber SL, Jonse RC, Holtz TH, Lopez AS, et al. An out- break of eosinophilic meningitis caused by Angiostrongylus cantonensis in travel- ers returning from the Caribbean. N Engl J Med. 2002;346:668–75. DOI: 10.1056/ NEJMoa012462 5. Leone S, De Marco M, Ghirga P, Nicas- tri E, Esposito M, Narciso P. Knowledge about Avian Inﬂ uenza, European Region Addresses were chosen systematically by using stan- dard random route procedures, begin- ning with a randomly selected initial address. The survey was conducted 9. Zhou P, Chen N, Zhang RL, Lin RQ, Zhu XQ. Food-borne parasitic zoonoses in China: perspective for control. Trends Parasitol. 2008;24:190–6. DOI: 10.1016/j. pt.2008.01.001 Address for correspondence: Xing-Quan Zhu, Laboratory of Parasitology, College of Veterinary Medicine, South China Agricultural University, 483 Wushan St, Tianhe District, Guangzhou, Guangdong Province 510642, People’s Republic of China; email: xingquanzh@scau.edu.cn 1956 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 14, No. 12, December 2008
https://openalex.org/W3142893186	https://wne.fa.ru/jour/article/download/307/297	Russian	null	Strict Rules of Free Competition (about The Sveriges Riksbank Prize in Economic Sciences in Memory of Alfred Nobel 2020)	Mir novoj èkonomiki	2,021	cc-by	6,746	ОРИГИНАЛЬНАЯ СТАТЬЯ CC BY 4.0 DOI: 10.26794/2220-6469-2021-15-1-50-59 УДК 330.88(045) JEL В20 Strict Rules of Free Competition (about The Sveriges Riksbank Prize in Economic Sciences in Memory of Alfred Nobel 2020) Y P V Y. P. Voronov Institute of Economics and Industrial Engineering, Siberian Branch of Russian Academy of Sciences, Novosibirsk, Russia https://orcid.org/0000-0002-7835-5827 Y. P. Voronov Institute of Economics and Industrial Engineering, Siberian Branch of Russian Academy of Sciences, Novosibirsk, Russia https://orcid.org/0000-0002-7835-5827 Строгие правила свободной конкуренции (о Нобелевской премии по экономике 2020 г.) Ю. П. Воронов р Институт экономики и организации промышленного производства, Сибирское отделение Российской академии наук, Новосибирск, Россия https://orcid.org/0000-0002-7835-5827 © Воронов Ю. П., 2021 Мир новой экономики • Т. 15, № 1’2021 АННОТАЦИЯ Изучение результатов экономических исследований экономистов, удостоенных нобелевских премий, представляет собой принципиально важную задачу для определения наиболее перспективных направлений развития российской экономической науки. В анализе достижений нобелевских лауреатов 2020 г. автор показывает связи между работами лауреатов и их предшественников, многие из которых были также удостоены Нобелевской премии по экономике. Выделены четыре линии развития достижений предшественников: асимметрия информации, ограниченная рацио- нальность, дизайн рыночных механизмов и новый институционализм. В статье не рассматриваются проблемы теории аукционов, касающиеся ее формального обоснования, являющиеся частью математической экономики и теории игр. Основное внимание автор уделяет результатам эмпирического анализа фактически реализуемых аукционов. Этот анализ разделен на три части, соответствующие трем этапам каждого аукциона: стартовые проблемы (недоверие, репутация участников и пр.), ход аукциона (поведение участников и результаты, в частности «проклятие победителя». Основные выводы статьи состоят в том, что премия, полученная за «усовершенствование теории аукционов», вписана в более широкий контекст экономических исследований и что лауреаты решали проблемы поддержания свободной конкуренции и снижения вероятности монополизации рынка. ур р р Ключевые слова: Нобелевская премия по экономическим наукам; аукционы; асимметрия информации; ограниченная рациональность; дизайн рыночных механизмов; теория игр; математическая экономика; «хищническое ценообра- зование»; монополизация рынка; свободная конкуренция; репутация участников аукциона; сговор Для цитирования: Воронов Ю. П. Строгие правила свободной конкуренции (о Нобелевской премии по экономике 2020 г.). Мир новой экономики. 2021;15(1):50-59. DOI: 10.26794/2220-6469-2021-15-1-50-59 ORIGINAL PAPER ЭКОНОМИЧЕСКАЯ ТЕОРИЯ / ECONOMIC THEORY ЭКОНОМИЧЕСКАЯ ТЕОРИЯ / ECONOMIC THEORY Введение «аукцион», правда, не встречался, но, по существу, и лауреаты премии 1995 г. (Р. Лукас), 1996 г. (У. Викри и Дж. Миррлис) и 2007 г. (Л. Гурвич, Э. Маскин и Р. Майерсон) активно работали с темой аукционов. «аукцион», правда, не встречался, но, по существу, и лауреаты премии 1995 г. (Р. Лукас), 1996 г. (У. Викри и Дж. Миррлис) и 2007 г. (Л. Гурвич, Э. Маскин и Р. Майерсон) активно работали с темой аукционов. «аукцион», правда, не встречался, но, по существу, и лауреаты премии 1995 г. (Р. Лукас), 1996 г. (У. Викри и Дж. Миррлис) и 2007 г. (Л. Гурвич, Э. Маскин и Р. Майерсон) активно работали с темой аукционов. Более конкретная на этот раз формулировка достижений лауреатов 2020 г., привязка их именно к аукционам, может создать впечатление, что их ис- следования относятся к частной проблеме, к одной из относительно экзотических рыночных процедур, применяющихся в немногих конкретных ситуациях. Однако это не так. Сами лауреаты вписывают свои работы в существенно более широкий контекст и выработку новых рыночных механизмов. «аукцион», правда, не встречался, но, по существу, и лауреаты премии 1995 г. (Р. Лукас), 1996 г. (У. Викри и Дж. Миррлис) и 2007 г. (Л. Гурвич, Э. Маскин и Р. Майерсон) активно работали с темой аукционов. Более конкретная на этот раз формулировка достижений лауреатов 2020 г., привязка их именно к аукционам, может создать впечатление, что их ис- следования относятся к частной проблеме, к одной из относительно экзотических рыночных процедур, применяющихся в немногих конкретных ситуациях. Однако это не так. Сами лауреаты вписывают свои работы в существенно более широкий контекст и выработку новых рыночных механизмов. Политическая экономия XIX в. была наукой, прин- ципиально отличающейся от современной эконо- мической теории. Тогда было достаточно показать или даже упомянуть, что рынок склонен к моно- полизации. Сейчас же дотошные люди начинают задавать вопросы относительно того, каким же образом конкурентный рынок это себе позволя- ет. Такая мысль приходит в связи со следующим событием. Более конкретная на этот раз формулировка достижений лауреатов 2020 г., привязка их именно к аукционам, может создать впечатление, что их ис- следования относятся к частной проблеме, к одной из относительно экзотических рыночных процедур, применяющихся в немногих конкретных ситуациях. Однако это не так. Сами лауреаты вписывают свои работы в существенно более широкий контекст и выработку новых рыночных механизмов. В 2020 г. два профессора Стэнфордского уни- верситета (США) — Пол Милгром и Роберт Уил- сон — стали лауреатами Нобелевской премии по экономическим исследованиям. Введение Формулировка Нобелевского комитета: «за усовершенствование теории аукционов и разработку новых форматов аукционов». Последнее время практически основным кри- терием выбора новых нобелевских лауреатов-эко- номистов стало фактическое внедрение в хозяй- ственную практику результатов экономических исследований. Но это не исключает и тот факт, что очередные нобелевские лауреаты по экономике вписаны в канву постоянно развивающейся эко- номической науки мира. А, как известно, науки без теории не бывает. Биографии новых нобелевских лауреатов не отличаются резкими изменениями или потрясе- ниями. Пол Милгром получил степень бакалавра в Мичиганском университете, магистра по ста- тистике и доктора по специальности «бизнес» — в Стэнфордском. ABSTRACT Studying the research results by Nobel prize-winning economists is a fundamentally important task for determining the most promising areas of development of Russian economic science. The author traces the connections between all these works and their predecessors’ work, many of whom were also awarded the Nobel prize in economics. Three lines of development of the predecessors’ achievements are identified: information asymmetry, limited rationality, the design of market mechanism and new institutionalism. The article does not address the problems of auction theory related to its formal justification, which is part of mathematical economics and game theory. However, it is noted that this is an © Воронов Ю. П., 2021 Мир новой экономики • Т. 15, № 1’2021 51 Ю. П. Воронов Ю. П. Воронов Ю. П. Воронов essential part of the laureates’ achievements. The article’s main conclusions are that the prize received for “improving the theory of auctions” fits into the broader economic research context. The winners solved the problems of maintaining free competition and reducing the market’s likelihood of monopolization. The author pays special attention to the results of the empirical analysis of actually implemented auctions. This analysis is divided into three parts, corresponding to the three stages of each auction: starting problems (distrust, the reputation of participants, etc.), the course of the auction (behaviour of participants and results, in particular, the “winner’s curse”. Keywords: Nobel prize on economics; auctions, asymmetry of information; restricted rationality; market mechanism design; game theory; mathematical economics; “predatory pricing”; market monopolization; free competition; the reputation of auction participants; collusion For citation: Voronov Y. P. Strict rules of free competition (about the sveriges riksbank prize in economic sciences in memory of Alfred Nobel 2020). Mir novoi ekonomiki = The World of New Economy. 2021;15(1):50-59. DOI: 10.26794/2220-6469- 2021-15-1-50-59 ЭКОНОМИЧЕСКАЯ ТЕОРИЯ / ECONOMIC THEORY участника рынка ограничена потому, что он не может быть «идеальным вычислителем». фом, Майклом Спенсом и Джозефом Стиглицем. Предшественник лауреатов — также и лауреат Но- белевской премии по экономике 2001 г. «за анализ рынков с асимметричной информацией» Майкл Спенс 1. Он подробно анализировал результаты торгов, когда участники располагают различной информацией, в частности анализировал резуль- таты аукционной торговли. Но дело не только в ограниченных возможностях человека предвидеть будущее [3]. В своем поведении многие участники рынка часто руководствуются принципом удовлетворенности, а вовсе не мак- симизации прибыли. Думаю, многим читателям приходилось встречаться с людьми, которые, до- бившись определенного уровня достатка, перестают заботиться о его дальнейшем повышении. Собственно, путь двух лауреатов к премии 2020 г. начался со статьи Роберта Уилсона, написанной именно в русле концепции асимметричной инфор- мации [1]. Он прорабатывал конкретную ситуацию, когда в начале торгов информация между участни- ками распределена неравномерно, а по ходу дела эта неравномерность устраняется. Затем Р. Уилсон попытался уйти в формализацию не только аукци- онов и других видов рыночных сделок, но и долго временных партнерств [2], но аукционная торговля впоследствии заняла у него основное внимание. Способствовало такому возврату, без сомнения, то, что его идеи стал развивать Пол Милгром. Один очень близкий предшественник Пола Ми- лгрома и Роберта Уилсона из числа нобелевских лауреатов — Райнхард Зельтен [4], получивший премию в 1994 г.3 Концепция Р. Зельтена может рассматриваться как дальнейшее развитие идей ограниченной рациональности Г. Саймона. Линия третья — новые форматы аукционов как часть дизайна рыночных механизмов В 2007 г. лауреатами Нобелевской премии стали Эрик Маскин 4 и Роджер Майерсон 5, которые вне- сли большой вклад в теорию аукционов, рассма- тривая их как часть более общей проблемы раз- работки (дизайна) рыночных механизмов. К тому времени Пол Милгром и Роберт Уилсон были уже не только исследователями, но и признанными разработчиками новых форматов аукционов. 1 Спенс Майкл (р. 1943 г.) — профессор Калифорнийского уни- верситета (Беркли). 2 Саймон Герберт Александер (1916–2001) — профессор Уни- верситета Карнеги-Меллона, лауреат Нобелевской премии по экономике 1978 г. Линия первая — асимметрия информации Р. Уилсон все три этих степени получил в Гар- вардском университете. Научным руководителем у него был выдающийся специалист по теории при- нятия решений Говард Райффа. П. Милгром и Р. Уилсон получили премию, прежде всего, потому, что они практически внедряли до- стижения теории аукционов. Но в формулировке о присуждении премии на первый план выдвига- ются достижения лауреатов по разработке теории аукционов. Такое решение отчасти связано с тем, что аукционы занимают все более заметное место в системе рыночных механизмов. Но самая глав- ная причина в том, что аукционы связаны с не- сколькими линиями современных экономических теорий. Одна из них — концепция асимметричной информации, развитая лауреатами Нобелевской премии по экономике 2001 г. Джорджем Акерло- Оба лауреата сейчас преподают в Стэнфордском университете и работают на соседних кафедрах. Как пошутил Р. Уилсон: «И на работе, и дома расстояние между нами не больше 40 метров». То, что лауреаты — из США, стало обычным явле- нием. Вероятность получения Нобелевской премии по экономике для американцев вдвое выше, чем для граждан остального мира. Сама тема, которой занимались лауреаты, также не нова для Нобелевского комитета. В обоснованиях достижений предшествующих лауреатов термин wne.fa.ru ЭКОНОМИЧЕСКАЯ ТЕОРИЯ / ECONOMIC THEORY 3 Р. Зельтен (1930–2016) — единственный немецкий эконо- мист, удостоенный Нобелевской премии по экономике. Его достижения остались в тени Джона Нэша, вместе с которым он эту премию получил. 4 Маскин Эрик (р. 1950 г.) — профессор Принстонского универ- ситета, лауреат Нобелевской премии по экономике 2007 г. 5 Майерсон Роджер (р. 1951 г.) — профессор Чикагского уни- верситета, лауреат Нобелевской премии по экономике 2007 г. Линия вторая — ограниченная рациональность Начало этому направлению исследований поло- жил лауреат Нобелевской премии по экономике Герберт Саймон 2, отметивший, что человек вну- три организации объективно имеет ограничения на свое рациональное поведение. В экономической теории активно обсуждаются проблемы самых замысловатых рыночных ме- ханизмов, весьма далеких от обычной торговой сделки. Весьма примечателен в связи с этим переход предшественников П. Милгрома и Р. Уилсона от трактовки рыночного механизма как результата естественного хода событий к пониманию его как цели проектирования. Исходно экономисты вносили в термин «раци- ональный» совсем иной смысл, далекий от того, что подразумевают под ним обычные люди. Раци- ональное поведение, рассматриваемое экономиче- ской теорией, довольно «туповато» и состоит лишь в стремлении использовать ресурсы, которыми располагает человек, с наилучшей выгодой для себя. При этом, как отметил Пол Милгром, неявно допускается, что (согласно теории) у рационального человека есть один неограниченный ресурс — его интеллект и умение предвидеть будущие события. В теории проектирования рыночных механиз- мов было показано, почему аукцион оказывается обычно наиболее эффективным способом рас- пределения ресурсов частных (не общественных) товаров среди данной совокупности покупателей. Эта теория демонстрирует, в частности, что ме- ханизм аукционов приносит максимальный из На этот недостаток теории указал Герберт Сай- мон, предложив концепцию ограниченной реаль- ности, основанную на следующих предпосылках. Субъекты рынка имеют ограниченные способности четкого определения целей и предвидения послед- ствий принимаемых ими решений. Рациональность 3 Р. Зельтен (1930–2016) — единственный немецкий эконо- мист, удостоенный Нобелевской премии по экономике. Его достижения остались в тени Джона Нэша, вместе с которым он эту премию получил. 1 Спенс Майкл (р. 1943 г.) — профессор Калифорнийского уни- верситета (Беркли). 4 Маскин Эрик (р. 1950 г.) — профессор Принстонского универ- ситета, лауреат Нобелевской премии по экономике 2007 г. 2 Саймон Герберт Александер (1916–2001) — профессор Уни- верситета Карнеги-Меллона, лауреат Нобелевской премии по экономике 1978 г. 5 Майерсон Роджер (р. 1951 г.) — профессор Чикагского уни- верситета, лауреат Нобелевской премии по экономике 2007 г. Мир новой экономики • Т. 15, № 1’2021 53 Ю. П. Воронов Ю. П. Воронов ющий один из тезисов Гегеля: «Все существующие рыночные институты целесообразны, и нужно объ- яснять причины их существования». ожидаемых доходов продавцу. В отечественной литературе критерий дохода продавца (во многих случаях это — государство) полагается определяю- щим. Но в работах лауреатов используются и другие критерии, в частности максимум эффективности проданного права (лицензии). Наряду с утверждением необходимости анали- зировать и объяснять то, что существует, неоин- ституционализм как относительно новое (и века не прошло) направление экономической мысли базируется на нескольких постулатах. Линия четвертая — новые институционалисты Большинство исследователей считают П. Милгро- ма и Р. Уилсона продолжателями относительно нового направления в экономической науке — не- оинституционализма. Создателями этого направ- ления считаются нобелевские лауреаты по эконо- мике Рональд Коуз 6 и Дуглас Норт 7. Сам термин «неоинституционализм» ввел в на- учный оборот Оливер Уильямсон 8. Считается, что он же окончательно оформил это направление эко- номической мысли в цельное учение. Основной труд этого направления — монография Дугласа Норта «Институты, институциональные изменения и экономические преобразования» [6]. Но начало неоинституциональному направлению экономи- ческой мысли положила работа Р. Коуза «Природа фирмы» [7]. В ней были поиски ответа на неожи- данный вопрос: «Почему существуют фирмы?». Р. Коуз отвечал на него так: «Потому что внутри них ниже трансакционные издержки». Вопрос, на который Р. Коуз отвечал, со временем ушел в тень, внимание стали уделять исключительно теории трансакционных издержек, автором которой спра- ведливо считается Р. Коуз. Значительное внимание неоинституционали- сты уделяют анализу изменений структуры эко- номических агентов: домашних хозяйств, частных компаний и государства, которые неоклассиками воспринимались как данность и отдельно не из- учались. Признавая ограничения, которые учитывались неоклассической теорией (а именно — физиче- ские, порождаемые редкостью ресурсов, и тех- нологические, отражающие уровень развития знаний и мастерства экономических агентов), неоинституционалисты вводят еще один тип ограничений, связанных с институциональной структурой общества. Они утверждают, что в свя- зи с принятием новой разновидности ограниче- ний можно будет лучше понять и объяснить про- цессы реального мира, в котором экономические агенты действуют в условиях неопределенности и риска, высоких трансакционных издержек, не- четко определенных прав собственности, не- надежных договоров. Тем не менее игнорируется более важный эле- мент неоинституциональной концепции, развива- 6 Рональд Гарри Коуз (1910–2013) — английский экономист, профессор Чикагского университета, лауреат Нобелевской пре- мии по экономике 1991 г. «за открытие и прояснение точного смысла трансакционных издержек и прав собственности в ин- ституциональной структуре и функционировании экономики». 7 Дуглас Сесил Норт (1920–2015) — американский эконо- мист, профессор Вашингтонского университета, лауреат Но- белевской премии по экономике 1993 г. Получил ее совместно с Робертом Фогелем «за возрождение исследований в области экономической истории, благодаря приложению к ним эконо- мической теории и количественных методов, позволяющих объяснять экономические и институциональные изменения». 8 Оливер Итон Уильямсон (1932–2020) — профессор Кали- форнийского университета, лауреат Нобелевской премии по экономике в 2009 г. Мотивация поведения в неоинституционализ- ме более многообразная, чем в других течениях экономической мысли. Линия вторая — ограниченная рациональность Первый — субъекты экономики способны принимать решения о своих действиях индивидуально. Второй — ры- ночные институты появляются под влиянием по- ведения и интересов конкретных субъектов рынка, которые стремятся избежать хаоса. Информация, которой пользуются субъекты экономики, всегда асимметрична. У одной стороны сделки она больше, у другой — меньше. Это свойство экономической информации служит основой для недобросовестного (оппортунистического) поведения, которое, согла- сно концепции неоинституционализма, в равной мере должно учитываться теорией, как и поведение рациональное. В перспективе при разработке новых форматов аукционов рассматривается использование искус- ственного интеллекта [5]. ЭКОНОМИЧЕСКАЯ ТЕОРИЯ / ECONOMIC THEORY условных экономик. Но наиболее интересно то, что связано с обработкой данных и анализом факти- ческих аукционов [10]. Традиционной неоклассической теории, по мнению О. Уильямсона [8], присуща технологиче- ская парадигма. Неоинституционализм опирается в большей мере на представление об экономике как о системе взаимовыгодных отношений между людьми и компаниями. Реализация таких отно- шений невозможна иначе, чем через институци- ональную среду. Помимо серии формальных обоснований, те- ория аукционов включает в себя систему анализа эффективности различного типа аукционов. В ней рассматриваются три этапа аукциона: стартовые позиции участников, поведение их в ходе аукциона и оценка ими результатов по завершении аукци- она [11]. Представителей неоинституционализма более чем других обвиняют в «экономическом импери- ализме», т. е. в том, что они усматривают эконо- мические явления и процессы во всех сторонах общественной жизни. С такими критиками можно и поспорить. Если экономисты видят в проявле- ниях дружеских чувств возможность получения беспроцентного кредита, это не мешает психологам исследовать дружеские чувства другими методами и с помощью иного понятийного аппарата. 9 Уильям Викри (1914–1996) — американский экономист, профессор Колумбийского университета, лауреат Нобелевской премии по экономике 1996 г. Проблема старта — недоверие При любой торговой сделке в той или иной мере покупатель сомневается в свойствах покупаемого им товара или услуги. Благодаря формализации процедуры торгов в аукционной торговле уро- вень этих сомнений возможно оценить по ходу и по результатам аукциона. Один из элементов теории аукционов — оценка исходного недоверия покупателя. Этот «недостаток» я считаю обязательным для экономиста. Пусть уж психологи и социологи от- воевывают занятую нами территорию. Этот «недостаток» я считаю обязательным для экономиста. Пусть уж психологи и социологи от- воевывают занятую нами территорию. Поведение участников рынка зависит от част- ной информации, которой они владеют. Лауреаты премии 2020 г. провели множество эмпирических исследований, доказывающих, что при разработке формата аукциона в обязательном порядке нужно максимально снимать подозрения покупателей в том, что какая-то часть информации от них спе- циально скрыта [12]. Подозрения повышают осто- рожность участников и сдерживают их от высоких цен спроса. Уровень стартового недоверия может быть выявлен в ходе специальных исследований и опросов. Линия четвертая — новые институционалисты У классиков и неокласси- ков основной, а зачастую и единственный мотив поведения — максимизация выгоды, а в неоин- ституционализме анализируется механизм (ход) принятия решений, его условия и предпосылки. Мотив становится всего лишь одним из элементов предпосылок, которые изменчивы. wne.fa.ru Проблемы в ходе аукциона — возможность сговора Второй элемент теории аукционов базируется на анализе поведения участников в ходе аукци- она. Если в одном случае покупатели определя- ют свои предложения простой добавкой фикси- рованной суммы, а в другом они имеют в голо- ве процентные надбавки, то правила аукциона для тех и других должны быть разными. Пра- вила аукциона ограничивают действия участ- ников, что позволяет легче формализовать их поведение. Формализация действий участников аукциона представляет собой мощное средство ограничения возможностей крупных игроков, уравнивания их права с правами прочих участ- ников аукциона. При использовании простых Сговор и недоверие — две проблемы, повыша- ющие вероятность монополизации рынка. Потому меры, направленные на снижение этой вероятности, формируют препятствия деятельности будущих и уже закрепившихся монополистов, способствуют поддержанию свободной конкуренции. форматов аукциона эту цель не всегда удается достичь. Начну с отрицательных примеров, а именно — с залоговых аукционов 1995 г., которые лишили наше государство существенной части его собст- венности. Тема эта, к сожалению, чрезмерно поли- тизирована. Создается впечатление, что речь идет о приватизации в целом, а это не так. Залоговые аукционы начались после Указа Президента РФ Б. Н. Ельцина в августе 1995 г.13 Их было всего 12, проведенных до конца того же года, и коснулись они только нескольких крупных государственных компаний. Прирост цены продажи относительно стартовой был минимальным: контрольный па- кет акций «Сиданко» был продан с приростом 4%, «Юкоса» — 2,6%, «Норильского никеля» — 0,6% 14. Вместе с тем развитие такой теории полезно для практических приложений даже в том случае, если она на практике неприменима. В России положение с репутацией участников рынка можно признать катастрофическим. А ведь репутация является ос- новной частью так называемых нематериальных активов, которые имеют денежную оценку. В 1990-е гг. доля нематериальных активов рос- сийских компаний составляла 5%. За 30 лет она поднялась до 12%. Но это очень далеко от средне- мирового уровня (63%) 11. В РФ распространилась мировая практика оценки репутации. Любой руко- водитель или владелец фирмы может обратиться в эту систему 12 и получить стоимостную оценку репутации своей организации. Но особого спроса на это нет. Основным свидетельством сговора и являются такие малые величины прироста, говорящие об отсутствии конкуренции в ходе аукциона. Нет кон- куренции — следовательно, рынок полностью или частично монополизирован. Сговор — это способ взаимной поддержки тех, кто замещает свободную конкуренцию монополией или олигополией. Пол Милгром и Роберт Уилсон связывали про- блему оценки репутации с форматом проведения аукциона. Одно дело, когда в аукционе участвуют компании с безупречной репутацией, и совсем другое, если некоторые из них вызывают у других участников недоверие к самому аукциону и его возможным результатам. Понятно, что для разработки таких форматов аукционов, которые снижают вероятность сговора участников, нужна заинтересованная сторона и за- каз на такую разработку. Но по отсутствию в России до сих пор нормального научного анализа даже залоговых аукционов 1995 г. можно догадаться, что госзаказ на разработку форматов аукционов с низкой вероятностью сговора в нашей стране еще не созрел. 10 Основателями теории репутации и ее роли на рынке являют- ся лауреаты Нобелевской премии по экономике 2004 г. Финн Кидланд и Эдвард Прескотт. 11 URL: http://emco-eqs.webflow.io/ 12 URL: https://www.rep-in.ru/ 10 Основателями теории репутации и ее роли на рынке являют- ся лауреаты Нобелевской премии по экономике 2004 г. Финн Кидланд и Эдвард Прескотт. Что такое ТЕОРИЯ аукционов? Прежде чем ответить на этот вопрос, приведу не- сколько относящихся к нему фактов. В своих рабо- тах Пол Милгром и Роберт Уилсон в части, отме- ченной как основание для получения ими Нобе- левской премии по экономике, развивали резуль- таты исследований Уильяма Викри, который стал нобелевским лауреатом в 1996 г.9 Они были его последователями настолько, что когда У. Викри умер (через несколько дней после получения им Нобелевской премии), нобелевскую лекцию за него читал Пол Милгром. Корень недоверия участников не только аукцио- на, но и любой рыночной сделки кроется в том, что в любой информации могут содержаться элементы убеждения. Поэтому концепция асимметричной информации со всей очевидностью должна быть дополнена положением, что любая рыночная ин- формация может быть токсичной, т. е. способной понуждать участника рынка к принятию неверного решения. Исходной установкой Уильяма Викри было то, что аукционы — часть системы рыночных механиз- мов, удерживающих экономику от монополизации и спекулятивных пузырей. Можно приводить множество примеров сов- ременных аукционов, один удивительнее другого, в каждом из них — множество процедурных тон- костей. Как и многие экономисты, Пол Милгром ис- ходит из общего положения о том, что если нечто существует и, как нам кажется, не имеет прямого отношения к рыночным отношениям, то это — недо- работка исследователей и нужно найти эту связь [13]. В рамках исследований мотивов принятия реше- ний (как на аукционах, так и в рыночной экономике вообще) лауреаты занялись мотивом поддержа- ния репутации компании как достойного участия Как и многие экономисты, Пол Милгром ис- ходит из общего положения о том, что если нечто существует и, как нам кажется, не имеет прямого отношения к рыночным отношениям, то это — недо- работка исследователей и нужно найти эту связь [13]. Теория аукционов во многом формальная и вы- ражена многими математическими уравнениями [9]. Формализованная часть теории аукционов зна- чительна, она органично входит в математическую экономику, работающую с моделями некоторых В рамках исследований мотивов принятия реше- ний (как на аукционах, так и в рыночной экономике вообще) лауреаты занялись мотивом поддержа- ния репутации компании как достойного участия Мир новой экономики • Т. 15, № 1’2021 55 Ю. П. Воронов Ю. П. Воронов Ю. П. Воронов рынка 10. Назначая цену или определяя параметры ценовой политики, фирма, заботящаяся о своей репутации, ориентируется не столько на текущую прибыль, сколько на будущее поведение партнеров и конкурентов. форматов аукциона эту цель не всегда удается достичь. 12 URL: https://www.rep-in.ru/ 11 URL: http://emco-eqs.webflow.io/ Подведение итогов, «проклятие победителя» Третий элемент теории аукционов (в ее эмпи- рической части) — подведение итогов аукциона. Проигравшие заведомо аукционом недоволь- ны, но, оказывается, что часто бывает недоволен и победитель аукциона. Такую ситуацию лауреаты 13 Указ Президента РФ от 31.08.1995 № 889 «О порядке переда- чи в залог акций, находящихся в федеральной собственности». 14 Рассчитано по данным: URL: http://nationalization.ru/ privatizaciya-v‑90/kriminal-zalogovyh-aukcionov/. wne.fa.ru ЭКОНОМИЧЕСКАЯ ТЕОРИЯ / ECONOMIC THEORY обозначили как «проклятие победителя» и под- робно ее проанализировали. Она может быть по- нятна по результатам опроса многих победителей аукционов, которые часто переживают, что они переплатили. Один из таких активов — право пользования ра- диоэфиром. Участники аукционов по продаже лицензий на право использования данного спектра радиоча- стот на данной территории делятся на несколько групп. В лицензиях заинтересованы владельцы радиостанций, провайдеры сотовой связи. Многие государства мира используют наработки П. Милгро- ма и Р. Уилсона, продавая на аукционах лицензии на право использования диапазонов радиочастот. При одних форматах аукционов таких жа- лоб много, при других — существенно меньше. Факт победы может считаться как плохим, так и хорошим известием. Используемый формат аукциона тем лучше, чем меньше сожалений у его победителя. На эту закономерность первым обратил внимание Роберт Уилсон [14]. У победи- теля оценка его будущего дохода была наиболее оптимистичной. После победы он получает массу отрицательной информации, узнав, что все про- чие дали меньшую оценку, чем он. На основании этой информации он корректирует прежнюю оценку своего будущего дохода от выигрыша и впадает в пессимизм. В США аукционы на радиочастоты проходят с 1994 г. Первый из них и был дебютом П. Милгрома и Р. Уилсона как разработчиков аукционных проце- дур. С того времени было проведено 87 аукционов, т. е. 10 аукционов каждые три года. На этих аукцио- нах было заработано около 60 млрд долл. Последний масштабный аукцион прошел в 2008 г. на частоты 700 МГц. Первый аукцион по 3G-частотам прошел в Индии в 2010 г., выручка составила 14 млрд долл. В 2015 г. там состоялся аукцион на частоты от 0,8 до 2,1 ГГц, продолжавшийся 19 дней. Выручка государ- ства — 17 млрд долл. То есть везде такие аукционы приносят доход государству. Пол Милгром «проклятие победителя» не трак- тует просто как феномен, симптом эффективности (или неэффективности) выбранного формата аук- циона. Он объясняет его, используя две катего- рии — убеждение и открытость информации [15]. Интересно, что этот подход лауреата во много пересекается с идеями, высказанными ровно век назад русским академиком В. М. Бехтеревым [16]. Подведение итогов, «проклятие победителя» Последний проводил границу между внушени- ем и убеждением, полагая, что убеждение состоит в объяснении человеку, как фактически обстоят дела и противопоставлял его внушению, которое есть инструмент воздействия не только на сознание, но и на подсознание человека. В России такие аукционы не пользуются попу- лярностью — ни на рынке частот для радиосвязи, ни для сотовой связи. Так, в 2015 г. был проведен аукцион продолжительностью семь дней по продаже лицензий на право использования радиочастот для сетей связи четвертого поколения (4G). На следую- щие торги — в 2016 и 2018 гг. — не было подано ни одной заявки (https://digital.gov.ru/ru/events/38690/). В 2020 г. полосы миллиметрового диапазона для создания пилотных зон сетей 5G были без какого- либо конкурса предоставлены совместному пред- приятию «Мегафона» и «Ростелекома», созданному специально для их получения. Пол Милгром проводит аналогичную границу между убеждением и открытостью информации по иному критерию. Убеждение — есть целевое воздействие на сознание участников рынка, в от- крытости информации такой цели не содержится, во всяком случае, явно. «Проклятие победителя» есть результат убеждения (а, возможно, и внушения), даже если продавец не ставил себе задачу внушить или убедить покупателя в достоинствах приобре- таемого им товара, услуги или права. Таким образом, аукционы по продаже радиоча- стот для сотовой связи новых поколений в России закончились, а сговор стал незамеченным. Насе- ление страны не очень волнует, кому достанутся радиочастоты. Но уход конкуренции из этой сферы должен был стать событием, очень важным для отечественной экономической науки. К сожалению, этого не произошло. рынок, либо блокируют вхождение на него новых игроков [17]. которая сбила цены ради того, чтобы вытеснить с рынка конкурентов, которые не выдержат про- даж по таким низким ценам. Иногда цены в роз- нице ниже не только оптовых, но и себестоимости производства товара. Крупная компания может компенсировать свои убытки за счет продаж дру- гих товаров или торговли на других рынках, а бо- лее слабые конкуренты поймут, что таких цен они не выдержат и уйдут. Тогда крупная компания- монополист может спокойно повышать цены, не беспокоясь о том, что кто-либо затронет ее инте- ресы. Возникает проблема — как отделять «хищни- чество» от конкурентного ценообразования? Для этого нужно знать, располагает ли фирма активами, которые необходимы для того, чтобы «пересидеть» конкурента, и согласна ли она длительное время нести убытки. Еще один способ отделения «хищничества» от нормальной конкуренции связан с предложенным лауреатами разделением «хищнического цено образования» на три класса, которым соответствуют три типа моделей. Термин «predatory pricing» принято перево- дить на русский язык как «хищническое цено- образование». В таком переводе скрываются два подвоха. Во-первых, «pricing» — это не столько «ценообразование» (в смысле формирования цен на рынке), сколько «назначение цены от- дельным продавцом». Со словом «predatory» еще сложнее: при полной расшифровке речь идет о «хищнике», который охраняет свою территорию или спрятался и ждет жертву. Лично я считаю наиболее близким перевод этого термина как «предательский», за которым стоят иные цели, чем торговля. Но термин уже закрепился как «хищнический», поэтому его придется использо- вать и после этих разъяснений. Такое назначение цены повышает вероятность монополизации рынка или сохранение монополии одного участ- ника рынка — «хищника». В большинстве стран оно считается нарушением антимонопольного законодательства и юридически преследуется. Первому соответствуют модели репутации [18]. Когда уже работающий участник рынка, встречаясь с потоком желающих прийти на этот рынок, откры- вает ценовую войну, он приобретает репутацию «крутого», что отвращает приход новых конкурентов. Второму классу соответствуют сигнальные мо- дели. Новичок не знает, слаб или нет действующий участник рынка, т. е. высоки или нет его издер- жки. И принимая решение о входе на рынок, он анализирует его цену. Здесь П. Милгром выделил два возможных состояния равновесия: раздельное и совместное. В первом участник выдерживает низ- кую цену и несет убытки. При втором оба участника несут убытки. Сигнальная модель может быть, например, та- кой: 1. Действующий участник рынка выбирает цену. 2. Низкие доходы для новичка, если у участника рынка низкие затраты. 3. Игра по захвату рынка. Частный случай «хищнической» цены — так на- зываемая «блокирующая цена» (deterred price). «Хищники» на рынке Современная рыночная экономика характерна тем, что основная часть сделок внутри нее состоит в торговле правами на какую-то деятельность. Тот, кто пытается привязать современную экономику к товарным потокам, обрекает себя на неудачу. Взамен них появляются нематериальные активы. Если на рынке снизились цены, для покупателей это хорошо. Если речь идет о потребительском товаре, статистические органы бодро рапортуют о том, что инфляция низкая. Но на самом деле на этот рынок пришла крупная компания-«хищник», Мир новой экономики • Т. 15, № 1’2021 57 Ю. П. Воронов Ю. П. Воронов Ю. П. Воронов рынок, либо блокируют вхождение на него новых игроков [17]. ВЫВОДЫ В схеме «хищника», захватывающего рынок, цены снижаются для того, чтобы затем установить монополию. И определить это намерение можно только по последующим событиям, когда после устранения конкурентов цены повышаются. Но и тогда на рынок может прийти новый игрок, ко- торый собьет цены с той же целью. Итак, несмотря на то, что Нобелевская премия по экономике получена Полом Милгромом и Робер- том Уилсоном за развитие теории аукционов, их исследования охватывают куда более широкую область [21]. Экономисты, которые претендовали на полу- чение Нобелевской премии в 2020 г., интересны не столько сами по себе, сколько темами своих исследований, но Нобелевский комитет посчи- тал их менее важными по сравнению с теорией аукционов. Например, Стивен Берри и Джеймс Левинсон (Йельский университет) и Ариэль Пейкс (Гарвардский университет) создали широко ис- пользуемую логистическую модель оценки спро- са BLP (по первым буквам их фамилий). Дэвид Дики (Университет Северной Каролины) и Уэйн Фуллер (Университет Айовы) разработали тест Дики-Фуллера, с помощью которого оценивают временные ряды на вероятность роста. Забавно, что в 2003 г. премию получили Роберт Фрай Энгл и Клайв Гренджер, которые вели свои исследова- ния, используя этот тест. Теперь же премию по- лучили еще и Пол Милгром с Робертом Уилсоном, для которых также важна проверка временного ряда на стационарность. Кроме того, последующее после захвата рынка повышение цен не может быть длительным, иначе это привлечет на данный рынок новых игроков, которые могут оказаться более сильными в фи- нансовом отношении [20]. Характерно, что модели «хищнического цено образования» не отличаются особой сложностью. Их строят для того, чтобы дать общую качественную оценку самого факта «хищничества». Упрощения сознательны и объяснимы. Если приближать модели к реальности, придется вводить чрезмерно большое число параметров, отображающих эту реальность. Такие модели интересны еще и потому, что за частной проблемой «хищнического» ценообразова- ния стоит уже упоминавшаяся масштабная пробле- ма экономической теории, а именно — склонность рыночной экономики к монополизации отдельных рынков и, в этом смысле, отрицание возможности длительного существования свободной конкуренции. «Хищники» займут монопольное положение на всех рынках, и время свободной конкуренции закончится. Такие модели интересны еще и потому, что за частной проблемой «хищнического» ценообразова- ния стоит уже упоминавшаяся масштабная пробле- ма экономической теории, а именно — склонность рыночной экономики к монополизации отдельных рынков и, в этом смысле, отрицание возможности длительного существования свободной конкуренции. «Хищники» займут монопольное положение на всех рынках, и время свободной конкуренции закончится. Очевидно, в год, когда мировая экономика пере- живает небывалые изменения, Нобелевский коми- тет исходил из того, что от исследований трендов будет мало пользы. ВЫВОДЫ Предшественник нобелевских лауреатов Р. Зель- тен делит на три вида стратегии поведения человека на рынке и не только: Прогнозировалась в лауреаты и профессор Гарвардского университета Клаудия Голдин. Она занимается гендерным экономическим неравенст- вом, но актуальность этой темы поблекла на фоне событий, происходящих в 2020 г. 1. Рутина, когда он поступает так, как делал это обычно. 2. Воображение, когда человек придумывает нечто оригинальное, ранее не встречавшееся. СПИСОК ИСТОЧНИКОВ/REFERENCES рынок, либо блокируют вхождение на него новых игроков [17]. Она назначается «хищником», который уже убрал кон- курентов и защищает захваченный им рынок. Она назначается такой, чтобы новичку (возможно, еще более крупному «хищнику») было бы неинтересно заходить на этот рынок. В такой модели низкая цена говорит о том, что у участника рынка низкие затраты. Ориентируясь на низкие доходы, новичок начинает продавать товар по более низкой цене. В такой модели низкая цена говорит о том, что у участника рынка низкие затраты. Ориентируясь на низкие доходы, новичок начинает продавать товар по более низкой цене. Третьему классу соответствуют модели «боль- шого кармана “хищника”» [19]. В модели «хищни- чества» «толстый кошелек» действуют две фирмы, новичок и участник. Они несут одинаковые из- держки в период противостояния и отличаются только размерами ликвидных активов. Побеждает тот, у кого эти активы больше. Эти исследования имеют мощного потенци- ального заказчика — антимонопольную службу. Однако в подавляющем большинстве стран мира нет прямой связи науки с деятельностью этой службы. Признание какой-то компании «хищником» требует юридического оформления, которое находится за пределами научных теорий и их эмпирическо- го подтверждения. Юристы могут отрицать саму связь между ценовой войной и недобросовестной конкуренцией. Кроме того, на практике чаще всего встречается не чистая монополия, а олигополия, когда несколько «хищников» либо захватывают У таких моделей много недостатков: требуется обширная информация, при равновесии на рынке ценовые войны отсутствуют, есть сомнительное допущение, что новичок не в состоянии привлечь больше активов. Но самое главное — они могут использоваться исключительно для пояснения происходящих процессов, и, на мой взгляд, бес- wne.fa.ru ЭКОНОМИЧЕСКАЯ ТЕОРИЯ / ECONOMIC THEORY 3. Рассуждение, когда он приходит к решению на основании построенных им логических кон- струкций. полезны как рабочий инструмент. Творческие приемы предпринимателей содержат иногда столько выдумки, что никакая теория их охватить не в состоянии. 3. Рассуждение, когда он приходит к решению на основании построенных им логических кон- струкций. 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Статья поступила 10.11.2020; после рецензирования 30.11.2020; принята к публикации 15.12.2020. Автор прочитал и одобрил окончательный вариант рукописи. The article was received on 10.11.2020; revised on 30.11.2020 and accepted for publication on 15.12.2020. The author read and approved the final version of the manuscript. ИНФОРМАЦИЯ ОБ АВТОРЕ / ABOUT THE AUTHOR Юрий Петрович Воронов — кандидат экономических наук, ведущий научный сотрудник Лаборатории моделирования и анализа экономических процессов, Институт экономики и организации промышленного производства СО РАН, Новосибирск, Россия Yuri P. Voronov — Cand. Sci. (Econ.), Leading Researcher, Laboratory for Modeling and Analysis of Economic Processes, Institute of Economics and Organization of Industrial Production SB RAS, Novosibirsk, Russia corpus-cons@ngs.ru Юрий Петрович Воронов — кандидат экономических наук, ведущий научный сотрудник Лаборатории моделирования и анализа экономических процессов, Институт экономики и организации промышленного производства СО РАН, Новосибирск, Россия Yuri P. Voronov — Cand. Sci. (Econ.), Leading Researcher, Laboratory for Modeling and Analysis of Economic Processes, Institute of Economics and Organization of Industrial Production SB RAS, Novosibirsk, Russia corpus-cons@ngs.ru Статья поступила 10.11.2020; после рецензирования 30.11.2020; принята к публикации 15.12.2020. Автор прочитал и одобрил окончательный вариант рукописи. The article was received on 10.11.2020; revised on 30.11.2020 and accepted for publication on 15.12.2020. The author read and approved the final version of the manuscript. wne.fa.ru
https://openalex.org/W3197050755	https://ccforum.biomedcentral.com/track/pdf/10.1186/s13054-021-03676-5	English	null	Acute kidney injury in ECMO patients	Critical care	2,021	cc-by	7,550	© Ostermann and Lumlertgul 2021, corrected publication 2024. Open Access This article is licensed under a Creative Commons Attribu- tion 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedica- tion waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Pathophysiology of AKI in ECMOh Pathophysiology of AKI in ECMO The underlying mechanisms of AKI in patients treated with ECMO are complex and multifactorial (Table 1). Abstract This article is one of ten reviews selected from the Annual Update in Intensive Care and Emergency Medicine 2021. Other selected articles can be found online at https://www.biomedcentral.com/collections/annualupdate2021. Further information about the Annual Update in Intensive Care and Emergency Medicine is available from https://link. springer.com/bookseries/8901. practice and design future studies for prevention and management of this high-risk group. Acute kidney injury in ECMO patients Marlies Ostermann1* and Nuttha Lumlertgul1,2,3 Ostermann and Lumlertgul Critical Care (2021) 25:313 https://doi.org/10.1186/s13054-021-03676-5 Ostermann and Lumlertgul Critical Care (2021) 25:313 https://doi.org/10.1186/s13054-021-03676-5 Critical Care Introduction Extracorporeal membrane oxygenation (ECMO) is a life- saving therapy for patients with severe respiratory and/or cardiovascular failure. There are two main configurations: (1) veno-arterial ECMO (VA-ECMO) in patients with refractory cardiogenic shock or combined cardiorespira- tory failure, and (2) veno-venous ECMO (VV-ECMO) in patients with potentially reversible causes of respiratory failure. Over the past decade, use of ECMO has increased substantially in critical care units, emergency depart- ments, interhospital transfers, operating rooms, and dur- ing cardiopulmonary resuscitation (CPR) [1].h Patient factors and critical illness *Correspondence: Marlies Ostermann Marlies.Ostermann@gstt.nhs.uk 1 Department of Critical Care, King’s College London, Guy’s and St Thomas’ NHS Foundation Trust, London, UK 2 Division of Nephrology and Excellence Centre for Critical Care Nephrology, King Chulalongkorn Memorial Hospital, Bangkok, Thailand 3 Critical Care Nephrology Research Unit, Chulalongkorn University, Bangkok, Thailand Prior to ECMO initiation, hemodynamic instability, low cardiac output, high intra-thoracic pressure, exposure to nephrotoxic agents, severe hypoxemia, hypercapnia, sys- temic inflammation/immune-mediated effects, and neu- rohormonal dysregulation can contribute to AKI [8]. In patients with heart failure, cardiac dysfunction, increased Incidence of AKI in ECMOh The reported incidence of AKI in patients treated with ECMO varies from 26 to 85% due to differences in patient characteristics, AKI definition, and clinical settings. The pooled estimated incidence of severe AKI requiring RRT is 45% [6]. AKI is more common in VA-ECMO than in VV-ECMO (61% vs. 46%) and is most often present on the day of ECMO cannulation [6, 7]. The Extracorporeal Membrane Oxygenation for Severe Acute Respiratory Distress Syndrome (EOLIA) and Conventional Ventila- tory Support versus Extracorporeal Membrane Oxygena- tion for Severe Adult Respiratory Failure (CESAR) trials demonstrated a lower incidence of AKI and use of RRT in patients receiving VV-ECMO compared with standard treatment [2]. The in-hospital mortality ranges from 21 to 37% in patients receiving VV-ECMO compared to 40–60% in patients treated with VA-ECMO [2–4]. Despite improv- ing survival in recent years, adverse effects are common including acute kidney injury (AKI), infection, throm- bosis, and bleeding [5]. AKI is a frequent complica- tion among patients treated with ECMO, resulting in increased morbidity and mortality [6]. Understanding the impact of AKI, its contributing factors, and renal replacement therapy (RRT) is essential to inform clinical © Ostermann and Lumlertgul 2021, corrected publication 2024. Open Access This article is licensed under a Creative Commons Attribu- tion 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedica- tion waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. ECMO‑related factors ll Following ECMO cannulation, an improvement in oxy- genation helps restore the microcirculation in previ- ously hypoxic and hypoperfused organs and tissues, often in association with a degree of ischemia–reperfusion injury and production of reactive oxygen species (ROS) [13]. Continuous flow during VA-ECMO reduces pulsa- tility, which may compromise renal cortical blood flow and upregulate the RAAS inducing systemic vasocon- striction [14]. Circuit-related factors contributing to the development of AKI include hemolysis, rhabdomyolysis from local ischemia, hemorrhage, renal microthrombo- sis, and cannula-related complications (e.g., malposition of the cannula leading to venous obstruction, choles- terol embolism following cannulation, aortic dissection) [15, 16]. Hemolysis may occur due to a combination of shear stress from blood travelling through the blood pump, negative intra-circuit pressures, and contact with the non-biological and non-endothelialized surface of ECMO membranes [15]. This leads to elevated plasma free hemoglobin, release of free iron, oxidative stress, and filtered heme pigments causing tubular obstruction [17]. Blood exposure to artificial surfaces also induces the release of inflammatory cytokines, complement and leukocyte activation, and hypercoagulability. Finally, 74 although VA-ECMO improves oxygenation and periph- eral circulation, limited LV off-loading combined with low ejection fraction can result in LV overdistension and worsening pulmonary edema. IMV invasive mechanical ventilation, PEEP positive end-expiratory pressure, VA veno-arterial, RAAS renin–angiotensin–aldosterone system, ANP atrial natriuretic peptide IMV invasive mechanical ventilation, PEEP positive end-expiratory pressure, VA veno-arterial, RAAS renin–angiotensin–aldosterone system, ANP atrial natriuretic peptide intra-abdominal pressure (IAP), and renal congestion contribute to impaired renal blood flow and cardiore- nal syndrome [9]. AKI might also occur in the context of other critical illness-related complications including bleeding, limb ischemia, infection, and coagulopathy [8]. Ostermann and Lumlertgul Critical Care (2021) 25:313 Ostermann and Lumlertgul Critical Care (2021) 25:313 Page 2 of 10 Table 1 Risk factors for acute kidney injury (AKI) during extracorporeal membrane oxygenation (ECMO) IMV invasive mechanical ventilation, PEEP positive end-expiratory pressure, VA veno-arterial, RAAS renin–angiotensin–aldosterone system, ANP atrial natriuretic peptide Factors Pathophysiological mechanisms Patient-related variables Hypoperfusion Loss of autoregulation Hypoxia Hypercapnia Nephrotoxins Systemic inflammation Cardiorenal syndrome Increased intrathoracic pressure Increased intra-abdominal pressure Neuro-hormonal effects IMV-related factors Biotrauma ECMO-related factors PEEP Hemodynamic variables Continuous flow (VA-ECMO) Ischemia– reperfusion injury Hormonal variables RAAS dysregulation ANP downregulation Circuit-related factors Blood shear stress Rhabdomyolysis Hemolysis and oxidative stress Embolism Aortic dissection Systemic inflammation Systemic inflammation Renal macro/microcirculatory dysfunction Bioincompatibility Blood/air/surface interaction Hypercoagulable state Table 1 Risk factors for acute kidney injury (AKI) during extracorporeal membrane oxygenation (ECMO) Table 1 Risk factors for acute kidney injury (AKI) during extracorporeal membrane oxygenation (ECMO) IMV invasive mechanical ventilation, PEEP positive end-expiratory pressure, VA veno-arterial, RAAS renin–angiotensin–aldosterone system, ANP atrial natriuretic peptide Factors Pathophysiological mechanisms Patient-related variables Hypoperfusion Loss of autoregulation Hypoxia Hypercapnia Nephrotoxins Systemic inflammation Cardiorenal syndrome Increased intrathoracic pressure Increased intra-abdominal pressure Neuro-hormonal effects IMV-related factors Biotrauma ECMO-related factors PEEP Hemodynamic variables Continuous flow (VA-ECMO) Ischemia– reperfusion injury Hormonal variables RAAS dysregulation ANP downregulation Circuit-related factors Blood shear stress Rhabdomyolysis Hemolysis and oxidative stress Embolism Aortic dissection Systemic inflammation Systemic inflammation Renal macro/microcirculatory dysfunction Bioincompatibility Blood/air/surface interaction Hypercoagulable state system (RAAS), and suppression of atrial natriuretic peptide (ANP) release [9]. Lung-protective ventilation limits lung injury and has potential to reduce the risk of AKI [12]. However, permissive hypoxia and hypercapnia might ensue and decrease renal blood flow [9]. RRT and ECMO Indications Fluid overload is highly prevalent and associated with higher mortality and pro-longed ECMO duration [23]. According to a recent survey, fluid overload management (43%) or prevention (16%) are the predominant triggers for RRT initiation during ECMO, followed by AKI (35%), and electrolyte disturbances (4%) [24]. Risk factors for AKI Invasive mechanical ventilation is associated with altered hemodynamics and release of pro-inflammatory cytokines (e.g., tumor necrosis factor [TNF]-α, inter- leukin [IL]-1β, IL-6 and IL-8) [10]. Plasma cytokine concentrations are predictive of AKI development and renal non-recovery [11]. The application of positive end- expiratory pressure (PEEP) has several beneficial effects for lung recruitment and decrease in left ventricular (LV) pre- and afterload. However, increasing PEEP and/ or tidal volumes may elevate intrathoracic pressure, reduce venous return, decrease cardiac output, and increase right ventricular (RV) afterload, resulting in elevated sys- temic venous pressure, venous congestion, and reduc- tion of renal perfusion. In addition, fluid retention may develop as a result of activation of the sympathetic nerv- ous system (SNS) and renin–angiotensin–aldosterone Reported risk factors for AKI during ECMO are older age, pre-existing comorbidities (e.g., cirrhosis), post car- diotomy shock as indication for ECMO, late implanta- tion of ECMO, reduced LV ejection fraction (LVEF), intraoperative transfusion, high lactate, high plasma free hemoglobin, increased bilirubin, and high neutrophil to lymphocyte ratio [18]. Red blood cell distribution width >14.1%, a marker of inflammation and anemia, has also been found to be associated with an increased risk of severe AKI [19]. During ECMO, high inotropic equiva- lents, ECMO pump speed, and ECMO duration are linked to AKI development [20]. Higher pump speeds are associated with hemolysis, leukocyte and platelet destruction, and complement activation [21]. To prevent heme pigment-associated AKI, pump revolutions/min Ostermann and Lumlertgul Critical Care (2021) 25:313 Ostermann and Lumlertgul Critical Care (2021) 25:313 Page 3 of 10 Page 3 of 10 RRT, the demand-capacity concept has been proposed as a method to guide the decision-making process. Accord- ingly, RRT should be considered if the degree of fluid overload and AKI-related metabolic derangements are likely to overwhelm the kidneys’ capacity to compensate, and pharmacological measures (diuretic therapy, sodium bicarbonate) are unlikely to be effective [30]. The expert committee of the 21st Acute Dialysis Quality Initiative (ADQI) meeting concluded that there was no evidence of benefit for pre-emptive use of RRT in patients treated with ECMO [9]. Therefore, the decision to initiate RRT in patients receiving ECMO should be based on usual abso- lute and relative indications for critically ill patients. (RPM) should be limited to safe levels to avoid exces- sive negative pressures. Risk factors for AKI AKI patients who required RRT whilst receiving ECMO were more likely to be treated with VA-ECMO, had more organ dysfunction at the time of ECMO insertion, and required more transfusions [22]. Modality RRT options include continuous RRT (CRRT), prolonged intermittent renal replacement therapy (PIRRT), inter- mittent hemodialysis (IHD), and peritoneal dialysis. Each modality has advantages and disadvantages (Table 2). CRRT and peritoneal dialysis are suitable for patients with hemo- dynamic instability although CRRT enables more precise fluid and electrolyte management. Meanwhile, PIRRT and IHD allow planned circuit downtime. It is possible to pro- vide CRRT, PIRRT and IHD via integration into the ECMO circuit or separately. When choosing CRRT, any mode of clearance can be delivered, namely slow continuous ultra- filtration (SCUF), continuous veno-venous hemofiltration (CVVH), continuous veno-venous hemodialysis (CVVHD), and continuous veno-venous hemodiafiltration (CVVHDF). Timingh g Theoretically, early initiation of RRT may help resolve fluid overload faster and achieve better sodium removal per unit volume than diuretics in ECMO patients. In general ICU patients, recent randomized controlled trials (RCTs) not only failed to demonstrate the survival benefits of early over standard initiation strategy, but also showed increased harm in the early-initiation group including an increased risk of dialysis dependence at 90 days and adverse events [25–27]. A post-hoc sub-analysis of The Artificial Kidney Initiation in Kidney Injury (AKIKI) trial in acute respira- tory distress syndrome (ARDS) also demonstrated similar outcomes between early and standard initiation strate- gies [28]. Another study using propensity-score matching compared early versus late initiation of CRRT after ECMO (median time from ECMO to CRRT initiation 1 vs. 15 days) and found no difference in survival [29]. Techniquesh There are three ways to provide RRT with ECMO: using an in-line hemofilter, connecting a RRT device to the ECMO circuit (integrated system), or using a separate f In light of the fact that serum creatinine, AKI stage and urine output are poor markers to guide initiation of Table 2 Advantages and disadvantages of each renal replacement modality during extracorpo-real membrane oxygenation (ECMO) IHD intermittent hemodialysis, PIRRT prolonged intmeromviettmenetn thsemodialysis, CRRT continuous renal replacement therapy, PD peritoneal dialysis Modality Advantages Disadvantages IHD Integration in ECMO circuit possible Reduced filter downtime Lower costs than CRRT Need for more rapid fluid removal Risk of hemodynamic instability Disequilibrium syndrome PIRRT Integration in ECMO circuit possible Reduced filter downtime Lower costs than CRRT Slower volume and solute removal than IHD Risk of hemodynamic instability in high-risk patients CRRT Integration in ECMO circuit possible Continuous fluid and solute removal Allows more precise control of fluid balance Better hemodynamic stability Patient immobilization Increased risk of hypothermia High costs PD Better hemodynamic stability Technically simple Lower cost No addition of anticoagulation Less experience in adult patients Requires specific intraperitoneal catheters Risk of peritonitis Risk of hyperglycemia May interfere with diaphragmatic movements IHD intermittent hemodialysis, PIRRT prolonged intmeromviettmenetn thsemodialysis, CRRT continuous renal replacement therapy, PD peritoneal dialysis Ostermann and Lumlertgul Critical Care (2021) 25:313 Page 4 of 10 Table 3 Advantages and disadvantages of RRT techniques during extracorporeal membrane oxygenation (ECMO) [31, 33] RRT renal replacement therapy, UF ultrafiltration Techniques Advantages Disadvantages In-line hemofilter Low cost Generates large volumes of UF No need for separate anticoagulation Small priming volume No pressure monitoring Requires external infusion device to control UF and deliver replacement fluid Less precise UF Limited solute clearance Flow turbulence and risk of hemolysis Independent RRT access (parallel system) Allows fine-tune adjustment of solute and fluid removal Able to provide RRT independent of ECMO Allows use of regional anticoagulation Simplified circuit changing without need for perfusionist Mode of solute clearance not restricted Need for separate vascular access Risk of mechanical and infectious complications Higher extracorporeal blood volume Technically more complex to manage two separate circuits RRT connected to ECMO circuit (integrated system) Allows fine-tune adjustment of solute and fluid removal Mode of solute clearance not restricted No need for separate vascular access Avoids complications related to line insertion Pressure alarms (low pressure alarms if connected pre-pump and high pressure alarms when connected post-pump) Requires a RRT machine capable of adjusting alarm settings Risk of air entrapment if access line is connected before centrifugal pump Flow turbulence with risk of hemolysis and thrombus formation Generation of shunt within ECMO circuit Recirculation RRT renal replacement therapy, UF ultrafiltration by adding replacement fluid (CVVH) or dialysis fluid (CVVHD) through standard infusion pumps. Fig. 1 Options of combining extracorporeal membrane oxygenation (ECMO) and continuous renal replacement therapy (CRRT) circuits. a An in-line hemofilter is integrated into the ECMO circuit. Replacement fluid is directly administered into the ECMO circuit. Alternatively, dialysis fluid can be supplied in a counter-current position. Replacement/dialysis fluid rates and ultrafiltration rates can be controlled via infusion pumps. b The CRRT device is connected to the patient through a separate catheter independent of the ECMO circuit. c The access (inlet) and the return (outlet) lines of the CRRT device are connected before the centrifugal blood pump (low-pressure part) of the ECMO circuit. d Both the access and the return lines of the CRRT device are connected after the blood pump. e The access line of the CRRT device is connected after the blood pump (high-pressure), while the CRRT return line is connected before the centrifugal blood pump. f The access line of the CRRT device is connected directly after the membrane oxygenator, while the return line is connected directly before the oxygenator (See figure on next page.) Techniquesh Ultrafiltra- tion rate is regulated by connecting a standard infusion device to the effluent port of the hemofilter. However, the amount of removed fluid is less accurate and prone to error up to 800 ml/day [34]. A more precise method is to weigh the actual volume of ultrafiltration using a scale or a volumetric measuring device but this method is labor- intensive. Since hemofilters are not designed for use with high pressure systems and the maximal volume of the infusion pump is limited at 1 l/h, convective and diffusive clearance are less effective than with CRRT using con- ventional membranes. The hemofilter blood flow rate can be adjusted via a stopcock or a flow-restrictor. Neverthe- less, the generated turbulent flow might cause hemolysis and trigger thrombus formation. Most importantly, there is no pressure monitoring with this technique, which may lead to delayed detection of hemolysis, filter rupture or clot formation. RRT access from the ECMO circuit (parallel system) [31, 32] (Table 3). In the absence of evidence-based data, practice is based on expert opinion, availability of machines, local expertise, and staff organization. A 2013 survey of 65 ECMO centers showed that 50.8% of centers used independent CRRT circuits while 21.5% used in-line hemofilter [24]. The results of a recent survey of ECMO centers in France and Switzerland are awaited. In‑line hemofilter It is possible to provide RRT by incorporating a hemofil- ter into the ECMO circuit [9, 15, 31] (Fig. 1a). The hemo- filter is placed after the pump pre-oxygenator so that the oxygenator can trap air and clots. The positive pres- sure from the ECMO circuit will forward the blood flow through the hemofilter. Then, blood is returned from the filter to the ECMO circuit before the pump. The blood flow rate in the hemofilter is the difference between the total ECMO blood flow rate and the actual flow delivered to the patient, which is measured by placing an ultra- sonic probe on the arterial return line from the ECMO circuit. This technique is mainly used for ultrafiltration via the SCUF mode. CVVH or CVVHD can be delivered Parallel system (independent RRT access) Setting up an independent RRT device is simple as it does not require ECMO circuit manipulation (Fig. 1b). Dose Ostermann and Lumlertgul Critical Care (2021) 25:313 Page 5 of 10 Blood pump Membrane oxygenator Blender CO2 Pressure range: -20 to -100 mmHg Pressure range: +150 to +350 mmHg Hemofilter Dialysis fluid R l Effluent fluid Return line Access line a Blood pump Membrane oxygenator Blender CO2 CO2 CO2 Pressure range: -20 to -100 mmHg Pressure range: +150 to +350 mmHg Hemofilter Dialysis fluid Replacement fluid Effluent fluid Return line Access line Membrane oxygenator Blender Pressure range: -20 to -100 mmHg Pressure range: -20 to -100 mmHg Pressure range: +150 to +350 mmHg Pressure range: +150 to +350 mmHg RRT machine Blood pump Membrane oxygenator Blender RRT machine Access line Return line Blood pump a b c Fig. 1 (See legend on previous page.) Access line Membrane oxygenator b CO2 CO2 Membrane oxygenator Blender Pressure range: -20 to -100 mmHg Pressure range: -20 to -100 mmHg Pressure range: +150 to +350 mmHg Pressure range: +150 to +350 mmHg RRT machine Blood pump Membrane oxygenator Blender RRT machine Access line Return line Blood pump c Fig. 1 (See legend on previous page.) RRT machine Access line Return line Membrane oxygenator Fig. 1 (See legend on previous page.) Fig. 1 (See legend on previous page.) Ostermann and Lumlertgul Critical Care (2021) 25:313 Page 6 of 10 e f CO2 Pressure range: -20 to -100 mmHg Pressure range: +150 to +350 mmHg Access line Return line Membrane oxygenator Blender RRT machine Blood pump Membrane oxygenator Blender CO2 Pressure range: -20 to -100 mmHg Pressure range: +150 to +350 mmHg RRT machine Access line Return line Blood pump e Membrane oxygenator Blender CO2 Pressure range: -20 to -100 mmHg Pressure range: +150 to +350 mmHg RRT machine Access line Return line Blood pump e f CO2 Pressure range: -20 to -100 mmHg Pressure range: -20 to -100 mmHg Pressure range: +150 to +350 mmHg Pressure range: +150 to +350 mmHg Access line Access line Return line Return line Membrane oxygenator CO2 Membran oxygenator Blender RRT machine Blood pump rane Blender RRT machine Blood pump Membrane oxygenator Blender CO2 Pressure range: -20 to -100 mmHg Pressure range: +150 to +350 mmHg RRT machine Access line Return line Blood pump Fig. Integrated system (combining RRT machine into the ECMO circuit)h There are few studies comparing the efficacy between the different techniques. Compared with an in-line hemofil- ter technique, integrating a RRT machine into the ECMO circuit was shown to provide more accurate fluid man- agement [37]. A recent study concluded that independent RRT access was associated with fewer effective sessions and shorter filter lives in comparison with the integrated system [38]. However, the average prescribed RRT dose was 40 ml/kg/h, which is higher than currently recom- mended. In addition, the CVVH modality was used with 33% of replacement fluid given pre-filter, which might result in a relatively high filtration fraction. Regional cit- rate anticoagulation was not used [39]. Another study also reported longer filter life for the integrated system compared with the parallel technique [35]. With regards to clearance, a recent study demonstrated similar efficacy for solute clearance and ultrafiltration between the paral- lel and the integrated methods [40]. There are several ways to incorporate a RRT device into the ECMO circuit using two high-flow Luer lock three- way taps as connectors to the RRT access (inlet) line and return (outlet) line [9, 31, 33, 35] (Fig. 1c–f). The pres- sure before the ECMO pump is negative (range: − 20 to − 100 mmHg) and post-pump is positive (range: + 150 to + 350 mmHg), which might interfere with the RRT circuit. If a centrifugal pump is used, the RRT access line should be placed post-pump (either before or after the oxygenator) to avoid air entrapment (Fig. 1d, e). The RRT return line should be connected before the oxygen- ator (either pre- or post-pump) to avoid air embolism, clot formation and venous admixture. Careful attention should be paid to the inherently set pressure limits of the ECMO circuit and RRT devices from different manufac- turers. The default access pressure of the RRT machine is typically negative. High post-pump pressure may trigger pressure alarms at the entry point of the RRT machine although current RRT machines can tolerate higher pres- sures or allow adjustment of alarm settings up to + 350 to + 500 mmHg. To overcome the alarm limits of the RRT machine, a long monitoring extension line attached to the RRT access tubing or reducing the blood flow rates can help to lower the pressure from the ECMO circuit [8]. Technical aspects Mediator removal Al h h i d Although raised cytokine concentrations have been demonstrated in patients with AKI receiving ECMO and might be implicated in multiorgan dysfunction [41], there is insufficient evidence to recommend blood puri- fication therapy outside the setting of AKI for patients receiving ECMO. Therefore, the use of RRT and/or hemoadsorption with the sole intention of clearing pro- or anti-inflammatory mediators during ECMO is not rec- ommended [9]. Integrated system (combining RRT machine into the ECMO circuit)h Another option is to use clamps on the connectors or flow restrictors placed outside the tubing to adjust the RRT circuit pressure on the access or, alternatively, on the return line to avoid extreme pressures [36]. However, this may cause turbulence in blood flow and trigger hemolysis or thrombosis. When withdrawing blood post-oxygen- ator and returning blood before pump, re-circulation in the RRT circuit (shunt within a shunt) and RRT under- dosing may occur. Alternatively, the access and return lines may be safely connected through Luer locks imme- diately before and after the oxygenator to avoid pressure alarms (Fig. 1f). Parallel system (independent RRT access) 1 continued Blender Return line e f CO2 Pressure range: -20 to -100 mmHg Pressure range: -20 to -100 mmHg Pressure range: +150 to +350 mmHg Pressure range: +150 to +350 mmHg Access line Access line Return line Return line Membrane oxygenator CO2 Membran oxygenator Blender RRT machine Blood pump rane Blender RRT machine Blood pump Membrane oxygenator CO2 Pressure range: -20 to -100 mmHg Pressure range: +150 to +350 mmHg Blood pump Fig. 1 continued Membrane oxygenator e f CO2 Pressure range: -20 to -100 mmHg Pressure range: +150 to +350 mmHg Access line Access line Return line R t Membrane oxygenator Blender RRT machine Blood pump Blender RRT machine 150 to 350 mmHg Blood pump Membrane oxygenator f Access line Return line Membran oxygenator rane Fig. 1 continued Ostermann and Lumlertgul Critical Care (2021) 25:313 Page 7 of 10 and modality can be adjusted, and fluid balance can be controlled in a precise manner as per usual protocol. Regional anticoagulation can also be added and opti- mised to prolong filter longevity. However, the need to insert a separate vascular access whilst systemically anti- coagulated poses a risk for line-related mechanical and infectious complications. This technique also requires an additional extracorporeal blood volume, which might interfere with ECMO performance [9, 15, 31]. method is more effective than using an in-line hemofilter and provides more precise ultrafiltration control and sol- ute clearance by any modality of choice. Use of the heater on the CRRT device is optional. There is also no need for routine additional anticoagulation. However, every connection and disconnection requires support from an ECMO specialist/perfusionist and might pose a risk of air embolism/clot to both devices. Drug dosing ECMO d y g The long-term renal prognosis in ECMO survivors is uncertain. Previous studies showed high rates of libera- tion from dialysis at hospital discharge [4, 48]. However, only 42% of AKI stage 3 survivors had complete renal recovery [7]. In a cohort of post-cardiotomy patients with cardiogenic shock receiving VA-ECMO, all but 2 patients recovered from AKI stage 3 at 6 months [18]. However, it should be noted that creatinine at discharge might be falsely low due to loss of muscle mass and malnutri- tion following prolonged hospitalization. Therefore, low serum creatinine levels may lead to erroneous glomeru- lar filtration rate (GFR) results and mislead clinicians, resulting in inappropriate drug dosing and inadequate follow-up. ECMO and RRT can significantly alter the pharmacoki- netics of medications such as antibiotics and sedatives, yet little is known about the optimal regimen for patients treated with both RRT and ECMO. Generally, ECMO increases the volume of distribution and reduces drug clearance. The ECMO circuit might act as a reservoir and redistribute the sequestered drug back into the patient leading to prolonged effects, especially of lipophilic med- ications with a large volume of distribution (e.g., voricon- azole, propofol, fentanyl, midazolam) [8]. In contrast, the ECMO membrane and tubing may adsorb some drugs and reduce plasma concentrations. The presence of RRT increases the risk of both under- and over-dosing further. A preliminary analysis showed that standard dosing of meropenem (1 g 8-hourly) is likely to maintain sufficient trough concentrations (> 2 mg/l) to treat highly suscepti- ble gram-negative pathogens but might be inadequate for higher trough targets [43]. Individualizing drug regimens in patients receiving concomitant ECMO and RRT using therapeutic drug monitoring is suggested where possible until more pharmacokinetic data become available. It is established that AKI survivors are at increased risk for long-term mortality, end-stage kidney dis- ease, chronic kidney disease (CKD), and poorer qual- ity of life. However, only a few studies have explored the long-term outcomes of ECMO patients with AKI. In children, two large ECMO studies independently reported a 20-year experience and showed no incidence of end-stage kidney disease in the absence of primary renal disease [38, 48]. In contrast, analysis of a VA- ECMO cohort of adult population showed an 85% inci- dence of major adverse kidney events, comprised death, end-stage kidney disease, and reduced GFR at 1 year [49]. Anticoagulation Systemic infusion of unfractionated heparin is the stand- ard anticoagulation in patients receiving RRT and ECMO unless contraindicated. However, significant clotting or excessive bleeding precluding the use of systemic heparin may require the addition of regional citrate anticoagula- tion to ensure effective RRT delivery [42]. Nevertheless, significant citrate dilution might occur. If the RRT access line is connected from the post-oxygenator limb and the return line is connected to the pre-oxygenator limb, Combining RRT into the ECMO circuit has several benefits. No additional vascular access is needed and complications related to line insertion are avoided. This Ostermann and Lumlertgul Critical Care (2021) 25:313 Page 8 of 10 (SOFA) score, blood loss, transfusion requirement, hemodynamic instability, liver failure, low Glasgow coma score, and fluid overload [7, 46, 47]. infused citrate will be partially delivered and mixed with pre-oxygenator blood. This may reduce clotting in the oxygenator. Calcium should be infused via a separate central venous access to reduce clotting in the system. Published: 31 August 2021 21. Lou S, MacLaren G, Best D, Delzoppo C, Butt W. Hemolysis in pediatric patients receiving centrifugal-pump extracorporeal membrane oxygena- tion: prevalence, risk factors, and outcomes. Crit Care Med. 2014;42:1213–20. Short‑term outcomes AKI and RRT have been shown to be independently asso- ciated with mortality but it is uncertain whether they directly increase the risk of dying or merely represent the acuity and severity of the illness [19]. The pooled esti- mated hospital and/or 90-day mortality rates of patients with AKI and severe AKI requiring RRT while on ECMO were 62.0% and 68.4%, respectively [6]. The likelihood of dying in hospital of ECMO patients receiving RRT is three times that of those without RRT [6]. Importantly, mortality has decreased by > 20% since 2016 compared with data from before 2015, possibly due to better patient selection, timing, and clinical application [44]. Risk factors for 1-year major adverse kidney events included lower GFR at baseline, higher AKI stage at ECMO cannulation, and number of red blood cell trans- fusions. Moreover, the median GFR decline was 20 ml/ min/1.73 m2, and half of AKI survivors had a GFR decline of more than 30%. Decline of GFR by > 30% is associated with > 5 times increased risk of end-stage kidney disease [50]. Therefore, the risk of serious long-term renal out- comes should not be underestimated in ECMO patients with AKI. Analysis of a national Taiwan database includ- ing 3200 adult patients receiving ECMO with up to 10-year follow-up data revealed higher rates of all- cause mortality, end-stage kidney disease and CKD in patients with RRT-requiring AKI compared with non-dialy- sis-requiring AKI patients [45]. Prolonged CRRT use (> 7 days vs. ≤ 6 days) was associated with an increased risk of end-stage kidney disease, ventilator dependence, and readmission rate but not survival after discharge [51]. AKI requiring RRT is associated with other complica- tions, including sepsis, need for fasciotomy/amputation, respiratory failure, intra-aortic balloon pump (IABP) usage, massive blood transfusion, and failure to wean from ECMO [45]. Acute respiratory failure can also be worsened following AKI due to fluid overload, pulmonary edema, increased inflammatory mediators and increased risk of intercurrent sepsis. Other risk factors for mor- tality include age, oliguria, AKI stage 3, RRT duration hypercapnia, high sequential organ failure assessment Ostermann and Lumlertgul Critical Care (2021) 25:313 Ostermann and Lumlertgul Critical Care (2021) 25:313 Page 9 of 10 Acknowledgements g The authors would like to thank the critical care team at Guy’s & St Thomas’ Hospital for providing care to patients who need ECMO and renal replace- ment therapy. 11. Murugan R, Wen X, Shah N, Lee M, Kong L, Pike F, et al. Plasma inflamma- tory and apoptosis markers are associated with dialysis dependence and death among critically ill patients receiving renal replacement therapy. Nephrol Dial Transp. 2014;29:1854–64. 11. Murugan R, Wen X, Shah N, Lee M, Kong L, Pike F, et al. Plasma inflamma- tory and apoptosis markers are associated with dialysis dependence and death among critically ill patients receiving renal replacement therapy. Nephrol Dial Transp. 2014;29:1854–64. Availability of data and materials 14. Ootaki C, Yamashita M, Ootaki Y, Kamohara K, Weber S, Klatte RS, et al. Reduced pulsatility induces periarteritis in kidney: role of the local renin- angiotensin system. J Thorac Cardiovasc Surg. 2008;136:150–8. Material used in this manuscript is available from the corresponding author at reasonable request. g y g ; 15. Askenazi DJ, Selewski DT, Paden ML, Cooper DS, Bridges BC, Zappitelli M, et al. Renal replacement therapy in critically ill patients receiving extracor- poreal membrane oxygenation. Clin J Am Soc Nephrol. 2012;7:1328–36. 16. Villa G, Katz N, Ronco C. Extracorporeal membrane oxygenation and the kidney. Cardiorenal Med. 2015;6:50–60. 15. Askenazi DJ, Selewski DT, Paden ML, Cooper DS, Bridges BC, Zappitelli M, et al. Renal replacement therapy in critically ill patients receiving extracor- poreal membrane oxygenation. Clin J Am Soc Nephrol. 2012;7:1328–36. Ethics approval and consent to participate Not applicable. 17. Ricci Z, Pezzella C, Romagnoli S, Iodice F, Haiberger R, Carotti A, et al. High levels of free hemoglobin in neonates and infants undergoing surgery on cardiopulmonary bypass. Interact Cardiovasc Thorac Surg. 2014;19:183–7. 18. Lepère V, Duceau B, Lebreton G, Bombled C, Dujardin O, Boccara L, et al. Risk factors for developing severe acute kidney injury in adult patients with refractory postcardiotomy cardiogenic shock receiving venoarterial extracorporeal membrane oxygenation. Crit Care Med. 2020;48:e715–21. Funding g No funding was received. Publications costs were funded by Dr Ostermann’s research fund. 13. Kilburn DJ, Shekar K, Fraser JF. The complex relationship of extracorporeal membrane oxygenation and acute kidney injury: Causation or associa- tion? Biomed Res Int. 2016;2016:1094296. Conclusion Husain-Syed F, Ricci Z, Brodie D, Vincent JL, Ranieri VM, Slutsky AS, et al. Extracorporeal organ support (ECOS) in critical illness and acute kidney injury: from native to artificial organ crosstalk. Intensive Care Med. 2018;44:1447–59. 8. Husain-Syed F, Ricci Z, Brodie D, Vincent JL, Ranieri VM, Slutsky AS, et al. Extracorporeal organ support (ECOS) in critical illness and acute kidney injury: from native to artificial organ crosstalk. Intensive Care Med. 2018;44:1447–59. 9. Joannidis M, Forni LG, Klein SJ, Honore PM, Kashani K, Ostermann M, et al. Lung-kidney interactions in critically ill patients: consensus report of the acute disease quality initiative(ADQI) 21 workgroup. Intensive Care Med. 2020;46:654–72. 9. Joannidis M, Forni LG, Klein SJ, Honore PM, Kashani K, Ostermann M, et al. Lung-kidney interactions in critically ill patients: consensus report of the acute disease quality initiative(ADQI) 21 workgroup. Intensive Care Med. 2020;46:654–72. 10. Gurkan OU, O’Donnell C, Brower R, Ruckdeschel E, Becker PM. Dif- ferential effects of mechanical ventilatory strategy on lung injury and systemic organ inflammation in mice. Am J Physiol Lung Cell Mol Physiol. 2003;285:L710–8. Authors’ contributions MO and NL reviewed the existing literature. NL wrote the first draft. MO made edits to the draft. All authors read and approved the final draft. 12. Brower RG, Matthay MA, Morris A, Schoenfeld D, Thompson BT, Wheeler A. Ventilation with lower tidal volumes as compared with traditional tidal volumes for acute lung injury and the acute respiratory distress syndrome. N Engl J Med. 2000;342:1301–8. Competing interests MO has received research funding and speaker honoraria from Fresenius Medical Care and Baxter. NL reports no conflict of interest. 19. Lee SW, Yu MY, Lee H, Ahn SY, Kim S, Chin HJ, et al. Risk factors for acute kidney injury and in-hospital mortality in patients receiving extracorpor- eal membrane oxygenation. PloS ONE. 2015;10:e0140674. 20. Salis S, Mazzanti VV, Merli G, Salvi L, Tedesco CC, Veglia F, et al. Cardio- pulmonary bypass duration is an independent predictor of morbid- ity and mortality after cardiac surgery. J Cardiothorac Vasc Anesth. 2008;22:814–22. Declarations yg 16. Villa G, Katz N, Ronco C. Extracorporeal membrane oxygenation and the kidney. Cardiorenal Med. 2015;6:50–60. 16. Villa G, Katz N, Ronco C. Extracorporeal membrane oxygenation and the kidney. Cardiorenal Med. 2015;6:50–60. Conclusion 3. Gao S, Liu G, Yan S, Lou S, Gao G, Hu Q, et al. Outcomes from adult veno-arterial extracorporeal membrane oxygenation in a cardiovascular disease center from 2009 to 2019. Perfusion. 2021; https://doi.org/10. 1177/0267659121993365. [Epub ahead of print]. AKI is extremely common and associated with worse short-term and long-term outcomes in patients receiv- ing ECMO, especially when RRT is required. The most common indication for RRT initiation in these patients is fluid control. RRT should be initiated when the anticipated demand from fluid overload and metabolic derangements exceeds the capacity of the kidneys to compensate. The modality and techniques of provid- ing RRT in patients receiving ECMO depend on local practice and expertise. Provision of RRT as an arma- mentarium of multiorgan support therapy requires a multidisciplinary team engagement (such as intensiv- ists, nephrologists, cardiologists, cardiac, thoracic and vascular surgeons, perfusionists, dedicated nurses, pharmacists, dietitians, and others) from admission, through ECMO cannulation and RRT initiation, until after discharge. Further research should determine the optimal technique to combine ECMO and RRT, optimal drug dosing and long-term renal prognosis. 4. Deatrick KB, Mazzeffi MA, Galvagno SMJ, Boswell K, Kaczoroswki DJ, Rabinowitz RP, et al. Breathing life back into the kidney—continuous renal replacement therapy and veno-venous extracorporeal membrane oxygenation. ASAIO J. 2021;67:208–12. 5. Lafç G, Budak AB, Yener A, Cicek OF. Use of extracorporeal membrane oxygenation in adults. Heart Lung Circ. 2014;23:10–23. 5. Lafç G, Budak AB, Yener A, Cicek OF. Use of extracorporeal membrane oxygenation in adults. Heart Lung Circ. 2014;23:10–23. 6. 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Combes A, Peek GJ, Hajage D, Hardy P, Abrams D, Schmidt M, et al. ECMO for severe ARDS: systematic review and individual patient data meta- analysis. Intensive Care Med. 2020;46:2048–57. Ostermann and Lumlertgul Critical Care (2021) 25:313 Ostermann and Lumlertgul Critical Care (2021) 25:313 Page 10 of 10 Page 10 of 10 23. Swaniker F, Kolla S, Moler F, Custer J, Grams R, Barlett R, et al. Extracor- poreal life support outcome for 128 pediatric patients with respiratory failure. J Pediatr Surg. 2000;35:197–202. 43. Shekar K, Fraser JF, Taccone FS, Welch S, Wallis SC, Mullany DV, et al. The combined effects of extracorporeal membrane oxygenation and renal replacement therapy on meropenem pharmacokinetics: a matched cohort study. Crit Care. 2014;18:565. 24. Fleming GM, Askenazi DJ, Bridges BC, Cooper DS, Paden ML, Selewski DT, et al. A multi-center international survey of renal supportive therapy during ECMO: the kidney intervention during extracorporeal membrane oxygenation (KIDMO) group. ASAIO J. 2012;58:407–14. 44. Mitra S, Ling RR, Tan CS, Shekar K, MacLaren G, Ramanathan K. Concurrent use of renal replacement therapy during extracorporeal membrane oxygenation support: a systematic review and meta-analysis. J Clin Med. 2021;10:241. oxygenation (KIDMO) group. ASAIO J. 2012;58:407–14. yg g p 25. Bagshaw SM, Wald R, Adhikari NKJ, Bellomo R, da Costa BR, Dreyfuss D, et al. Timing of initiation of renal-replacement therapy in acute kidney injury. N Engl J Med. 2020;383:240–51. 45. Chen SW, Lu YA, Lee CC, Chou AH, Wu VC, Chang SW, et al. Long-term outcomes after extracorporeal membrane oxygenation in patients with dialysis-requiring acute kidney injury: a cohort study. PLoS ONE. 2019;14:e0212352. j y g 26. Barbar SD, Clere-Jehl R, Bourredjem A, Hernu R, Montini F, Bruyère R, et al. Timing of renal-replacement therapy in patients with acute kidney injury and sepsis. N Engl J Med. 2018;379:1431–42. 46. Haneya A, Diez C, Philipp A, Bein T, Mueller T, Schmid C, et al. Impact of acute kidney injury on outcome in patients with severe acute respiratory failure receiving extracorporeal membrane oxygenation. Crit Care Med. 2015;43:1898–906. 27. Gaudry S, Hajage D, Schortgen F, Martin-Lefevre L, Pons B, Boulet E, et al. Initiation strategies for renal-replacement therapy in the intensive care unit. N Engl J Med. 2016;375:122–33. 47. Schmidt M, Bailey M, Kelly J, Hodgson C, Cooper DJ, Scheinkestel C, et al. Impact of fluid balance on outcome of adult patients treated with extra- corporeal membrane oxygenation. Intensive Care Med. 2014;40:1256–66. 28. Gaudry S, Hajage D, Schortgen F, Martin-Lefevre L, Verney C, Pons B, et al. Timing of renal support and outcome of septic shock and acute respira- tory distress syndrome. 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https://openalex.org/W2584116200	https://europepmc.org/articles/pmc5286452?pdf=render	English	null	Correction: Corrigendum: Microtubule stabilising peptides rescue tau phenotypes in-vivo	Scientific reports	2,017	cc-by	226	www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports The Acknowledgements section in this Article is incomplete. “This work was supported by the Wessex Medical Trust, the Alzheimer’s Society and the Henry Smith’s foun- dation. Thanks to Prof. St. Johnstone (University of Cambridge, UK) for providing the dtau antibody and Dr. Peter Davies (Albert Einstein College of Medicine; Bronx, NY) for providing the PHF-1 antibody”. Scientific Reports \| 7:40833 \| DOI: 10.1038/srep40833 Corrigendum: Microtubule stabilising peptides rescue tau phenotypes in-vivoi cientific Reports 6:38224; doi: 10.1038/srep38224; published online 02 December 2016; updated on 01 February 017 should read: “This work was supported by the Wessex Medical Trust, the Alzheimer’s Society and the Henry Smith’s founda- tion. Thanks to Prof. St. Johnstone (University of Cambridge, UK) for providing the dtau antibody and Dr. Peter Davies (Albert Einstein College of Medicine; Bronx, NY) for providing the PHF-1 antibody. Thanks to the ARUK South Coast Network for contributing to publication costs”. This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ © The Author(s) 2017
https://openalex.org/W2049692703	https://europepmc.org/articles/pmc3178495?pdf=render	English	null	Program for expectant and new mothers: a population-based study of participation	BMC public health	2,011	cc-by	8,284	RESEARCH ARTICLE Open Access Abstract Background: The Manitoba Healthy Baby Program is aimed at promoting pre- and perinatal health and includes two components: 1) prenatal income supplement; 2) community support programs. The goal of this research was to determine the uptake of these components by target groups. Methods: Data on participation in each of the two program components were linked to data on all hospital births in Manitoba between 2004/05 through 2007/08. Descriptive analyses of participation by maternal characteristics were produced. Logistic regression analyses were conducted to identify factors associated with participation in the two programs. Separate regressions were run for two groups of women giving birth during the study period: 1) total population; 2) those receiving provincial income assistance during the prenatal period. Results: Almost 30% of women giving birth in Manitoba received the Healthy Baby prenatal income supplement, whereas only 12.6% participated in any community support programs. Over one quarter (26.4%) of pregnant women on income assistance did not apply for and receive the prenatal income supplement, despite all being eligible for it. Furthermore, 77.8% of women on income assistance did not participate in community support programs. Factors associated with both receipt of the prenatal benefit and participation in community support programs included lower SES, receipt of income assistance, obtaining adequate prenatal care, having completed high school and having depressive symptoms. Having more previous births was associated with higher odds of receiving the prenatal benefit, but lower odds of attending community support programs. Being married was associated with lower odds of receiving the prenatal benefit but higher odds of participating in community support programs. Conclusions: Although uptake of the Healthy Baby program in Manitoba is greater for women in groups at risk for poorer perinatal outcomes, a substantial number of women eligible for this program are not receiving it; efforts to reach these women should be enhanced. infant morbidity and mortality [11] as well as longer- term health, cognitive and behavioural problems [12-20]. infant morbidity and mortality [11] as well as longer- term health, cognitive and behavioural problems [12-20]. Fortunately, a great deal is known regarding not only risk factors, but also some of the protective factors asso- ciated with perinatal outcomes. Good prenatal nutrition can have a positive impact on birth weight, gestation and intrauterine growth [21] as well as on neurological development [22-24]. * Correspondence: marni_brownell@cpe.umanitoba.ca 1Department of Community Health Sciences, Faculty of Medicine, University of Manitoba, 408-727 McDermot Avenue, Winnipeg, Manitoba, R3E 3P5, Canada Full list of author information is available at the end of the article © 2011 Brownell 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 Adequate prenatal care can also have a positive impact on perinatal outcomes through medical, nutritional and educational interventions [25]. There is also abundant evidence on the positive effects of breastfeeding on health in infancy and early child- hood [26,27]. The type of parenting an infant receives also has a tremendous impact on health and develop- ment; warm and responsive parental care is a protective Marni D Brownell1,2, Mariette Chartier1,2, Wendy Au2 and Jennifer Schultz2 Marni D Brownell1,2, Mariette Chartier1,2, Wendy Au2 and Jennifer Schultz2 Brownell et al. BMC Public Health 2011, 11:691 http://www.biomedcentral.com/1471-2458/11/691 Brownell et al. BMC Public Health 2011, 11:691 http://www.biomedcentral.com/1471-2458/11/691 Brownell et al. BMC Public Health 2011, 11:691 Population and Data Source This study took place in Manitoba, a province of 1.2 million people in the geographic centre of Canada. All data came from the Manitoba Population Health Research Data Repository which houses population- based information on health and social service utiliza- tion for all residents of Manitoba. Due to comprehen- sive universal health care coverage, virtually all contacts with the health care system are captured [39,40]. The databases used in this study included: hos- pital discharge abstracts (which include up to 25 ICD10-CA diagnosis codes); physician visit records (which include an ICD-9-CM diagnosis code); the population registry (which includes demographic infor- mation on all residents registered for health care); newborn screening forms (which assess biological and social risk factors for families with a newborn, and are completed by Public Health Nurses on about 85% of all live births in the province); small-area census infor- mation (linked by residential postal codes); and social allowance management information (which includes information on all residents receiving provincial income assistance). Individual-level information from these data sources was linked across data sets using encrypted identifying numbers. The validity of the data in the Repository is well-documented [39,41-46]. Success of prenatal and infant programs is at least partially dependent on the uptake of these programs by target groups; if not all of the most vulnerable women and families participate in the programs, then it is diffi- cult to evaluate their success. Evidence from the Sure Start program in the UK suggests that positive benefits of the program are limited to the less socially deprived participants, with some adverse effects evident in the most disadvantaged families [36,37]. Relatively little information is available on uptake of prenatal and infant programs. An evaluation of the Canada Prenatal Nutri- tion Program claims that the program is effective at reaching “at-risk” populations, however this conclusion is based on demographic descriptions of participants, with no information on who is not participating in the program [38]. The study examined all Manitoba women having a live birth in hospital from April 1, 2004 through March 31, 2008 (N = 56,560). Background infant morbidity and mortality [11] as well as longer- term health, cognitive and behavioural problems [12-20]. Fortunately, a great deal is known regarding not only risk factors, but also some of the protective factors asso- ciated with perinatal outcomes. Good prenatal nutrition can have a positive impact on birth weight, gestation and intrauterine growth [21] as well as on neurological development [22-24]. Adequate prenatal care can also have a positive impact on perinatal outcomes through medical, nutritional and educational interventions [25]. There is also abundant evidence on the positive effects of breastfeeding on health in infancy and early child- hood [26,27]. The type of parenting an infant receives also has a tremendous impact on health and develop- ment; warm and responsive parental care is a protective The time extending from conception to a child’s first birthday is a crucial one in terms of child development and life-long health [1-3]. Maternal factors such as stressful life circumstances, low socioeconomic status, poor nutrition and health, and smoking and alcohol/ drug use during pregnancy can adversely influence birth outcomes and newborn health [4-10]. In turn, outcomes such as low birth weight, preterm births and intrauter- ine growth restriction have an impact on neonatal and Brownell et al. BMC Public Health 2011, 11:691 http://www.biomedcentral.com/1471-2458/11/691 Page 2 of 10 programs, and that not all those eligible for or targeted by the Healthy Baby program would be participating. factor in infancy which leads to secure attachments with parents and healthy neurological and psychological development [28]. Methods Based on this evidence, a number of prenatal and early childhood programs have been developed to improve birth and infant outcomes. These programs can improve outcomes by advocating for prenatal care, encouraging cessation of smoking and alcohol use, providing supple- mental incomes, promoting breastfeeding and positive parenting practices, and by decreasing stress through the provision of social and emotional support. Nutrition intervention programs and programs offering income supplements have both demonstrated positive effects on birth outcomes [29-34]. There is also evidence that high- risk mothers and their infants, such as those experiencing a high degree of stress or living in low income situations, benefit from social support programs [35]. Population and Data Source Program data from the Healthy Baby program came from the Healthy Child Manitoba Office and included information on: 1) women receiving the prenatal benefit of up to $81 (CAD) per month during the second and third trimesters of pregnancy; 2) women participating in prenatal and/or postnatal community support programs, which varied in content across com- munities, but common goals included encouraging early and regular prenatal care, promoting healthy infant development, and improving nutrition. The program components are promoted through doctors’ offices, pos- ters within communities (e.g., bus shelters, community centre bulletin boards) and websites. These data were linked to birth records in the hospital abstract database to identify women participating in either component of the Healthy Baby program who had a live birth during the study period. The current study made use of a unique opportunity to link together population-based information on all births in the Canadian province of Manitoba, with pre- natal and infant program participation data, to describe uptake of the program. In 2001, the Healthy Baby pro- gram was introduced province-wide in Manitoba by the Healthy Child Manitoba Office. The goal of this pro- gram was to promote prenatal and perinatal health. The Manitoba Healthy Baby program consists of two compo- nents: 1) a prenatal benefit, which is a targeted income supplement for low income women, and; 2) community support programs, which are educational and supportive groups available to all women from the prenatal period through to an infant’s first birthday. The goal of this study was to determine whether groups of women tar- geted by the Health Baby program (e.g., low income) were participating in each of the components of the program and to identify maternal factors associated with participation in the program. We hypothesized that par- ticipation by target groups would be greater for the pre- natal benefit component than the community support The study protocol was approved by the University of Manitoba Health Research Ethics Board (H2008:187), the Manitoba Health Information Privacy Committee, Manitoba Family Services and Consumer Affairs, and the Healthy Child Manitoba Office. Brownell et al. BMC Public Health 2011, 11:691 http://www.biomedcentral.com/1471-2458/11/691 Brownell et al. BMC Public Health 2011, 11:691 http://www.biomedcentral.com/1471-2458/11/691 Page 3 of 10 Page 3 of 10 Variables Used in Analyses Independent variables for two groups of women: 1) all women giving birth during the study period; 2) all women giving birth dur- ing the study period who received income assistance for at least one month during pregnancy. for two groups of women: 1) all women giving birth during the study period; 2) all women giving birth dur- ing the study period who received income assistance for at least one month during pregnancy. Several characteristics of women giving birth that are potentially related to participation in either component of the Healthy Baby program were identified as covari- ates: 1) mother’s age at the time of the baby’s birth was dichotomized as < 20 and 20+ years; 2) parity was cate- gorized as 0, 1, 2 or 3+ previous births; 3) adequacy of prenatal care was defined using date of initiation and number of prenatal visits according to the index created by Alexander and Kotelchuck [47]; 4) region of resi- dence was categorized as urban (Winnipeg, population = 675,000, and Brandon, population = 45,000)) and rural (the rest of Manitoba), with rural divided geogra- phically into south and mid, and north Manitoba; 5) a composite measure of area-level SES comprising infor- mation from the 2006 Canada census on employment, education, lone-parent families and income [48,49], based on areas of approximately 400 people; 6) indica- tion of receipt of income assistance for at least one month during pregnancy; 7) marital status; 8) high school completion; and, 9) maternal depressive symp- toms. Table 1 shows the percent of women in each of the study populations with each of these characteristics. Dependent variables PNC = Prenatal Care SES = Socioeconomic status. Composite measure based on area-level education, employment, family structure and income. IA = income assistance Results Of the 56,560 hospital births in the study period, 16,540 (29.2%) were to women who received the Healthy Baby prenatal benefit. There was little variation in these per- centages over the 4 years of the study period. Figure 1 shows the percent of births to women who received the prenatal benefit by groups that were targeted by the Healthy Baby program. Nearly three-quarters (72.4%) of women receiving income assistance during pregnancy received the prenatal benefit, compared to 21.8% of women not receiving income assistance (Figure 1a). Over 50% of women living in the lowest rural (50.2%) and urban (52.7%) income areas received the prenatal benefit, compared to 13.6% in the rural highest and 6.4% in the urban highest income areas (Figure 1b). 57.0% of mothers who were teens received the prenatal benefit, compared to 26.8% of mothers who were 20 year or older when they gave birth (Figure 1c). Regression analyses were repeated including only those women who received income assistance during pregnancy (Table 3). Maternal factors associated with increased odds of both prenatal benefit receipt and com- munity support program participation for women receiving income assistance during pregnancy were receipt of adequate prenatal care, living in a lower SES area, and being rated as depressed by a Public Health Nurse during the early postnatal period. Living in north- ern compared to urban Manitoba was associated with reduced odds of receiving the prenatal benefit and parti- cipating in community support programs for this group of women. Being married was associated with reduced odds of receiving the prenatal benefit, but increased odds of participating in community support programs. Living in rural south/mid compared to urban Manitoba was also associated with reduced odds of prenatal bene- fit receipt but increased odds of community support program participation. Being a teen mom was associated with reduced odds of receiving the prenatal benefit, but was not associated with community support program participation. Number of children was not associated with prenatal benefit receipt, but having fewer children was associated with increased odds of participation in y y g g Of the 48,007 hospital births to women living in communities where we had information on commu- nity support program participation, 6063 (12.8%) were to women who participated in at least one community support program during pregnancy and/or up to one year postnatally. Analysis The statistical method used in our analysis was General- ized Linear Models (GLM) with a binomial distribution. Several predictors (see “Variables Used in Analysis”) which were believed to be associated with a mother’s uptake of the prenatal benefit and/or her participation in community support programs were entered into the regression models and parameter estimates and 95% confidence intervals for each of the predictors were pro- duced. Exponentiation of the parameter estimates allowed us to calculate the odds ratios for each of the predictors. Separate regressions were run for the two groups of women giving birth defined above. Models were run separately for prenatal benefit receipt and community support program participation (four models in total). Women living in First Nations communities, and two small communities in southern Manitoba, were excluded from analyses of community support program participation because their participation was not known. A small percentage of women participating in commu- nity support programs declined to share information on their participation, so were also removed from the ana- lysis of support program participation. These exclusions resulted in the removal of 8553 births (15.1% of total The two outcomes studied were receipt of the prenatal benefit and participation in community support pro- grams. We looked at factors associated with participa- tion in either component of the Healthy Baby program Table 1 Characteristics of Study Populations Table 1 Characteristics of Study Populations All Women Giving Birth (N = 56560) Women Giving Birth who received IA (N = 8183) Variable All (%) PB (%) CSP (%) All (%) PB (%) CSP (%) Teen mother 8.6 16.7 11.2 19.4 18.6 20.9 Parity 0 39.0 36.7 52.1 26.4 26.9 33.9 Parity 1 31.6 24.5 25.8 26.9 26.5 24.2 Parity 2 15.5 16.5 11.8 19.9 19.7 16.3 Parity 3 13.9 22.3 10.4 26.9 26.9 25.6 adequate PNC 28.2 22.0 34.0 18.2 20.5 24.0 urban 55.0 50.8 54.9 68.3 72.6 76.3 north 11.4 16.7 3.2 12.6 9.4 5.7 Mid/south 33.6 32.5 41.9 19.1 18.1 17.9 Mean SES (high value = low SES) 0.33 1.00 0.30 1.00 1.03 0.97 on IA during pg 14.6 36.3 27.4 100 100 100 married 69.6 36.2 65.6 21.8 15.4 21.5 high school 61.0 42.8 69.1 27.8 29.6 30.9 Depressive symptoms 9.8 11.9 16.5 30.3 28.6 24.2 PB t l b fit i t Brownell et al. Analysis BMC Public Health 2011, 11:691 http://www.biomedcentral.com/1471-2458/11/691 Page 4 of 10 Regression analysis results identifying factors asso- ciated with receipt of the prenatal benefit and/or partici- pation in community support programs for all women giving birth are given in Table 2. population) from the community support program ana- lysis. Thus, for the full population of women giving birth, there were 56560 births in the analysis of prenatal benefit receipt and 48007 births in the analysis of com- munity support program participation. For the analysis of women receiving income assistance during pregnancy, there were 8183 births in the analysis of prenatal benefit receipt and 7398 births in the analysis of community support program participation. For regressions where the outcome was receipt of prenatal benefit, then prena- tal community support program participation was entered as another covariate in the models; where com- munity support program participation was the outcome, prenatal benefit receipt was entered into the models. All analyses were performed using SAS version 9 [50]. For all women giving birth (total study population), maternal factors associated with increased odds of both prenatal benefit receipt and community support pro- gram participation were being a teen mother, living in south/mid rural compared to urban Manitoba, living in a low SES area, receiving income assistance during preg- nancy, having completed high school, and being rated as depressed by a Public Health Nurse during the early postnatal period. Living in north Manitoba was asso- ciated with lower odds of prenatal benefit receipt and community support program participation compared to women living in urban Manitoba. For two maternal fac- tors, the associations with prenatal benefit receipt were different than the associations with community support program participation. Having fewer children and being married were both associated with decreased odds of prenatal benefit receipt but increased odds of commu- nity support program participation. Receipt of adequate prenatal care was associated with increased odds of community support program participation, but was not associated with prenatal benefit receipt. Prenatal com- munity support program participation was associated with increased odds of prenatal benefit receipt, and receipt of the prenatal benefit was associated with increased odds of community support program participation. Results Figure 2 shows the percent of births to women participating in community support pro- grams by groups targeted by the Health Baby pro- gram. Just over one fifth (22.1%) of women receiving income assistance during pregnancy participated in any Community Support Program, compared to 10.8% of women not receiving income assistance (Figure 2a). Less than one fifth of women living in the lowest rural (12.9%) and urban (18.5%) income areas partici- pated in community support programs (Figure 2b), compared to 10.5% and 5.1% for the highest rural and urban income areas respectively. Just over one fifth of teen mothers (21.2%) participated in community sup- port programs (Figure 2c), compared to 12.1% of those 20 or older. Brownell et al. BMC Public Health 2011, 11:691 http://www.biomedcentral.com/1471-2458/11/691 Page 5 of 10 a) b) c) 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% IA no IA 647 women with missing IA information were excluded from graph 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% 15 and younger 16-17 18-19 20-29 30 and older 261 women with missing age information were excluded from graph 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% R1 R2 R3 R4 R5 U1 U2 U3 U4 U5 1211 women with missing income information were excluded from graph e of prenatal benefit by women at risk for poorer perinatal outcomes. a) By receipt of income assistance. b) By area-level other’s age at current birth. Results a) 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% IA no IA 647 women with missing IA information were excluded from graph 647 women with missing IA information were excluded from graph b) 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% R1 R2 R3 R4 R5 U1 U2 U3 U4 U5 1211 women with missing income information were excluded from graph b) 1211 women with missing income information were excluded from graph 1211 women with missing income information were excluded from graph c) 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% 15 and younger 16-17 18-19 20-29 30 and older 261 women with missing age information were excluded from graph Figure 1 Uptake of prenatal benefit by women at risk for poorer perinatal outcomes a) By receipt of income assistance b) By area lev c) Figure 1 Uptake of prenatal benefit by women at risk for poorer perinatal outcomes. a) By receipt of income assistance. b) By area-level income. c) By mother’s age at current birth. community support programs whereas having more children was associated with decreased odds. Having completed high school was associated with increased odds of receiving the prenatal benefit, but was not significantly associated with participation in community support programs. As was found for the analysis includ- ing all women giving birth, for women receiving income assistance during pregnancy participation in prenatal Brownell et al. BMC Public Health 2011, 11:691 http://www.biomedcentral.com/1471-2458/11/691 Page 6 of 10 t i t d ith Di i 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% IA noIA 647 womenwithmissingIAinformationwereexcludedfromgraph 30% 40% 50% 60% 70% 80% 90% 100% a) b) c) 0% 10% 20% 30% 40% R1 R2 R3 R4 R5 U1 U2 U3 U4 U5 1074womenwithmissing incomeinformationwereexcludedfromgraph 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% 15and younger 16Ͳ17 18Ͳ19 20Ͳ29 30andolder 180womenwithmissingageinformationwereexcludedfromgraph pation in community support programs by women at risk for poorer perinatal outcomes. a) By receipt of income area-level income. c) By mother’s age at current birth. Results 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% IA noIA 647 womenwithmissingIAinformationwereexcludedfromgraph 30% 40% 50% 60% 70% 80% 90% 100% a) b) c) 0% 10% 20% 30% 40% R1 R2 R3 R4 R5 U1 U2 U3 U4 U5 1074womenwithmissing incomeinformationwereexcludedfromgraph 40% 50% 60% 70% 80% 90% 100% 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% IA noIA 647 womenwithmissingIAinformationwereexcludedfromgraph a) 647 womenwithmissingIAinformationwereexcludedfromgraph 30% 40% 50% 60% 70% 80% 90% 100% b) 0% 10% 20% 30% 40% R1 R2 R3 R4 R5 U1 U2 U3 U4 U5 1074womenwithmissing incomeinformationwereexcludedfromgraph b) c) 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% 15and younger 16Ͳ17 18Ͳ19 20Ͳ29 30andolder 180womenwithmissingageinformationwereexcludedfromgraph c) Figure 2 Participation in community support programs by women at risk for poorer perinatal outcomes. a) By receipt of income assistance. b) By area-level income. c) By mother’s age at current birth. community support programs was associated with increased odds of prenatal benefit receipt and receipt of the prenatal benefit was associated with increased odds of community support program participation. Discussion Close to one third of all births (29%) in Manitoba were to women who received the Healthy Baby prena- tal benefit during pregnancy, and the benefit was Brownell et al. BMC Public Health 2011, 11:691 http://www.biomedcentral.com/1471-2458/11/691 Page 7 of 10 Table 2 Odds Ratios (95% CI) for Factors Associated with Receipt of Prenatal Benefit and Participation in Community Support Programs, All Women Giving Birth in Manitoba, 2004/05-2007/08 Variable PB CSP OR (95% CI) OR (95% CI) Mother’s Age (< 20 vs 20+) 1.31 (1.21, 1.41) 1.18 (1.06, 1.31) Parity (0 vs 3+ children) 0.6 (0.56, 0.65) 2.04 (1.84, 2.26) Parity (1 vs 3+ children) 0.52 (0.48, 0.56) 1.14 (1.02, 1.27) Parity (2 vs 3+ children) 0.67 (0.62, 0.73) 0.97 (0.86, 1.09) PNC (Adequate vs Inadequate) 1.02 (0.96, 1.07) 1.18 (1.11, 1.26) Region (North vs urban) 0.73 (0.68, 0.79) 0.85 (0.73, 1.00) Region (South/Mid vs urban) 1.17 (1.11, 1.23) 2.16 (2.03, 2.29) SES index (higher = lower SES) 1.52 (1.49, 1.55) 1.24 (1.06, 1.29) IA during pregnancy 3.47 (3.26, 3.71) 2.29 (2.09, 2.51) Marital status (married vs not) 0.2 (0.19, 0.21) 1.09 (1.00, 1.09) High School Completion 1.23 (1.17, 1.31) 1.81 (1.68, 1.95) Maternal Depressive Symptoms 1.21 (1.12, 1.30) 1.49 (1.38, 1.62) Prenatal CSP (yes vs no) 4.47 (3.93, 5.09) //// //// Prenatal CSP (unknown vs no) 2.26 (1.36, 3.82) //// //// Receipt of PB //// //// 2.10 (1.95, 2.26) Bolded values indicate statistical significance at p < 0.05 CSP = Community Support Programs PB = Prenatal Benefit PNC = Prenatal Care IA = Income Assistance Table 2 Odds Ratios (95% CI) for Factors Associated with Receipt of Prenatal Benefit and Participation in Community Support Programs, All Women Giving Birth in Manitoba, 2004/05-2007/08 Despite the relatively higher uptake of the prenatal benefit compared to the community support programs, there is still room for improvement in prenatal benefit participation. Over one quarter of women receiving income assistance, a group all eligible to receive the benefit, did not apply to receive it, and many teen mothers and women living in low income areas also did not receive the benefit. Thus, efforts to increase enrol- ment by target groups in the prenatal benefit program are warranted. Furthermore, efforts to enhance partici- pation in community support programs are required, given that a majority of the target population did not participate in these programs. Discussion The regression analyses conducted in this study identified a number of factors associated with participation in both of the components of the Healthy Baby program, providing program devel- opers and policy-makers with important evidence of how to improve participation. When looking at the entire population of women giv- ing birth, we found that although women with more children were more likely to receive the prenatal benefit, they were less likely to attend community support pro- grams. Not all Healthy Baby community support pro- grams offer child care services for children not involved in the program (children over 1 year of age) and this may be a barrier for participation by mothers with more than one child. The financial cost and difficulties of transporting multiple children to the programs may also be a barrier. Strategies to reduce the barriers to partici- pation in community support programs for women with multiple children should be considered. Obtaining adequate prenatal care was associated with greater participation in community support programs for both the entire population of women giving birth and for the income assistance population, and with pre- natal benefit receipt for the income assistance popula- tion. One of the ways pregnant women learn about the Healthy Baby program is through their prenatal care provider, so any efforts to increase early initiation of prenatal care will not only potentially benefit the preg- nancy [51-53], but also increase the likelihood that women will find out about and participate in the Healthy Baby program. Women at risk for inadequate prenatal care are also the same groups who have lower uptake of both components of the Healthy Baby pro- gram [52,54]. Strategies that have proven effective for increasing prenatal care elsewhere, such as public health outreach to disadvantaged groups [55], should be con- sidered in Manitoba. received by a majority of the women the program was designed to reach. Almost three-quarters of women receiving income assistance during pregnancy and over half of women in low income areas received the prenatal benefit. Furthermore, over half of teen mothers received the benefit, compared to about one quarter of mothers 20 years of age and older. In con- trast, less than 13% of all births in Manitoba were to women who participated in Healthy Baby community support programs, and relatively small proportions of target groups participated in these programs. Discussion Just over one-fifth of women receiving income assistance during pregnancy and the same proportion of teen-aged mothers participated in community support programs, and less than one-fifth of women from low income areas participated. received by a majority of the women the program was designed to reach. Almost three-quarters of women receiving income assistance during pregnancy and over half of women in low income areas received the prenatal benefit. Furthermore, over half of teen mothers received the benefit, compared to about one quarter of mothers 20 years of age and older. In con- trast, less than 13% of all births in Manitoba were to women who participated in Healthy Baby community support programs, and relatively small proportions of target groups participated in these programs. Just over one-fifth of women receiving income assistance during pregnancy and the same proportion of teen-aged mothers participated in community support programs, and less than one-fifth of women from low income areas participated. For both the entire population of women and the income assistance population, unmarried women were more likely to receive the prenatal benefit, but less likely to participate in community support programs. The lower participation in support programs associated with Brownell et al. Discussion BMC Public Health 2011, 11:691 http://www.biomedcentral.com/1471-2458/11/691 Page 8 of 10 Page 8 of 10 Table 3 Odds Ratios (95% CI) for Factors Associated with Receipt of Prenatal Benefit and Participation in Community Support Programs, Women Receiving Income Assistance During Pregnancy, 2004/05-2007/08 Variable PB CSP OR (95% CI) OR (95% CI) Mother’s Age (< 20 vs 20+) 0.75 (0.64, 0.88) 0.96 (0.81, 1.14) Parity (0 kids vs 3+ kids) 0.99 (0.83, 1.18) 1.44 (1.21, 1.72) Parity (1 kid vs 3+ kids) 0.88 (0.76, 1.02) 0.90 (0.77, 1.06) Parity (2 kids vs 3+ kids) 0.89 (0.76, 1.04) 0.79 (0.66, 0.94) PNC (Adequate vs Inadequate) 1.67 (1.43, 1.94) 1.27 (1.11, 1.46) Region (North vs urban) 0.4 (0.35, 0.47) 0.77 (0.61, 0.98) Region (South/Mid vs urban) 0.74 (0.65, 0.84) 1.29 (1.1, 1.51) SES (higher = lower SES) 1.13 (1.07, 1.19) 1.15 (1.08, 1.22) Marital status (married vs not) 0.25 (0.22, 0.28) 1.27 (1.10, 1.47) High School Completion 1.54 (1.36, 1.75) 1.11 (0.98, 1.27) Maternal Depressive Symptoms (yes vs no) 1.17 (1.00, 1.36) 1.39 (1.21, 1.60) Prenatal CSP (yes vs no) 3.35 (2.66, 4.22) //// //// Prenatal CSP (unknown vs no) 3.09 (1.34, 7.12) //// //// Receipt of HBPB //// //// 2.56 (2.18, 3.00) Bolded values indicate statistical significance at p < 0.05 CSP = Community Support Programs PB = Prenatal Benefit PNC = Prenatal Care IA = Income Assistance Table 3 Odds Ratios (95% CI) for Factors Associated with Receipt of Prenatal Benef Support Programs, Women Receiving Income Assistance During Pregnancy, 2004/05 Table 3 Odds Ratios (95% CI) for Factors Associated with Receipt of Prenatal Benefit and Participation in Community Support Programs, Women Receiving Income Assistance During Pregnancy, 2004/05-2007/08 not being married could suggest that unmarried women feel stigmatized or less welcome in the support groups. Anecdotal reports suggest that at least at some commu- nity program sites, married, middle-class mothers “take over” the program, which may make attendance for lower income, unmarried mothers less appealing. If uni- versal access to the community support programs is to continue, strategies to make programs welcoming to women from a broad range of socio-demographic back- grounds should be explored. the prenatal benefit presents an opportunity for mental health interventions. Both prenatal and postnatal mater- nal depression have been implicated in impaired fetal and infant development [58-60]. Acknowledgements and Funding g g A number of individuals contributed to this study. The authors wish to acknowledge our Advisory Group for their advice and insight regarding the Healthy Baby Program and interpretation of results (Cynthia Carr, Brenda Comaskey, Shannon Dennehy, Darlene Girard, Maureen Heaman, Tamara Hes, Deb Malazdrewicz, Chelsea Ruth, Rob Santos, Harvey Stevens and Joanne Waskin). The following colleagues also contributed to the study: Dan Chateau provided advice on statistical analysis and interpretation; Randy Fransoo, Pat Martens and Greg Finlayson provided feedback on draft versions of the manuscript; Charles Burchill, Shelley Derksen, Pat Nicol, Bill Peeler, Heather Prior, and Oke Ekuma assisted with data preparation and programming support; Angela Bailly assisted with table and graph preparation. 8. Mick E, Biederman J, Faraone SV, Sayer J, Kleinman S: Case-control study of attention-deficit hyperactivity disorder and maternal smoking, alcohol use, and drug use during pregnancy. J Am Acad Child Adolesc Psychiatry 2002, 41(4):378-385. 9. Ramsay MC, Reynolds CR: Does smoking by pregnant women influence IQ, birth weight, and developmental disabilities in their infants? A methodological review and multivariate analysis. Neuropsychol Rev 2000, 10(1):1-40. 10. Rasmussen C, Horne K, Witol A: Neurobehavioral functioning in children with Fetal Alcohol Spectrum Disorder. Child Neuropsychology 2006, 12(6):453-468. 11. Mathews TJ, Menacker F, MacDorman MF: Infant mortality statistics from the 2001 period linked birth/infant death data set. Natl Vital Stat Rep 2003, 52(2):1-28. p p We are indebted to Child and Family Services, Manitoba Family Services and Consumer Affairs, and the Healthy Child Manitoba Office, as well as Health Information Management, Manitoba Health for provision of data. We acknowledge the Manitoba Centre for Health Policy for use of data contained in the Population Health Research Data Repository under project # 2008-012 and Manitoba Health for providing funding for this project (HIPC#2008/2009-14). The results and conclusions are those of the authors and no official endorsement by the Manitoba Centre for Health Policy, Manitoba Health, or other data providers is intended or should be inferred. We are indebted to Child and Family Services, Manitoba Family Services and Consumer Affairs, and the Healthy Child Manitoba Office, as well as Health Information Management, Manitoba Health for provision of data. We acknowledge the Manitoba Centre for Health Policy for use of data contained in the Population Health Research Data Repository under project # 2008-012 and Manitoba Health for providing funding for this project (HIPC#2008/2009-14). Discussion For example, maternal postpartum depression has been associated with lower social engagement, more negative emotional responses and greater stress reactivity in 9-month-old infants [58]. Studies have also found that maternal depression can have long-lasting impacts on child development [61] underscoring the need for early intervention and the role the Healthy Baby program can play in identifying that need in some mothers. For the women giving birth who were on income assistance, high school completion was associated with receipt of the prenatal benefit. The completion of the three-page application form may be an onerous task for women with limited literacy skills. Providing assistance with the application process or simplifying the applica- tion form may encourage women with lower literacy skills to apply for the program. Having automatic enrol- ment in the benefit program for pregnant women receiving income assistance would increase the uptake by this group [56]. Although we did not have data on immigrant status, it is likely that language was also a barrier for completion of the application for some women; translation of the form into other languages may encourage immigrant women whose first language is not English to apply for the program [57]. The major strength of this research came from the ability to link population-based administrative data on women giving birth together with program participation data. This allowed us to determine not only who was participating in the program but also who was not parti- cipating. Together with the information on factors asso- ciated with program participation, this study provides important information to policy makers and those implementing programs for expectant and new mothers on improving uptake of programs. A limitation of the study was the lack of available data on participation in community support programs run by the Canada Prena- tal Nutrition Program (CPNP). CPNP runs the commu- nity support programs in First Nations communities and two small southern rural communities. Unlike the pro- grams run by Healthy Baby, CPNP does not collect per- son-level information on program participation, making it impossible not only to determine participation rates The finding that women with depressive symptoms, whether from the entire population or the income assis- tance population, were more likely to participate in community support programs and more likely to receive Page 9 of 10 Page 9 of 10 Brownell et al. BMC Public Health 2011, 11:691 http://www.biomedcentral.com/1471-2458/11/691 Manitoba Centre for Health Policy. Author details 1D f 15. Power C, Hertzman C, Jeffries BJ: Birth weight, childhood socioeconomic environment, and cognitive development in the 1958 British birth cohort study. BMJ 2002, 325(7359):305. 1Department of Community Health Sciences, Faculty of Medicine, University of Manitoba, 408-727 McDermot Avenue, Winnipeg, Manitoba, R3E 3P5, Canada. 2Manitoba Centre for Health Policy, University of Manitoba, 408-727 McDermot Avenue, Winnipeg, Manitoba R3E 3P5, Canada. 1Department of Community Health Sciences, Faculty of Medicine, University of Manitoba, 408-727 McDermot Avenue, Winnipeg, Manitoba, R3E 3P5, Canada. 2Manitoba Centre for Health Policy, University of Manitoba, 408-727 McDermot Avenue, Winnipeg, Manitoba R3E 3P5, Canada. 1Department of Community Health Sciences, Faculty of Medicine, University of Manitoba, 408-727 McDermot Avenue, Winnipeg, Manitoba, R3E 3P5, 2 Canada. 2Manitoba Centre for Health Policy, University of Manitoba, 408-727 McDermot Avenue, Winnipeg, Manitoba R3E 3P5, Canada. 16. Lahti J, Raikkonen K, Kajantie E, Heinonen K, Pesonen AK, Jarvenpaa AL, Strandberg T: Small body size at birth and behavioural symptoms of ADHD in children aged five to six years. J Child Psychol Psychiatry 2006, 47(11):1167-1174. Acknowledgements and Funding The results and conclusions are those of the authors and no official endorsement by the Manitoba Centre for Health Policy, Manitoba Health, or other data providers is intended or should be inferred. 12. Breslau N, Johnson EO, Lucia VC: Academic achievement of low birthweight children at age 11: The role of cognitive abilities at school entry. J Abnorm Child Psychol 2001, 29(4):273-279. 13. Hack M, Klein NK, Taylor HG: Long-term developmental outcomes of low birth weight infants. Future Child 1995, 5(1):176-196. 14. Huddy CL, Johnson A, Hope PL: Educational and behavioural problems in babies of 32-35 weeks gestation. Arch Dis Child Fetal Neonatal Ed 2001, 85(1):F23-F28. Manitoba Health, or other data providers is intended or should be inferred. Conclusions Received: 11 May 2011 Accepted: 6 September 2011 Published: 6 September 2011 Received: 11 May 2011 Accepted: 6 September 2011 Published: 6 September 2011 The Manitoba Healthy Baby prenatal benefit is reaching a reasonable proportion of women at risk for poor peri- natal outcomes, however there are opportunities to improve uptake of the program. The Manitoba Healthy Baby community support programs are not reaching the majority of women at risk for poor perinatal outcomes and efforts to increase participation are required. Identi- fying factors associated with uptake of these two compo- nents of the Healthy Baby program can help to inform those delivering the programs of where efforts are needed to increase participation. List of abbreviations CAD C di d ll List of abbreviations CAD: Canadian dollars; ICD10-CA: International Classification of Diseases, Version 10, Canadian enhancements; SES: Socioeconomic status; UK: United Kingdom. 6. Kramer MS: Determinants of low birth weight: Methodological assessment and meta-analysis. Bull World Health Organ 1987, 65(5):663-737. 7. Kramer MS, Olivier M, McLean FH, Willis DM, Usher RH: Impact of intrauterine growth retardation and body proportionality on fetal and neonatal outcome. Pediatrics 1990, 86(5):707-713. 7. Kramer MS, Olivier M, McLean FH, Willis DM, Usher RH: Impact of intrauterine growth retardation and body proportionality on fetal and neonatal outcome. Pediatrics 1990, 86(5):707-713. Authors’ contributions MB developed the design for the study, with input from the other authors. Analysis of the data was carried out by WA, with direction and input from the MB and MC. Interpretation of the data involved all authors. The manuscript was drafted by MB, with significant contributions from the other authors. All authors read and approved the final manuscript. MB developed the design for the study, with input from the other authors. Analysis of the data was carried out by WA, with direction and input from the MB and MC. Interpretation of the data involved all authors. The manuscript was drafted by MB, with significant contributions from the other authors. All authors read and approved the final manuscript. 17. Lawlor DA, Batty DG, Morton SM, Deary IJ, Macintyre S, Ronalds G, Leon DA: Early life predictors of childhood intelligence: Evidence from the Aberdeen children of the 1950s study. J Epidemiol Community Health 2005, 59(8):656-663. 18. Lawlor DA, Bor W, O’Callaghan MJ, Williams GM, Najman JM: Intrauterine growth and intelligence within sibling pairs: Findings from the Mater- University study of pregnancy and its outcomes. J Epidemiol Community Health 2005, 59(4):279-282. Discussion JS is a Research Coordinator at the Manitoba Centre for Health Policy. in these communities, but also to identify the factors in these communities that are associated with program participation. References 1. Barker DJ: The origins of the developmental origins theory. J Intern Med 2007, 261(5):412-417. 2. Power C, Hertzman C, Jeffries BJ: Birth weight, childhood socioeconomic environment, and cognitive development in the 1958 British birch cohort study. BMJ 2002, 325(7359):305. 3. Irwin LG, Siddiqi A, Hertzman C: A powerful equalizer. World Health Organization’s Commission on the social determinants of health. 2007 [http://whqlibdoc.who.int/hq/2007/a91213.pdf], Accessed March 17, 2010. 3. Irwin LG, Siddiqi A, Hertzman C: A powerful equalizer. World Health Organization’s Commission on the social determinants of health. 2007 [http://whqlibdoc.who.int/hq/2007/a91213.pdf], Accessed March 17, 2010. 4. DiFranza JR, Aligne CA, Weitzman M: Perinatal and postnatal environmental tobacco smoke exposure and children’s health. Pediatrics 2004, 113(Suppl 4):1007-1015. 4. DiFranza JR, Aligne CA, Weitzman M: Perinatal and postnatal environmental tobacco smoke exposure and children’s health. Pediatrics 2004, 113(Suppl 4):1007-1015. pp 5. Finch BK: Socioeconomic gradients and low birth-weight: Empirical and policy considerations. Health Serv Res 2003, 38(6 Pt 2):1819-1841. 6. Kramer MS: Determinants of low birth weight: Methodological assessment and meta-analysis. Bull World Health Organ 1987, 65(5):663-737. 5. Finch BK: Socioeconomic gradients and low birth-weight: Empirical and policy considerations. Health Serv Res 2003, 38(6 Pt 2):1819-1841. Authors’ information MB is a Senior Research Scientist with the Manitoba Centre for Health Policy and Associate Professor of Community Health Sciences, Faculty of Medicine, University of Manitoba. 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Scholl TO, Johnson WG: Folic acid: Influence on the outcom 24. Scholl TO, Johnson WG: Folic acid: Influence on the outcome of pregnancy. Am J Clin Nutr 2000, 71(5 Suppl):1295S-1303S. 47. Alexander GR, Kotelchuck M: Quantifying the adequacy of prenatal care: A comparison of indices. Public Health Rep 1996, 111(5):408-418. pregnancy. Am J Clin Nutr 2000, 71(5 Suppl):1295S-1303S. 25. Alexander GR, Korenbrot CC: The role of prenatal care in 25. Alexander GR, Korenbrot CC: The role of prenatal care in preventing low birth weight. Future Child 1995, 5(1):103-120. 48. Authors’ information Martens P, Frohlich N, Carriere K, Derksen S, Brownell M: Embedding child health within a framework of regional health: Population health status and sociodemographic indicators. Can J Public Health 2002, 93(Suppl 2): S15-S20. 26. 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Remler DK, Glied SA: What other programs can teach us: Increasing participation in health insurance programs. Am J Public Health 2003, 93:67-74. 34. Rodriguez-Bernal CL, Rebagliato M, Inguez C, Vioque J, Navarrete- Munoz EM, Murcia M, Bolumar F, Marco A, Ballester F: Diet quality in early pregnancy and its eff ects on fetal growth outcomes: The Infancia y Medio Ambiente (Childhood and Environment) Mother and Child Cohort Study in Spain. Am J Clin Nutr 2010, 91(6):1659-1666. 57. Aizer A: Public health insurance, program take-up, and child health. The Review of Economics and Statistics 2007, 89(3):400-415. 58. Feldman R, Granat A, Pariente C, Kanety H, Kuint J, Gilboa-Schechtman E: Maternal depression and anxiety across the postpartum year and infant social engagement, fear regulation, and stress reactivity. J Am Acad Child Adolesc Psychiatry 2009, 48(9):919-927. 35. Shaw E, Levitt C, Wong S, Kaczorowski J: Systematic review of the literature on postpartum care: Eff ectiveness of postpartum support to improve maternal parenting, mental health, quality of life, and physical health. Birth 2006, 33(3):210-220. 59. Reck C, Hunt A, Fuchs T, Weiss R, Noon A, Moehler E, Downing G, Tronick EZ, Mundt C: Interactive regulation of affect in postpartum depressed mothers and their infants: an overview. Psychopathology 2004, 37:272-280. 36. Belsky J, Melhuish E, Barnes J, Leyland AH, Romaniuk H, the National Evaluation of Sure Start Research Team: Effects of Sure Start local programmes on children and families” early findings from a quasi- experimental, cross sectional study. BMJ 2006, 332:1476-1481. 60. Weinstock M: Alterations induced by gestational stress in brain morphology and behaviour of the offspring. Prog Neurobiol 2001, 65:427-451. 37. Rutter M: Is Sure Start an effective preventive intervention? Child and Adol Mental Health 2006, 11(3):135-141. 61. 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Roos NP, Black C, Roos LL, Frohlich N, De Coster C, Mustard CA, Brownell M, Shanahan M, Fergusson P, Toll F, Carriere KC, Burchill C, Fransoo R, MacWilliam L, Bogdanovic B, Friesen D: Managing health services: How the Population Health Information System (POPULIS) works for policy makers. Medical Care 1999, 37(6 Suppl):JS27-JS41. doi:10.1186/1471-2458-11-691 Cite this article as: Brownell et al.: Program for expectant and new mothers: a population-based study of participation. BMC Public Health 2011 11:691. doi:10.1186/1471-2458-11-691 41. Oreopoulos P, Stabile M, Walld R, Roos LL: Short, medium, and long term consequences of poor infant health: An analysis using siblings and twins. J Hum Resour 2008, 43:88-138. 42. Roos LL, Gupta S, Soodeen R, Jebamani L: Data quality in an information- rich environment: Canada as an example. Can J Aging 2005, 24(Suppl 1):153-170.
https://openalex.org/W2621346069	https://bmcnurs.biomedcentral.com/track/pdf/10.1186/s12912-017-0223-5	English	null	The use of life stories and its influence on persons with dementia, their relatives and staff – a systematic mixed studies review	BMC nursing	2,017	cc-by	9,908	© The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Abstract Background: Dementia is an important predictor of nursing home admissions. Due to progressive dementia symptoms, over time it becomes difficult for persons with dementia to communicate their wishes and participate in decisions concerning their everyday lives. Their well-being, sense of dignity, integrity and personhood are at risk. The persons’ life stories have been highlighted as particularly important in dementia care and are referred to as seeing the person beyond the dementia. The aim of this study was to explore and describe the use of life stories and its influence on persons with dementia living in nursing homes, their relatives and staff. Methods: A systematic mixed studies review was conducted. The literature searches were performed in the following databases: CINAHL, PubMed and PsycINFO and the Cochrane library, as well as by hand searching references in the studies included. An updated search was performed eight months after the first search. Data was synthesised inspired by integrative analysis. Results: Three studies using quantitative design and two studies (presented in three papers) using qualitative design representing research from 2006 to 2015 were included in the review. Life stories generally had a positive influence on the persons with dementia, their relatives, and staff. The use of life stories might contribute to ‘Maintenance of the person with dementia as a whole person rather than a demented patient’. On the other hand, enabling persons with dementia to tell their own story could be a challenge. For the staff it could be challenging when sensitive information emerged uninvited. Involving relatives could also be difficult as to whose story were uncovered. Conclusions: The use of person’s life story might be of significance, but there is not enough evidence to make any statement about its importance as the research is scarce. Studies, including randomised controlled trials, are needed to measure the impact of life story work on the physiological and psychological aspects of persons with dementia, and also how it influences their relatives and staff. Keywords: Dementia, Life stories, Systematic mixed studies review, Nursing home need care and support in many areas of their lives [2] and dementia is an important predictor of nursing home admissions [4, 5]. The use of life stories and its influence on persons with dementia, their relatives and staff – a systematic mixed studies review Vigdis Abrahamsen Grøndahl1, Mona Persenius2, Carina Bååth2 and Ann Karin Helgesen1 Correspondence: vigdis.a.grondahl@hiof.no 1Faculty of Health- and Social Studies, Østfold University College, 1757 Halden, Norway Full list of author information is available at the end of the article Grøndahl et al. BMC Nursing (2017) 16:28 DOI 10.1186/s12912-017-0223-5 Grøndahl et al. BMC Nursing (2017) 16:28 DOI 10.1186/s12912-017-0223-5 Open Access Outcome Studies were included when the outcome focused on the consequences of the use of life stories for the person with dementia, relatives and/or staff. Type of studies d Peer-reviewed papers using qualitative, quantitative or mixed methods, and presenting research in English, Norwegian, Swedish or Danish, were included. There were no limits regarding date of publication beyond the coverage of the databases themselves. Reviews and books were excluded from the review. Life stories are also used as a part of reminiscence work in which the person’s life reviews, often supported by photographs, artefacts and music, were actively used therapeutically [38, 39]. A review of intervention studies that focused on the use of life stories in the care of per- sons with dementia, found positive changes in the inte- gration of self and that the quality of life was enhanced [38]. Life story works have been highlighted as particu- larly important in dementia care [40] and are referred to as seeing the person beyond the dementia [41]. Aim The aim of this study was to explore and describe the use of life stories and its influence on persons with de- mentia living in nursing homes, their relatives and staff. Participants and setting Studies comprising the use of life stories for persons with dementia living in nursing homes were included. Studies concerning the use of life stories for people living at home who received home care were excluded. Furthermore, studies focusing on life stories regarding people with men- tal health problems were excluded to ensure that the focus remained on persons with dementia. Methods A systematic mixed studies review with an integrated design was undertaken to integrate and synthesise results from quantitative, qualitative and mixed methods studies [42]. The design was chosen to explore and describe the use of life stories’ influence on the person with dementia, the relatives and staff. The research group strived to use the methodological guidance of the Cochrane Collaboration [43] in the search and also to structure the review together with the guidelines from “Preferred Reporting Items for Systematic Reviews and Meta-Analyses” (PRISMA) [44]. It has been argued that it is the day-to-day decisions that are really omnipresent for persons with dementia [19, 20] as the possibility of participating in decisions concerning their everyday life will most likely impact the person’s well-being and sense of dignity, integrity, and personhood [11, 20–22]. However, due to progressive dementia symptoms such as increased cognitive and physical impairment, over time it will become more dif- ficult for persons with dementia to express themselves, make choices, communicate their wishes and understand their present circumstances [23, 24]. Background Due to a greater prevalence of older people in society as a result of demographic changes, the incidence of per- sons with dementia has increased internationally [1, 2]. It is estimated that there will be 48.1 million persons with dementia globally by 2020. This is expected to reach 90.3 million by 2040 [3]. Persons with dementia Dementia is a collective term for several diseases that permanently and progressively reduce cognitive func- tions. Alzheimer’s disease is the most common cause of dementia, followed by vascular dementia, Lewy Body dementia and frontotemporal dementia [3, 6, 7]. As there is no medical curative treatment of dementia [2, 7], specific nursing care that positively affects the quality of life of the person is of the utmost importance [8, 9]. * Correspondence: vigdis.a.grondahl@hiof.no 1Faculty of Health- and Social Studies, Østfold University College, 1757 Halden, Norway Full list of author information is available at the end of the article * Correspondence: vigdis.a.grondahl@hiof.no 1Faculty of Health- and Social Studies, Østfold University College, 1757 Halden, Norway Full list of author information is available at the end of the article Grøndahl et al. BMC Nursing (2017) 16:28 Grøndahl et al. BMC Nursing (2017) 16:28 Page 2 of 11 Grøndahl et al. BMC Nursing (2017) 16:28 Kitwood’s work has been very influential in challenging the standard biomedical paradigm and offering an alterna- tive for understanding dementia [8, 10, 11]. His conceptu- alisation of dementia highlighted the dialectic interplay between neuropathology and the social-psychological con- text of the individual, and contributed to the development of person-centred care within dementia care [11–13]. Since Kitwood’s work, various descriptions of person- centred care have been developed [14, 15]. Other related concepts such as relationship-centred care [16], senses framework [17], and dementia care nursing [18] are also presented. These descriptions and concepts have much in common by supporting the person’s rights, values and be- liefs, and involving the person with dementia in decision making [8, 11, 14–16, 18]. Since no recent reviews were found that focused on how the life stories of persons with dementia influence persons with dementia, their relatives and staff, this study was carried out. Eligibility criteria Eligibility criteria were based on the aim of the review and were determined before the literature search started. The aim was explorative and descriptive, and the inclu- sion criteria reflect this. When this happens, relatives may be entitled, according to political documents, legislation and nurses’ ethical codes [25–29], to participate, together with the person with dementia, in order to take care of the his/her inter- ests. A recent dissertation [20, 30] has highlighted that rel- atives could be a link to good dementia care because by knowing the person’s life story and preferences they could share important information with nursing home staff. In a survey, the majority (98.7%) of relatives stated that they had good knowledge of the person’s habits and prefer- ences. However, about 30% of relatives had not been asked to provide written information about their near ones, which shows that there is room for improvement in this issue [31]. Life story work that involves recording aspects of a person’s past and present life, and then using this information to benefit the person in his/her present situ- ation [32], has been used in many countries within a range of health and social care settings [33–37]. Identification of relevant literature Information sources The literature searches were performed in three elec- tronic databases: CINAHL via EBSCO, PubMed via Grøndahl et al. BMC Nursing (2017) 16:28 Page 3 of 11 NCBI and PsycINFO via OVID, as well as the Cochrane Library, 26 January 2015. An updated search was performed on 1 September 2015. ‘Dementia’ covering Dementia, Frontotemporal Dementia, Dementia Vascular, Dementia Multi-Infarct, Levy body Disease, Dementia Senile, Dementia Presenile, was used as a key search term in combination with the Boolean operator ‘AND’ with the search terms ‘biographical approach’, ‘diary’, ‘narratives’, ‘life story’ or ‘life histories’, and used as appropriate for the database. The full electronic search strategy for Pubmed is shown to exemplify the search: ((dementia [MeSH Major Topic] AND biographical approach), (dementia [MeSH Major Topic] AND diary), (dementia [MeSH Major Topic] AND narratives), (dementia [MeSH Major Topic] AND life story), (dementia [MeSH Major Topic] AND life history)). The references in the selected studies were scru- tinised for further studies by looking for the key search phrases in the titles. Results The included studies represent research from 2006 to 2015 with three studies using quantitative design and two stud- ies (presented in three papers) using qualitative design. Three of the papers are from the United Kingdom, one from the United States, one from Northern Ireland and one from Australia. The number of persons with dementia who participated was 70. In addition, there were 33 rela- tives and 159 members of staff. Five of the papers specified the age of the person with dementia, all papers specified gender, while three papers described the cognitive ability of the person with dementia. The relatives’ age and gender were specified in, respectively, one and three out of four papers, and their relationship to the person with dementia was described in all four papers. Among the five papers that included staff, four papers described the staff’s profes- sions. The demographic characteristics of the participants included in the studies are shown in Table 1. Enable relatives to see their near one as a whole person The results showed that two of the papers described how the use of life stories might influence relatives. Family members appreciated that life story work made their near ones more visible, present and heard [48]. They could now focus on their near ones as persons who had lived a meaningful life and enjoyed accom- plishments throughout their lifetime [52]. Reviewing memories released relatives from the immediate dementia-related care in their everyday experience, thus enabling them to see their near ones as a whole person in new and different ways – even in ways not previously appreciated [52]. The relatives enjoyed participating in the process of implementing life stories [48] and they experienced great comfort and confidence in observing the ability of the nursing staff to recognise, value and incorporate life stories in everyday care [52]. The relatives also gained insight into their own grieving with regard to the process of their near ones’ dementia illness, something that helped them to cope with it more positively [52]. Appraisal and data extraction The quality of each paper included in the review was assessed by two authors independently, using the Critical Appraisal Skills Programme Tools (CASP) for qualita- tive, quantitative and RCT studies [45], modified by Nordström and Wilde-Larsson [46, 47]. A scoring sys- tem was used to rate the studies as low, medium or high quality. Papers with low quality scores were then reas- sessed by the two other authors. Any differences on quality ratings were discussed in the research group until agreement was reached. One study was excluded based on low quality. In total six papers were included in the synthesis. Information regarding aim, study design, research type, sample, intervention, outcomes and key results were extracted from the studies. See Table 1 for characteristics of included studies and quality assessment. After a life storybook process, persons with mild to moderate dementia scored significantly more positively on the outcome measures of cognition, depression, positive mood and communication than the control group [50]. Regarding quality of life as measured on the Quality of life-Alzheimer’s disease scale, to take part in the creation of their own life story book had significantly positive benefits for persons with dementia immediately after the life review session had been completed [51]. Their autobiographical memory also significantly im- proved. However, no difference was observed between the control and experimental group six weeks after having received the life storybook [51]. Methods of synthesis An integrative analysis inspired by Sandelowski et al. [42] was conducted. The aim provided direction for the analyses of the results from the included papers. Kellett et al. [52] found that persons with dementia benefited when life stories were used, as both staff and relatives were more capable of stimulating and provok- ing memories, as well as knowing how to calm them when necessary. Study selection A modified flow diagram (Fig. 1) shows the procedure for the identification and selection process. The elec- tronic database searches revealed 798 papers, with a fur- ther six papers identified through the reference lists. Duplicates were removed which resulted in 749 papers. All four authors independently read the titles. All titles were assessed according to their relevance to ensure that the eligibility criteria and the aim of the study were met and screened on a scale from 1 (relevant), 2 (maybe rele- vant) to 3 (not relevant). There were 706 papers with a score of 3 which were excluded. The papers with scores 1 and 2 on the title (49 papers) were divided into two equal parts and two authors read one half of the ab- stracts, the other two authors read the other half and gave each abstract a score from 1 to 3. Abstracts with score 1 or 2 were then read by all four authors, and the scores were subsequently discussed until consensus was Fig. 1 Flow diagram of the selection process. Source: modified version of flow diagram as reported by Moher et al. [44] Fig. 1 Flow diagram of the selection process. Source: modified version of flow diagram as reported by Moher et al. [44] Grøndahl et al. BMC Nursing (2017) 16:28 Page 4 of 11 and using life stories in care were also revealed, as described later. reached. As a result, 38 papers were excluded, leaving 11 papers. The 11 papers were assessed in full text by all four authors independently to ensure that the inclusion criteria were met. Four papers were excluded, leaving seven papers to be assessed for quality. Enable the voice of the person with dementia to be heard The results showed that five of the papers concerned how the use of life stories might influence the person with dementia. The life story enabled the voice of per- sons with dementia to be heard and to feel proud about themselves and their lives [48]. However, enabling per- sons with dementia to tell their own life story could be a challenge due to memory loss. Joint authorship is re- ferred to as a way of supporting the ability of persons with dementia to contribute to their own story [49]. Results Results from the integrative analyses of the six papers show that the use of life stories might contribute to ‘Maintenance of the person with dementia as a whole person rather than a demented patient’ as it enabled the voice of the person with dementia to be heard, enabled relatives to see their near one as a whole person and en- abled staff to understand persons with dementia and their relatives. However, several challenges in creating Page 5 of 11 Grøndahl et al. BMC Nursing (2017) 16:28 Table 1 Characteristics of included studies and quality assessment Authors/Country Title Aim Design Research type Sample, including participants’ characteristics Intervention Outcome Summary of relevant findings Quality assessment Buron 2010 [53] USA Life history collages. Effects on nursing home staff caring for residents with dementia. To compare changes in nursing staff knowledge of individual residents and their perceptions of knowing the person, staff- to- resident, communication, and staff –to-staff communication before and after exposure to life history collages. A pilot study. A pretest- posttest, quasi- experimental design. Quantitative A convenience sample of five residents with dementia (3 intervention, 2 control) and 36 nursing staff members (18 intervention, 18 control), resident in two nursing homes. Person with dementia: Age: 63–95 years Gender: 1 woman and 4 men Length of stay: 10– 11 months MMSE score: 1–11 Staff: Age: 19–62 years Gender: 30 women and 6 men Profession: 1 RN and 35 other Length of employment: 0- ≥10 years Interviews with each resident’s surrogate were conducted and tangible (e.g., photos) and intangible (e.g., verbal description) information about each resident in three areas were collected: family, job/careers and likes/dislikes/ interests. A graphic design artist used the information to create the essence of each resident’s participant’s life in the form of a life history collage that were placed on the residents’ room walls for a period of 4 weeks. Nursing staff knowledge - author developed questionnaire Nursing staff perceptions of individualized care - IC-KNOW - IC-COMMUNICATION-SR - IC-COMMUNICATION-SS The intervention group members’ knowledge of residents’ family, job/ careers, and likes/ dislikes/interests improved significantly at posttest and at 3 weeks post intervention. The perceptions of individualized care/ person centered care practices did not improve significantly. Medium Haight, Gibson, Michel 2006 [50] Northern Ireland The Northern Ireland life review/life storybook project for people with dementia. To test the effectiveness of a structured life review/ life storybook process. A controlled pilot study. Results For staff “opening possibilities” four characteristics: having a point of reference to communicate with resident and family, appreciating the resident and family member as people bound by a family history, developing insight and understanding into behavior and developing confidence to relate as a person, were found. For person with dementia “knowing how” two characteristics shared by staff and family: knowing how to stimulate and provoke memories and knowing how to calm the person with dementia using biography, were found. “Complementing the organization” concerning organization issues, three characteristics: promoting community, the challenges of integrating FBW in practice and the complexities of division of labour and Medium Table 1 Characteristics of included studies and quality assessment (Continued) Kellett, Moyle, McAllister, King, Gallagher 2010 [52]Australia Life stories and biography: a means of connecting family and staff to people with dementia. To assess the Family Biography Workshop (FBW) designed to support family and staff to co- construct the history of the person with dementia in residential care. A pilot study. Focus group interviews. Qualitative A purposeful sample of seven family careers and their relatives in care, seven care staff and one researcher in long term dementia care. Person with dementia: Gender: 1 woman and 6 men Relatives: Relationship to the person with dementia: 5 spouses and 2 children Staff: Profession: 8 other The FBW process consisting of a series of six weekly two-hour sessions, involved families and staff completing a set of exercises designed to help them build a biography of the life of the person with dementia. Between the sessions, incrementally, both family members and staff shared biographical materials with the residents, and thus, involving the person with dementia. The FBW was used to provide a structured process and a defined role for family caregivers to assist staff in personalizing nursing care. Family-staff caregiver - attitudes - perceptions of roles - conflict - subsequent management of stress For family caregivers “standing outside” four characteristics: freeing the family from the present, breaking free of the disease-saturated narrative, gaining insight into grieving and healing and learning something about my relative not previously appreciated, were found. For staff “opening possibilities” four characteristics: having a point of reference to communicate with resident and family, appreciating the resident and family member as people bound by a family history, developing insight and understanding into behavior and developing confidence to relate as a person, were found. Results For person with dementia “knowing how” two characteristics shared by staff and family: knowing how to stimulate and provoke memories and Medium aracteristics of included studies and quality assessment (Continued) Life stories and biography: a means of connecting family and staff to people with dementia. To assess the Family Biography Workshop (FBW) designed to support family and staff to co- construct the history of the person with dementia in residential care. A pilot study. Focus group interviews. Qualitative A purposeful sample of seven family careers and their relatives in care, seven care staff and one researcher in long term dementia care. Person with dementia: Gender: 1 woman and 6 men Relatives: Relationship to the person with dementia: 5 spouses and 2 children Staff: Profession: 8 other The FBW process consisting of a series of six weekly two-hour sessions, involved families and staff completing a set of exercises designed to help them build a biography of the life of the person with dementia. Between the sessions, incrementally, both family members and staff shared biographical materials with the residents, and thus, involving the person with dementia. The FBW was used to provide a structured process and a defined role for family caregivers to assist staff in personalizing nursing care. Family-staff caregiver - attitudes - perceptions of roles - conflict - subsequent management of stress For family caregivers “standing outside” four characteristics: freeing the family from the present, breaking free of the disease-saturated narrative, gaining insight into grieving and healing and learning something about my relative not previously appreciated, were found. For staff “opening possibilities” four characteristics: having a point of reference to communicate with resident and family, appreciating the resident and family member as people bound by a family history, developing insight and understanding into behavior and developing confidence to relate as a person, were found. For person with dementia “knowing how” two characteristics shared by staff and family: knowing how to stimulate and provoke memories and knowing how to calm the person with dementia using biography, were found. “Complementing the organization” concerning organization issues, three characteristics: promoting community, the challenges of integrating FBW in practice and the complexities of division of labour and responsibility, were found. Medium Table 1 Characteristics of included studies and quality assessment (Continued) Kellett, Moyle, McAllister, King, Gallagher 2010 [52]Australia Life stories and biography: a means of connecting family and staff to people with dementia. Results Pre- and posttest. Quantitative A convenience sample of 30 older people (15 intervention, 15 control) who had mild to moderate dementia, resident in six assisted-living facilities. Person with dementia: Age: 60–99 years Gender: 25 women and 6 men MMSE score: mean 17.84 at pretest A series of semistructured interviews guided by the tool The Life Review and Experiencing Form (LREF) was conducted during 8 weeks. A life storybook was constructed by care staff and the participants in dyads. The life storybook was an illustrated loose-leaf binder with photographs and explanatory captions by using the elderly person’s own words. The participants - cognition (MMSE) - depression (CSDD) - mood (AMS) - function (FIM) - communication (CS) - behaviors (MBS) Significant differences between experimental and control groups were found in 4 out of 6 measures: cognition, depression, positive mood and communication. Medium Page 6 of 11 Grøndahl et al. BMC Nursing (2017) 16:28 Table 1 Characteristics of included studies and quality assessment (Continued) Kellett, Moyle, McAllister, King, Gallagher 2010 [52]Australia Life stories and biography: a means of connecting family and staff to people with dementia. To assess the Family Biography Workshop (FBW) designed to support family and staff to co- construct the history of the person with dementia in residential care. A pilot study. Focus group interviews. Qualitative A purposeful sample of seven family careers and their relatives in care, seven care staff and one researcher in long term dementia care. Person with dementia: Gender: 1 woman and 6 men Relatives: Relationship to the person with dementia: 5 spouses and 2 children Staff: Profession: 8 other The FBW process consisting of a series of six weekly two-hour sessions, involved families and staff completing a set of exercises designed to help them build a biography of the life of the person with dementia. Between the sessions, incrementally, both family members and staff shared biographical materials with the residents, and thus, involving the person with dementia. The FBW was used to provide a structured process and a defined role for family caregivers to assist staff in personalizing nursing care. Family-staff caregiver - attitudes - perceptions of roles - conflict - subsequent management of stress For family caregivers “standing outside” four characteristics: freeing the family from the present, breaking free of the disease-saturated narrative, gaining insight into grieving and healing and learning something about my relative not previously appreciated, were found. Results Person with dementia: Age: 80–88 years Gender: 2 women and 2 men Relatives: Gender: 3 women Relationship to person with dementia: 1 spouse and 2 children Staff: Profession: 1 RN and 11 other A practice development approach with six key themes was applied to the LSW intervention. Participant were given the opportunity to develop the use of LSW in whichever way they choose; encouraging the use of creativity in practice. Three cases developed a life story book and one a pen picture. Person-centered outcomes Using LSW can enhance person-centred care. Three main themes: “from patient to person”, “can you hear me?” and “pride and enjoyment”, were found. For family careers: allowed them to uphold their relative’s personhood. For care staff: enabled them to see the person behind the patient. For the person with dementia: enabled their voice to be heard and enabled them to feel proud about themselves and their lives. Medium McKeown, Ryan, Ingleton, Clarke 2015 [49] United Kingdom ‘You have to be mindful of whose story it is’: the challenges of undertaking life story work with people with dementia and their family carers. To critically appraise some of the challenges that may emerge through the process of undertaking Life story work (LSW). Recommend how such challenged may be overcome or minimized. Contribute to what is currently a gap in the LSW literature. A multiple case study. Conversations, observations, interviews, and documentary analysis. Qualitative A sample of four people with dementia across four care settings for people with dementia, 12 multi- professional staff and three relatives. Person with dementia: Age: 80–88 years Gender: 2 women and 2 men Relatives: Gender: 3 women Relationship to person with dementia: 1 spouse and A practice development approach with eight principles was applied to the life story work intervention. Three cases developed a life story book and one a pen picture. Experiences of people with dementia, family careers and care staff in using life story work. Several challenges may occur during the process of undertaking LSW. Four main themes “personal disclosures”, “whose story is it?”, “quality of life story books”, and “under and overuse of life story books”, were found. Medium Table 1 Characteristics of included studies and quality assessment (Continued) McKeown, Clarke, Ingleton, Ryan, Repper 2010 [48] United Kingdom The use of life story work with people with dementia to enhance person- centred care. Results To explore the use of taking a Life Story Work (LSW) approach with people with dementia. To investigate the ways in which LSW: - is understood and developed in practice - is experienced by all participants - affects the delivery of care - affects outcomes of care A multiple case study. Conversations, observations, interviews, and documentary analysis. Qualitative A purposeful sample of four people with dementia across four care settings for people with dementia, 12 multi-professional staff and three relatives. Person with dementia: Age: 80–88 years Gender: 2 women and 2 men Relatives: Gender: 3 women Relationship to person with dementia: 1 spouse and 2 children Staff: Profession: 1 RN and 11 other A practice development approach with six key themes was applied to the LSW intervention. Participant were given the opportunity to develop the use of LSW in whichever way they choose; encouraging the use of creativity in practice. Three cases developed a life story book and one a pen picture. Person-centered outcomes Using LSW can enhance person-centred care. Three main themes: “from patient to person”, “can you hear me?” and “pride and enjoyment”, were found. For family careers: allowed them to uphold their relative’s personhood. For care staff: enabled them to see the person behind the patient. For the person with dementia: enabled their voice to be heard and enabled them to feel proud about themselves and their lives. Medium McKeown, Ryan, Ingleton, Clarke 2015 [49] United Kingdom ‘You have to be mindful of whose story it is’: the challenges of undertaking life story work with people with dementia and their family carers. To critically appraise some of the challenges that may emerge through the process of undertaking Life story work (LSW). Recommend how such challenged may be overcome or minimized. Contribute to what is currently a gap in the LSW literature. A multiple case study. Conversations, observations, interviews, and documentary analysis. Qualitative A sample of four people with dementia across four care settings for people with dementia, 12 multi- professional staff and three relatives. Person with dementia: Age: 80–88 years Gender: 2 women and 2 men Relatives: Gender: 3 women Relationship to person with dementia: 1 spouse and 2 children Staff: Profession: 1 RN and 11 other A practice development approach with eight principles was applied to the life story work intervention. Three cases developed a life story book and one a pen picture. Results Person with dementia: Age: 80–88 years Gender: 2 women and 2 men Relatives: Gender: 3 women Relationship to person with dementia: 1 spouse and 2 children Staff: Profession: 1 RN and 11 other A practice development approach with eight principles was applied to the life story work intervention. Three cases developed a life story book and one a pen picture. Experiences of people with dementia, family careers and care staff in using life story work. Several challenges may occur during the process of undertaking LSW. Four main themes “personal disclosures”, “whose story is it?”, “quality of life story books”, and “under and overuse of life story books”, were found. Medium Subramaniam,Woods, Whitaker 2014 [51] United Kingdom Life review and life story books for people with mild to moderate dementia: a randomised controlled trial. To evaluate the effect of different pathways for developing a life story book (LSB) for people with dementia. A preliminary, randomized single blind controlled trial, with two parallel arms Quantitative A sequential individual- based randomization sample of 23 people with dementia (11 intervention, 12 control) living in 11 care homes participated, 23 relatives and 68 care staff. Person with dementia: Age: 73–99 years Life review /life story book intervention. Persons with dementia received 12 individual sessions undertaking the life review process leading to their own life storybook The persons with dementia: Primary outcome: - quality of life (QOL-AD) Secondary outcome: - depression (GDS-12R) - autographical memory (AMIE) - relationship with their relatives For persons with dementia: A significant between- group difference was found immediately after the life review session had been completed in favor of LSB group. No differences in quality of life between the High Table 1 Characteristics of included studies and quality assessment (Continued) McKeown, Clarke, Ingleton, Ryan, Repper 2010 [48] United Kingdom The use of life story work with people with dementia to enhance person- centred care. To explore the use of taking a Life Story Work (LSW) approach with people with dementia. To investigate the ways in which LSW: - is understood and developed in practice - is experienced by all participants - affects the delivery of care - affects outcomes of care A multiple case study. Conversations, observations, interviews, and documentary analysis. Qualitative A purposeful sample of four people with dementia across four care settings for people with dementia, 12 multi-professional staff and three relatives. Results To assess the Family Biography Workshop (FBW) designed to support family and staff to co- construct the history of the person with dementia in residential care. A pilot study. Focus group interviews. Qualitative A purposeful sample of seven family careers and their relatives in care, seven care staff and one researcher in long term dementia care. Person with dementia: Gender: 1 woman and 6 men Relatives: Relationship to the person with dementia: 5 spouses and 2 children Staff: Profession: 8 other The FBW process consisting of a series of six weekly two-hour sessions, involved families and staff completing a set of exercises designed to help them build a biography of the life of the person with dementia. Between the sessions, incrementally, both family members and staff shared biographical materials with the residents, and thus, involving the person with dementia. The FBW was used to provide a structured process and a defined role for family caregivers to assist staff in personalizing nursing care. Family-staff caregiver - attitudes - perceptions of roles - conflict - subsequent management of stress For family caregivers “standing outside” four characteristics: freeing the family from the present, breaking free of the disease-saturated narrative, gaining insight into grieving and healing and learning something about my relative not previously appreciated, were found. For staff “opening possibilities” four characteristics: having a point of reference to communicate with resident and family, appreciating the resident and family member as people bound by a family history, developing insight Medium Page 7 of 11 Grøndahl et al. BMC Nursing (2017) 16:28 and their lives. McKeown, Ryan, Ingleton, Clarke 2015 [49] United Kingdom ‘You have to be mindful of whose story it is’: the challenges of undertaking life story work with people with dementia and their family carers. To critically appraise some of the challenges that may emerge through the process of undertaking Life story work (LSW). Recommend how such challenged may be overcome or minimized. Contribute to what is currently a gap in the LSW literature. A multiple case study. Conversations, observations, interviews, and documentary analysis. Qualitative A sample of four people with dementia across four care settings for people with dementia, 12 multi- professional staff and three relatives. Results (QCPR) Staff: - approaches to dementia (ADQ) - knowledge about residents (author developed questionnaire) Relatives: - relationship with the person with dementia (QCPR) LSB intervention and the LSB as gift intervention groups were found six weeks after receiving the LSB. For both groups QOL-AD had improved. At 12-week assessment, there was a significant improvement in scores for the LSB group compared with the LSB as a gift group. There was no significant differences in between the groups on depression. After twelve weeks relationship warmths, rated by the person with dementia, had improved in the LSB group. For staff: Six weeks after the LSB had been available, staff attitude had improved significantly. Staff knowledge regarding the residents improved significantly at twelve-week follow- up assessment. For relatives: Quality of relationship improved significantly after the LSBs were produced by either pathways. le 1 Characteristics of included studies and quality assessment (Continued) Gender: 16 women and 7 men Length of stay: 25–51 months CDR: Mild to moderate dementia Relatives: Age: 44–83 years Gender: 15 women and 8 men Relationship to person with dementia: 13 children and 10 other Staff: Age: 20–64 years Gender: 62 women and 6 men Profession: 3 RNs and 68 other Length of employment: 1–40 years (LSB). Initially the focus is on childhood and adulthood, then family, home and adulthood. Finally there is a summary. Life story book as gift i ntervention. The researcher worked closely with the participant’s relative, meeting them 5–6 times over 12 weeks. Together they developed a life storybook illustrated with photos and pictures to be given as a gift for their relative who was not involved in the process. (QCPR) Staff: - approaches to dementia (ADQ) - knowledge about residents (author developed questionnaire) Relatives: - relationship with the person with dementia (QCPR) LSB intervention and the LSB as gift intervention groups were found six weeks after receiving the LSB. For both groups QOL-AD had improved. At 12-week assessment, there was a significant improvement in scores for the LSB group compared with the LSB as a gift group. There was no significant differences in between the groups on depression. After twelve weeks relationship warmths, rated by the person with dementia, had improved in the LSB group. For staff: Six weeks after the LSB had been available, staff attitude had improved significantly. Staff knowledge regarding the residents improved significantly at twelve-week follow- up assessment. Results Experiences of people with dementia, family careers and care staff in using life story work. Several challenges may occur during the process of undertaking LSW. Four main themes “personal disclosures”, “whose story is it?”, “quality of life story books”, and “under and overuse of life story books”, were found. Medium Subramaniam,Woods, Whitaker 2014 [51] United Kingdom Life review and life story books for people with mild to moderate dementia: a randomised controlled trial. To evaluate the effect of different pathways for developing a life story book (LSB) for people with dementia. A preliminary, randomized single blind controlled trial, with two parallel arms Quantitative A sequential individual- based randomization sample of 23 people with dementia (11 intervention, 12 control) living in 11 care homes participated, 23 relatives and 68 care staff. Person with dementia: Age: 73–99 years Life review /life story book intervention. Persons with dementia received 12 individual sessions undertaking the life review process leading to their own life storybook The persons with dementia: Primary outcome: - quality of life (QOL-AD) Secondary outcome: - depression (GDS-12R) - autographical memory (AMIE) - relationship with their relatives For persons with dementia: A significant between- group difference was found immediately after the life review session had been completed in favor of LSB group. No differences in quality of life between the High High Page 8 of 11 Grøndahl et al. BMC Nursing (2017) 16:28 Table 1 Characteristics of included studies and quality assessment (Continued) Gender: 16 women and 7 men Length of stay: 25–51 months CDR: Mild to moderate dementia Relatives: Age: 44–83 years Gender: 15 women and 8 men Relationship to person with dementia: 13 children and 10 other Staff: Age: 20–64 years Gender: 62 women and 6 men Profession: 3 RNs and 68 other Length of employment: 1–40 years (LSB). Initially the focus is on childhood and adulthood, then family, home and adulthood. Finally there is a summary. Life story book as gift i ntervention. The researcher worked closely with the participant’s relative, meeting them 5–6 times over 12 weeks. Together they developed a life storybook illustrated with photos and pictures to be given as a gift for their relative who was not involved in the process. Enable staff to understand persons with dementia and their relatives persons with dementia, their relatives and staff. Further- more, among the included studies, the quality of four of the studies was rated as medium quality and only one was rated as high quality. The results showed that five of the papers were about how the use of life stories might influence staff. Introducing a life story collage increased the staffs’ knowledge of the persons with dementia regarding family, jobs/careers, likes, dislikes and interests. It improved the relationship between staff and the persons with dementia, and the staffs’ involvement [53]. Life story work helped the staff to see the person with dementia as more than a patient and it enhanced their understanding of the person for whom they were caring [48]. The use of the family biography workshop, which is a structured process to facilitate the involvement of staff, family members and friends of the person with dementia in co-constructing biographies of their lives, gave the staff better knowledge of how to stimulate and provoke memories, and how to calm the person by using his/her biography. The staff discovered points of reference to communicate with the person with dementia and family and view them as part of a family history. The staffs’ understanding and insight into behaviour increased and they felt empowered to provide relationship-centred care rather than task-oriented care [52]. Further, staff attitudes to persons with dementia mea- sured as hopefulness and person-centeredness, improved when a life story book was developed [51]. However, an- other study found that staff perceptions of individualised care or person-centred care practices did not improve sig- nificantly after introducing a life story collage [53]. The total sample covered in this review is small and the included studies provide little information about per- sons with dementia, their relatives and staff, as well as the overall context. No specific diagnoses of the persons with dementia were mentioned and only two of the studies provided information about the person’s MMSE score. Consequently, as little or nothing about the diagnoses and the extent of the person’s cognitive impairment are known, it is not possible to explore pos- sible relationships between diagnoses, degree of cognitive impairment, the use of life story and its influence on the persons with dementia, their relatives and staff. Enable staff to understand persons with dementia and their relatives This infor- mation could be of interest as previous research shows that the more severe the dementia symptoms, the less the care is individualised and the less the relatives are involved in the care [20]. The integrated and synthesised results from the quanti- tative and qualitative studies show that use of life stories might contribute to ‘Maintenance of the person with de- mentia as a whole person rather than a demented patient’. This result is in line with what is referred to as ‘seeing the person beyond the dementia’, which is one of the goals of dementia care [11, 20, 41]. As this group of persons are at risk of becoming objectified and seen as non-persons, especially as the disease progresses [11, 20], the use of life stories should be considered an important element of the care in order to preserve and enhance the dignity and well-being of the person with dementia. The latter has been highlighted as particularly important with regard to dementia care in several studies [20, 54, 55]. As no medical curative treatment of dementia exists [2, 7], individually tailored care is crucial in order to offer the in- creasing number of persons with dementia and their rela- tives [1] high-quality professional care in the future. McKeown et al. [49] found that life story work may be overused and underused, and that finding a balance is important. From the staff point of view it was sometimes a challenge when sensitive information and personal dis- closures emerged uninvited. The results also showed that involving relatives could be a challenge as to whose story is uncovered. Questions were raised about the quality of the life story work when it had been created by relatives [49]. Developing a life story book either with the participation of the person with dementia or via relatives without involving the person with dementia improved the staff’s knowledge of the person with dementia regarding details such as hobbies, favourite food and school [51]. The process of creating life story work was perceived by staff as being enjoyable [48]. As the results revealed several challenges both in cre- ating and using the life stories in care, life story work has to be taken into consideration with a high level of sensitivity by the staff. It is therefore both worrying and striking that among the staff, there were only a few RNs. Results For relatives: Quality of relationship improved significantly after the LSBs were produced by either pathways. Page 9 of 11 Grøndahl et al. BMC Nursing (2017) 16:28 Page 9 of 11 Discussions The results of this review show that research into the use of life stories’ and its influence on persons with dementia, their relatives and staff is still scarce. Even though the literature search resulted in 749 papers once the duplicates had been removed, it was found that the majority of studies focused on the importance of the phenomenon of life story work and, to a much lesser extent, on how to actually use it and its influence on Enable staff to understand persons with dementia and their relatives This might be a coincidence, but most likely is not. Ac- cording to previous research, nursing homes are facing major challenges regarding a serious lack of personnel with formal health education [56] and the utmost need for a high degree of expertise in order to provide high- quality care is emphasised [30]. Methodological considerations Using an integrated review method provides an oppor- tunity to present a comprehensive understanding of a phenomenon of relevance to health care [57]. Even Grøndahl et al. BMC Nursing (2017) 16:28 Page 10 of 11 though only three quantitative and two qualitative studies were included in this review, presenting the existing knowledge is valuable, in order to start creating a know- ledge base for using the persons’ life stories in dementia care, and also to uncover areas for further research. Competing interests The authors declare that they have no competing interest. Competing interests Consent for publication Not applicable. Ethics approval and consent to participate Not applicable. The four researchers worked systematically in pairs to minimize subjectivity. The quality of the papers was assessed using a reliable quality assessment tool [45]. Any uncertainties were discussed in the research team until consensus was reached. 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Dupuis SL, Gillies J, Carson J, Whyte C, Genoe R, Loiselle L, Sadler L. Moving beyond patient and client approaches: mobilizing ‘authentic partnerships’ in dementia care, support and services. Dementia. 2012;11:427–52. Acknowledgements We want to thank the librarian Annelie Ekberg-Andersson at Karlstad University for valuable help in the search process. Acknowledgements We want to thank the librarian Annelie Ekberg-Andersson at Karlstad University for valuable help in the search process. Acknowledgements W h k h l 14. Brooker D. Person-centred dementia care: making services better. London: Jessica Kingsley Publishers; 2007. 15. McCormack B, McCance T. Person-centred nursing: theory and practice. Wiley-Blackwell: Chichester, West Sussex; 2010. Funding Not applicable. Acknowledgements We want to thank the librarian Annelie Ekberg-Andersson at Karlstad University for valuable help in the search process. 16. Nolan M, Davies S, Brown J, Keady J, Nolan J. Beyond ‘person-centred’ care: a new vision for gerontological nursing. J Clin Nurs. 2004;13:45–53. 19. Menne HL, Whitlatch CJ. Decision-makin dementia. Gerontologist. 2007;47:810–9 Author details 1 1Faculty of Health- and Social Studies, Østfold University College, 1757 Halden, Norway. 2Faculty of Health, Science, and Technology, Department of Health Sciences, Karlstad University, Karlstad, Sweden. Different concepts have been used for describing the use of life stories, which made the search process challenging. To strengthen the validity, we elected to use five concepts based on our own knowledge of the field. These concepts were life histories, life story, narratives, diary and bio- graphical approach. No further concepts were identified during the search process. To strengthen the reliability, a search update was performed in September 2015. No fur- ther studies were found. References in the chosen papers were scrutinised, but searches for ‘grey literature’ were not conducted. Received: 5 July 2016 Accepted: 24 May 2017 Received: 5 July 2016 Accepted: 24 May 2017 Funding 16. Nolan M, Davies S, Brown J, Keady J, Nolan J. Beyond ‘person-centred’ care: a new vision for gerontological nursing. J Clin Nurs. 2004;13:45–53. g Not applicable. 17. Nolan M, Davies S, Brown J. Transitions in care homes: towards relationship- centred care using the ‘senses framework’. Qual Ageing Older Adults. 2006;7:5–14. 18. Adams T. Dementia care nursing: promoting well-being in people with dementia and their families. Basingstoke: Palgrave Macmillan; 2008. Authors’ contributions VAG MP CB d AKH d 20. Helgesen AK. Patient participation in everyday life in special care units for persons with dementia. Faculty of Social and Life Sciences: Karlstad University Studies; 2013. VAG, MP, CB and AKH designed the study, collected the data, drafted the manuscript and contributed to the interpretation of the results and critical review of the manuscript. 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Nurs Ethics. 2015;22:577–93. 26. Ministry of Health and Social Affairs. Act of patients rights. SFS 2014:821. Stockholm: Socialdepartementet; 2014. (in Swedish) Stockholm: Socialdepartementet; 2014. (in Swedish) 27. Ministry of Health and Care Services. Act of patients rights. 2 July 1999 no 63. Oslo: Helse- og omsorgsdepartementet; 1999. (in Norwegian) 56. Authors’ contributions VAG MP CB d AKH d Harrington C, Choiniere J, Goldmann M, Jacobsen FF, Lloyd L, McGregor M, Stamatopoulos V, Szebehely M. Nursing home staffing standards and staffing levels in six countries. J Nurs Scholarsh. 2012;44:88–98. 28. Norwegian Directorate of Health. Forgetful, but not forgotten! Concerning the present situation and future challenges in strengthening the services for persons with dementia. Oslo: Sosial- og helsedirektoratet; 2007. (in Norwegian) 57. Whittemore R, Knafl K. The integrative review: updated methodology. J Adv Nurs. 2005;52:546–53. 29. International Council of Nurses. The ICN code of ethics for nurses. 2012. https://www.icn.ch/about-icn/code-of-ethics-for-nurses. Accessed 1 Nov 2012. 30. Helgesen AK, Larsson M, Athlin E. How do relatives of persons with dementia experience their role in the patient participation process in special care units? J Clin Nurs. 2013;22:1672–81. 31. Helgesen AK, Athlin E, Larsson M. Relatives’ participation in everyday care in special care units for persons with dementia. Nurs Ethics. 2015;22:404–16. 32. Murphy C, Moyes M. Life story work. In: Marshall M, editor. State of the art in dementia care. London: Centre for Policy on Ageing; 1997. p. 149–53. 33. Hewitt H. Tell it like it is. Learn Disability Prac. 2003;6:18–22 34. Hansebo G, Kihlgren M. Nursing home care: changes after supervision. J adv Nurs. 2004;45:269–79. 35. Rose R, Philpot T. The child’s own story: life story work with traumatized children. London: Jessica Kingsley Publishers; 2005. 36. Meininger HP. Narrative ethics in nursing for persons with intellectual disabilities. Nurs Philos. 2005;6:106–18. disabilities. Nurs Philos. 2005;6:106–18. 37. McKeown J, Clarke A, Repper J. Life story work in health and social care: systematic literature review. J adv Nurs. 2006;55:237–47. 38. Moos I, Björn A. Use of the life story in the institutional care of people with dementia: a review of intervention studies. Ageing soc. 2006;26:431–54. 39. Woods B, Spector A, Jones C, Orrell M, Davies S. Reminiscence therapy for dementia. Cochrane Database Syst rev. 2005;2 40. Kindell J, Burrow S, Wilkinson R, Keady JD. Life story resources in dementia care: a review. Qual Ageing Older Adults. 2014;15:151–61. y y care: a review. Qual Ageing Older Adults. 2014;15:151–61. 41. Batson P, Thorne K, Peak J. Life story work sees the person beyond the dementia. J Dementia Care. 2002;10:15–7. 41. Batson P, Thorne K, Peak J. Life story work sees the person beyond the dementia. J Dementia Care. 2002;10:15–7. dementia. 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Critical appraisal skills Programme (CASP): making sense of evidence. 2006. http://www.casp-uk.net/. Accessed Sept 2014. 46. Nordström G, Wilde-Larsson B. Assessment tool – quantitative studies (not RCT). Department of Health Science, Faculty of Health, Science and Technology: Karlstad University; 2006. (in Swedish) (unpublished) Submit your next manuscript to BioMed Central and we will help you at every step: Submit your next manuscript to BioMed Central and we will help you at every step: 47. Nordström G, Wilde-Larsson B. Assessment tool – qualitative studies. Department of Health Science, Faculty of Health, Science and Technology: Karlstad University; 2006. (in Swedish) (unpublished) 47. Nordström G, Wilde-Larsson B. Assessment tool – qualitative studies. Department of Health Science, Faculty of Health, Science and Technology: Karlstad University; 2006. (in Swedish) (unpublished) • We accept pre-submission inquiries • Our selector tool helps you to ﬁnd the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit and we will help you at every step: 48. McKeown J, Clarke A, Ingleton C, Ryan T, Repper J. The use of life story work with people with dementia to enhance person-centred care. Int J Older People Nursing. 2010;5:148–58. 48. McKeown J, Clarke A, Ingleton C, Ryan T, Repper J. The use of life story work with people with dementia to enhance person-centred care. Int J Older People Nursing. 2010;5:148–58. 49. McKeown J, Ryan T, Ingleton C, Clarke A. You have to be mindful of whose story it is: the challenges of undertaking life story work with people with dementia and their family carers. Dementia. 2015;14:238–56. 49. 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https://openalex.org/W4251235548	https://openresearch-repository.anu.edu.au/bitstream/1885/211558/1/01_McGaughran_Effects_of_sample_age_on_data_2020.pdf	English	null	Effects of sample age on data quality from targeted sequencing of museum specimens: what are we capturing in time?	Research Square (Research Square)	2,019	cc-by	8,682	© The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. McGaughran BMC Genomics (2020) 21:188 https://doi.org/10.1186/s12864-020-6594-0 McGaughran BMC Genomics (2020) 21:188 https://doi.org/10.1186/s12864-020-6594-0 Open Access Abstract Background: Next generation sequencing (NGS) can recover DNA data from valuable extant and extinct museum specimens. However, archived or preserved DNA is difficult to sequence because of its fragmented, damaged nature, such that the most successful NGS methods for preserved specimens remain sub-optimal. Improving wet- lab protocols and comprehensively determining the effects of sample age on NGS library quality are therefore of vital importance. Here, I examine the relationship between sample age and several indicators of library quality following targeted NGS sequencing of ~ 1300 loci using 271 samples of pinned moth specimens (Helicoverpa armigera) ranging in age from 5 to 117 years. Results: I find that older samples have lower DNA concentrations following extraction and thus require a higher number of indexing PCR cycles during library preparation. When sequenced reads are aligned to a reference genome or to only the targeted region, older samples have a lower number of sequenced and mapped reads, lower mean coverage, and lower estimated library sizes, while the percentage of adapters in sequenced reads increases significantly as samples become older. Older samples also show the poorest capture success, with lower enrichment and a higher improved coverage anticipated from further sequencing. Conclusions: Sample age has significant, measurable impacts on the quality of NGS data following targeted enrichment. However, incorporating a uracil-removing enzyme into the blunt end-repair step during library preparation could help to repair DNA damage, and using a method that prevents adapter-dimer formation may result in improved data yields. Keywords: Historical DNA, Library quality, Museum genomics, NGS, Targeted capture Effects of sample age on data quality from targeted sequencing of museum specimens: what are we capturing in time? Angela McGaughran1,2 Background species, but also from historical samples and even ex- tinct species. Collectively, this has shed light on human adaptation [4], relationships among humans and other hominids [5–7], and the place of extinct species, such as moa and mammoth, in evolutionary history [8, 9]. The technological innovations underlying next gener- ation sequencing (NGS) have resulted in an unprece- dented ability to obtain DNA sequence data from specimens encompassing the vast diversity of biological life [1–3]. In recent times, NGS has opened up possibil- ities not just for recovering DNA data from extant However, ancient DNA (aDNA; > 500 years old) has proven difficult to work with because of its fragmented nature – after the death of an organism, DNA is de- graded by endogenous nucleases, as well as damaged by chemical and physical events [10]. In addition to short fragment length, aDNA is commonly characterised by an increased occurrence of purine residues before strand Correspondence: ang.mcgaughran@gmail.com 1Australian National University, Research School of Biology, Division of Ecology and Evolution, Acton, Canberra, ACT 2600, Australia 2CSIRO Land and Water, Integrated Omics Team, Black Mountain Laboratories, Canberra, ACT 2600, Australia Correspondence: ang.mcgaughran@gmail.com 1Australian National University, Research School of Biology, Division of Ecology and Evolution, Acton, Canberra, ACT 2600, Australia 2CSIRO Land and Water, Integrated Omics Team, Black Mountain Laboratories, Canberra, ACT 2600, Australia Relationship between sample age and NGS quality metrics I first checked to see whether older samples had lower starting material concentrations (i.e., lower concentra- tions after adapter fill-in), and therefore required a higher number of indexing PCR cycles during the library preparation procedures. Both effects were seen in the data (T269 = 3.83; P < 0.01; R = 0.23(0.11:0.34) and T269 = -5.56; P < 0.01; R = -0.32(−0.42:-0.21), for starting concentration, and number of PCR cycles, respectively) and can be visualised in Fig. 2. Next, I examined the impact of sample age on several aspects of library quality, with respect to the alignment of reads to the full genome. Sample age was positively correlated with the total number of sequenced reads, the mean genome coverage, and the estimated library size (Fig. 3, Table 1). Meanwhile, as samples got older, the percentage of adapters increased significantly but, inter- estingly, the total percentage of duplication decreased slightly. The percentage of unmapped reads was not re- lated to sample age (Fig. 3, Table 1). The first attempt at using temporal samples in a population-scale context was made nearly 30 years ago [32], and was followed by a suite of such studies harnes- sing the power of museum samples (reviewed in [33]). In more recent times, new genomic methods that can bet- ter cope with low concentrations of starting material have been developed. In particular, targeted enrichment has proven useful for working with degradation- vulnerable specimens because the bait sequences are short and the method involves an amplification step fol- lowing hybridisation. As a result, users can obtain sub- stantial amounts of sequence data despite working with low molarity, fragmented DNA [34–36]. Thus, since its first applications to museum samples in the early 2010s [2, 37–41], a January 2020 search on GoogleScholar in- dicates its now widespread use (search term ‘targeted en- richment museum’ brings up > 27,000 results, > 2200 of these are for 2019 only; https://scholar.google.com.au/ scholar; accessed 17/01/2020). To examine the effect of sample age on the success of the targeted capture, I next evaluated several metrics with respect to the mapping of reads against the targeted regions. Sample age was positively correlated with the percentage of reads that mapped to baits and the mean bait coverage, as well as the percentage of reads on tar- get, and was highly correlated (R > 0.33; P < 0.01) with the percentage of baits covered from 1x to 30x (Fig. DNA damage analysis The program, mapdamage [42], was used to assess and quantify the damage patterns associated with NGS of historical specimens. In particular, the frequency of cyto- sine to thymine (C →T) mutations from the 5′-ends, and guanine to adenine (G →A) mutations from the 3′- ends was examined, as these follow cytosine deamin- ation; a common artefact in preserved DNA [2, 14, 43, 44]. I found no signal of C to T substitutions and G to A substitutions at high frequency at the 5′- and 3′-ends of mapped reads, respectively (Fig. 1). NGS methods compound these issues through loss of DNA in various steps of the library preparation protocol [18, 19]. Despite this, only a small number of studies have aimed at improving wet-lab protocols for NGS (e.g., [20–22]) and current conversion efficiencies remain around 30–70% [23–25]. Thus, there is scope for im- proving the efficiency of NGS methods through library preparation procedure manipulation. p p p p In addition, the effects of sample age on NGS data quality are generally understudied in a quantitative framework. Though some studies have noted negative relationships between sample age and parameters such as read length and number of reads [3, 26–31], focused analyses of the ways in which sample age, alongside ap- proaches employed during library preparation, may affect estimators of sequencing quality are rare. Such in- formation is particularly pertinent to population-scale museum studies, where users would benefit from further understanding of the quantitative effects of sample age on sequencing quality and the adjustments to library preparation protocol that could improve sequence quality. Page 2 of 10 McGaughran BMC Genomics (2020) 21:188 McGaughran BMC Genomics (2020) 21:188 ages on several indicators of sequenced read quality and identify key areas in the library preparation protocol that users should consider carefully when planning their experiments. breaks [11], and an increased frequency of cytosine to thymine substitutions near the ends of fragments [12, 13]. These three features also appear in historical (i.e., hundreds vs. thousands of years old) samples [2, 14, 15], and those that have been subjected to harsh conditions (e.g., formalin fixation) during preservation [16]. Coupled with this, endogenous DNA is generally present in only small amounts in preserved specimens [17]. Relationship between sample age and NGS quality metrics 4, Table 2). Meanwhile, the degree of saturation (which provides an indication of whether a higher sequence depth will translate into a higher percentage of covered positions) decreased as samples got older, as did the de- gree of enrichment (calculated as: on-target reads per Kb/off-target reads per Kb) (Fig. 4, Table 2). Here, I use 271 pinned insect specimens of the pest moth, Helicoverpa armigera, to test the effects of sample age on NGS library quality following a targeted capture approach. I use a temporal gradient of samples (5 to 117 years) to compare the effects of different sample Page 3 of 10 McGaughran BMC Genomics (2020) 21:188 McGaughran BMC Genomics Fig. 1 Plots of: a the frequency of C to T base substitutions from the 5′ end of each read; b the frequency of G to A base substitutions from the 3′ end of each read; c the frequency of C to T base substitutions at read position 1 from the 5′ end of reads vs. sample age; and d the frequency of G to A base substitutions at read position 1 from the 3′ end of reads vs. sample age. The expected signal for DNA damage due to age of specimens is an increased frequency of C to T and G to A transitions at the 5′ and 3′ end of reads, respectively, as well as a correlation between the frequency of such transitions at the 1st read position and sample age Fig. 1 Plots of: a the frequency of C to T base substitutions from the 5′ end of each read; b the frequency of G to A base substitutions from the 3′ end of each read; c the frequency of C to T base substitutions at read position 1 from the 5′ end of reads vs. sample age; and d the frequency of G to A base substitutions at read position 1 from the 3′ end of reads vs. sample age. The expected signal for DNA damage due to age of specimens is an increased frequency of C to T and G to A transitions at the 5′ and 3′ end of reads, respectively, as well as a correlation between the frequency of such transitions at the 1st read position and sample age Fig. Relationship between sample age and NGS quality metrics 2 Plot demonstrating for each sample used in this study, the date of collection, the starting DNA concentration, and (via the colour scale of points, as indicated by the legend to the right) the number of PCR cycles required during the library preparation Fig. 2 Plot demonstrating for each sample used in this study, the date of collection, the starting DNA concentration, and (via the colour scale of points, as indicated by the legend to the right) the number of PCR cycles required during the library preparation Fig. 2 Plot demonstrating for each sample used in this study, the date of collection, the starting DNA concentration, and (via the colour scale of points, as indicated by the legend to the right) the number of PCR cycles required during the library preparation Page 4 of 10 McGaughran BMC Genomics (2020) 21:188 McGaughran BMC Genomics Discussion In this work I showed that age of samples has a signifi per dollar sequencing output. Meanwhile, the degree of enrichment (a direct measure of targeted capture suc Fig. 3 The correlation between sample age and various measures of sequencing quality, including adapter level, total duplication, total number of sequences, total number of unmapped reads, mean genome coverage, and estimated library size, when reads were aligned to the reference genome. In (a), each bar in the plot represents the strength and direction of correlation, and the R-value is displayed in/on the bars. In (b), the raw data points are plotted for each pair-wise comparison, along with the linear regression trend line. See Table 1 for the detailed statistics Fig. 3 The correlation between sample age and various measures of sequencing quality, including adapter level, total duplication, total number of sequences, total number of unmapped reads, mean genome coverage, and estimated library size, when reads were aligned to the reference genome. In (a), each bar in the plot represents the strength and direction of correlation, and the R-value is displayed in/on the bars. In (b), the raw data points are plotted for each pair-wise comparison, along with the linear regression trend line. See Table 1 for the detailed statistics Discussion Such signals are unlikely to have been masked by contamination in the current study, because assessments of damage were made using sequence reads that had already been aligned to the reference genome. In addition, the percentage of unmapped reads was not related to sample age, indicating that contamination does not correlate with the age of the sample. Though I cannot be absolutely certain, this suggests that USER en- zyme (Uracil Specific Excision Reagent, which functions to remove uracil residues and repair resulting abasic sites; see Methods), was effective in the current study, and its role in repairing DNA damage is well-supported in the literature [13, 50–52]. modern humans, herbarium plants, Columbian and wooly mammoths, horses, and polar bear [45]. The former study also found that fragment lengths did not decrease in a consistent manner over time [14]; instead they are hypothesised to rapidly reduce to a small aver- age size following death before stabilising due to auto- lytic processes [14, 46]. Consistent with the current study for more recent (his- toric) DNA, research shows negative relationships be- tween sample age and various measures of NGS read quality, including mean coverage, read length, missing data, and number of recovered loci [27, 29, 30]. For ex- ample, a 6% decrease in coverage of targeted regions with every 10 years of sample age and a lowering of mean read depth by 40x per 10 years was shown for formalin-fixed paraffin-embedded (FFPE) tissue samples up to 32 years old [26]. Meanwhile, a targeted capture experiment including 185 bird samples up to 142 years of age compared a subset of modern and historical sam- ples for each of five bird species and also found signifi- cant negative relationships between sample age and the number of sequenced reads [31]. This is particularly true for ancient DNA samples, where comparisons of samples with and without uracil- removal treatment have shown marked reductions in common signals of DNA damage. For example, in one study of 11 cave bear bones (25,000–50,000 years old), a comparison of DNA molecules from 87 treated clonal samples showed zero G/C →A/T substitutions, while 19 such substitutions were present in the 79 clones that re- ceived no uracil-removal treatment [51]. Marked reduc- tions in deamination damage patterns have been similarly shown for historical studies. For example, Gor- den et al. Discussion For example, a significant negative correlation between amounts of endogenous mitochondrial DNA and age has been shown in pri- mates, horses, and cows [14], and a recent meta-analysis showed a bulk loss of DNA over time in samples of Page 5 of 10 Page 5 of 10 McGaughran BMC Genomics (2020) 21:188 Table 1 Correlation of sample age and several library quality metrics when reads were aligned to the full Helicoverpa armigera reference genome Library quality metric Correlation with sample age Total number of sequences T269 = 3.66; P < 0.01; R = 0.2180 (0.1015:0.3286) Total duplication T269 = 2.95; P < 0.01; R = 0.1771 (0.0592:0.2901) Mean genome coverage T269 = 5.93; P < 0.01; R = 0.34 (0.23:0.44) Estimated library size T269 = 8.24; P < 0.01; R = 0.45 (0.35:0.54) Adapter level T269 = -8.82; P < 0.01; R = -0.47(−0.56:-0.38) Total number of unmapped reads T269 = 4.60; P < 0.01; R = 0.27 (0.16:0.38) Table 1 Correlation of sample age and several library quality metrics when reads were aligned to the full Helicoverpa armigera reference genome Library quality metric Correlation with sample age Total number of sequences T269 = 3.66; P < 0.01; R = 0.2180 (0.1015:0.3286) Total duplication T269 = 2.95; P < 0.01; R = 0.1771 (0.0592:0.2901) Mean genome coverage T269 = 5.93; P < 0.01; R = 0.34 (0.23:0.44) Estimated library size T269 = 8.24; P < 0.01; R = 0.45 (0.35:0.54) Adapter level T269 = -8.82; P < 0.01; R = -0.47(−0.56:-0.38) Total number of unmapped reads T269 = 4.60; P < 0.01; R = 0.27 (0.16:0.38) Table 1 Correlation of sample age and several library quality metrics when reads were aligned to the full Helicoverpa armigera reference genome and preserved as pinned specimens and, based on tech- niques in use in recent history, it is likely they were killed using ethyl acetate, which has been shown to pro- duce degraded, low molecular weight DNA [48, 49]. Analysis of the moth DNA extracts confirmed a high level of degradation (most fragments < 500 bp), and re- search suggests that samples treated with harsh preser- vation methods (e.g., ethyl acetate, bleach), are likely to have an accelerated rate of deamination [14]. As a result, there is a reasonable likelihood that the moth DNA used here had been subject to chemical, as well as time-based, degradation. Discussion per dollar sequencing output. Meanwhile, the degree of enrichment (a direct measure of targeted capture suc- cess) also decreased as samples got older. This is consist- ent with the coverage metrics, showing that older samples require more sequencing for a greater enrich- ment success. In this work, I showed that age of samples has a signifi- cant effect on data quality following targeted NGS in H. armigera. In particular, following mapping of reads all coverage-based metrics, both across the whole genome and across targeted regions, were significantly correlated with sample age, such that older samples showed poorer coverage when compared to younger samples. In addition, saturation (an indicator of whether additional sequencing would result in a higher capture coverage) decreased as samples got older; older samples are there- fore less cost-efficient than younger samples in terms of Previous work has clearly shown a general pattern of DNA degradation over time. Discussion [53] compared untreated and treated forensic bone samples up to 50 years of age and found G/C →A/ T substitutions of 3–15 and < 1%, respectively. Mean- while, Bi et al. [2] used an enzyme which stalls amplifica- tion of templates containing uracil and found C →T frequencies of ~ 0.6%. In contrast, studies of similarly- aged (~ 100 years) museum samples that used no uracil- based treatment display C →T transition rates nearly an order of magnitude higher (2–4% [14];), and as high as 30% in some cases ([31]; see above). In the current study, the maximum rate of C →T transitions at the first position in mapped 5′ reads was ~ 0.04%. In the current study, mapdamage analysis detected no signature of deamination in the mapped reads. Previous work has shown that deamination, particularly the fre- quency of C →T substitutions, is common in old sam- ples [13, 42, 45] and significantly positively correlated with age ([14, 47]; R2 = 0.45; P = 1.44 × 10−10; n = 71 in the latter study). Generally, rates of both C →T and G →A substitutions towards the termini of reads tend to exceed 20% in samples > 500 years and can exceed 10% in historical samples [14], increasing towards 30% prior to soft-clipping [31]. The benefit of removing deaminated sites is that, left intact, they can lead to sequencing errors, particularly in low-coverage sequencing experiments [13]. However, there is a potential trade-off to consider. Uracil removal will cut all of the affected DNA fragments, thus poten- tially resulting in samples in which the majority of frag- ments have been cut [13, 51]. Even if employing a The rate of hydrolytic deamination varies with temperature, pH, and salinity, thus different conditions during the original sample deposition or capture are likely to account for any differences among samples [14]. Though much of the preservation journey of the samples studied here is unknown, the moths were all field-caught McGaughran BMC Genomics (2020) 21:188 Page 6 of 10 McGaughran BMC Genomics method to repair the DNA fragment after cutting (see 82% in the oldest samples) which tended to have lower Fig. 4 The correlation between sample age and various measures of sequencing quality, including saturation, total number of mapped reads, mean coverage, total number of reads on target, enrichment, and total number of baits with 1x coverage, when reads were aligned to the targets. Discussion In (a), each bar in the plot represents the strength and direction of correlation, and the R-value is displayed in/on the bars. In (b), the raw data points are plotted for each pair-wise comparison, along with the linear regression trend line. See Table 2 for the detailed statistics. Note that the total number of baits at 5x, 10x, 20x, and 30x coverage are excluded from these plots as they were highly correlated with the total number of baits at 1x coverage Fig. 4 The correlation between sample age and various measures of sequencing quality, including saturation, total number of mapped reads, mean coverage, total number of reads on target, enrichment, and total number of baits with 1x coverage, when reads were aligned to the targets. In (a), each bar in the plot represents the strength and direction of correlation, and the R-value is displayed in/on the bars. In (b), the raw data points are plotted for each pair-wise comparison along with the linear regression trend line See Table 2 for the detailed statistics Note that Fig. 4 The correlation between sample age and various measures of sequencing quality, including saturation, total number of mapped reads, mean coverage, total number of reads on target, enrichment, and total number of baits with 1x coverage, when reads were aligned to the targets. In (a), each bar in the plot represents the strength and direction of correlation, and the R-value is displayed in/on the bars. In (b), the raw data points are plotted for each pair-wise comparison, along with the linear regression trend line. See Table 2 for the detailed statistics. Note that the total number of baits at 5x, 10x, 20x, and 30x coverage are excluded from these plots as they were highly correlated with the total number of baits at 1x coverage Fig. 4 The correlation between sample age and various measures of sequencing quality, including saturation, total number of mapped reads, mean coverage, total number of reads on target, enrichment, and total number of baits with 1x coverage, when reads were aligned to the targets. In (a), each bar in the plot represents the strength and direction of correlation, and the R-value is displayed in/on the bars. In (b), the raw data points are plotted for each pair-wise comparison, along with the linear regression trend line. See Table 2 for the detailed statistics. Discussion Note that the total number of baits at 5x, 10x, 20x, and 30x coverage are excluded from these plots as they were highly correlated with the total number of baits at 1x coverage method to repair the DNA fragment after cutting (see Methods), if the starting DNA is highly degraded, the post-USER fragments could be too short to generate a final library of meaningful length. Ultimately, the recom- mendation to use a uracil-removing or stalling enzyme should come down to an understanding of the level of fragmentation, the preservation method, and the likeli- hood of deamination, of ones’ samples. An alternative to such treatment is the removal of false variant calls bioin- formatically, e.g., by trimming the ends of reads; how- ever this may lead to a high loss of data if done conservatively. 82% in the oldest samples), which tended to have lower starting concentrations and therefore usually required a higher number of indexing PCR cycles. Adapter dimers form when the adapters self-ligate instead of ligating to the sample DNA, and such dimers can dominate during PCR, which has a tendency to amplify shorter fragments more efficiently than longer ones [54]. In addition, adapter dimers form clusters at high efficiency and therefore consume valuable flow cell space during se- quencing, resulting in a high proportion of wasteful adapters in the sequenced reads [55], as found here. Adapter concentrations can be optimised prior to library preparation and dimers can usually be removed by doing some form of bead or gel-based clean-up or titration In the current study, the percentage of adapter con- tamination was much higher in older samples (up to ~ McGaughran BMC Genomics (2020) 21:188 Page 7 of 10 Page 7 of 10 Table 2 Correlation of sample age and several library quality metrics when reads were aligned to the targeted regions from the Helicoverpa armigera reference genome Table 2 Correlation of sample age and several library quality metrics when reads were aligned to the targeted regions from the Helicoverpa armigera reference genome in the sequenced reads, thus improving final data quality. Over the past two centuries, museum collections have grown in size and importance [58, 59] and simultaneous advances in sequencing technologies have unleashed a new frontier in museum genomics [2, 60, 61]. Indeed, museums hold indispensable records of the past, and act as libraries of biological diversity in time and space. Sample selection A total of 271 pinned specimens of the insect pest moth, Helicoverpa armigera, were obtained from several mu- seums and/or government departments across Australia, including the Australian National Insect Collection (Canberra), the Department of Agriculture and Food (Western Australia), the Department of Agriculture and Fisheries (Queensland), the Agricultural Scientific Col- lections Trust (New South Wales), and Museum Victoria (Victoria); specimens were collected at various time points between 1903 and 2015 (Fig. 1). [56], but, in the case of old and fragmented samples, the adapter-dimer is often very similar in size to the ligated library (~ 120 cf ~ 150 bp) and can therefore be very dif- ficult to remove [24, 57]. Fortunately, new kit-based methods can prevent adapter-dimer formation during li- brary preparation (e.g., DimerFree technology from Tecan Genomics Inc., or Dimerator™technology from DiaCarter, which blocks PCR amplification of adapter- adapter products). Though such methods were not widely available at the time the lab work for this project was undertaken, I have since used the UltraLow Ovation Kit (Tecan Genomics Inc.) on historical moth specimens of the same species and enjoyed significantly reduced adapter-dimer levels (< 5%). Genomic DNA preparation A ‘salting-out’ protocol [62] was followed to extract gen- omic DNA for all pinned specimens of H. armigera. Though described more than 20 years ago, this method is still highly used and has recently been shown to pro- duce higher DNA yields than other extraction methods, including phenol chloroform and kit-based [63]. I also found that the more expensive kit-based extraction methods gave equivalent starting concentrations to salting-out in a small test (data not shown). Here, the salting-out protocol was followed with a modification to the first step, which involved soaking moth abdomens in buffer with Proteinase K (#19133, Qiagen) for 24 h. A salt solution was then added to the abdomen preparation (after the abdomen was removed and cleaned), as per the recommended protocol [62]. Discussion As wet-lab protocols, sequencing methods, and bioinfor- matic pipelines continue to improve and evolve, ancient and archival DNA samples will become even more valu- able resources for the study of diverse historical processes. g g Library quality metric Correlation with sample age Total number of reads T269 = 4.38; P < 0.011.726e−05; R = 0.26 (0.14:0.37) Mean coverage T269 = 4.47; P < 0.01; R = 0.26 (0.15:0.37) Total number of baits on target T269 = 3.94; P < 0.01; R = 0.23 (0.12:0.34) Total number of baits at 1x coverage T269 = 8.77; P < 0.01; R = 0.47 (0.37:0.56) Total number of baits at 5x coverage T269 = 7.47; P < 0.01; R = 0.41 (0.31:0.51) Total number of baits at 10x coverage T269 = 6.95; P = < 0.01; R = 0.39 (0.28:0.49) Total number of baits at 20x coverage T269 = 6.31; P < 0.01; R = 0.36 (0.25:0.46) Total number of baits at 30x coverage T269 = 5.84; P = < 0.01 R = 0.34 (0.23:0.44) Saturation T269 = -0.22; P < 0.01; R = 0.22(−0.33:-0.10) Enrichment T269 = 6.65; P = < 0.01; R = 0.38 (0.27:0.47) Library quality metric Correlation with sample age Total number of reads T269 = 4.38; P < 0.011.726e−05; R = 0.26 (0.14:0.37) Mean coverage T269 = 4.47; P < 0.01; R = 0.26 (0.15:0.37) Total number of baits on target T269 = 3.94; P < 0.01; R = 0.23 (0.12:0.34) Total number of baits at 1x coverage T269 = 8.77; P < 0.01; R = 0.47 (0.37:0.56) Total number of baits at 5x coverage T269 = 7.47; P < 0.01; R = 0.41 (0.31:0.51) Total number of baits at 10x coverage T269 = 6.95; P = < 0.01; R = 0.39 (0.28:0.49) Total number of baits at 20x coverage T269 = 6.31; P < 0.01; R = 0.36 (0.25:0.46) Total number of baits at 30x coverage T269 = 5.84; P = < 0.01 R = 0.34 (0.23:0.44) Saturation T269 = -0.22; P < 0.01; R = 0.22(−0.33:-0.10) Enrichment T269 = 6.65; P = < 0.01; R = 0.38 (0.27:0.47) Conclusions Based on the findings presented here and, as shown pre- viously, museum samples are a great resource for an- swering an array of evolutionary questions, but there are inherent challenges linked to DNA degradation. Here, I identify two major considerations users should carefully consider when following standard library preparation protocols during targeted capture experiments of histor- ical samples. First, after careful consideration of the like- lihood of sample deamination, USER enzyme or similar incorporated into the blunt end-repair step could be a good option for removing and repairing DNA damage associated with historical specimens. Second, adapter contamination can be extremely high in sequence reads of older samples, thus users may find it particularly help- ful to consider a method that guarantees prevention of adapter-dimer formation. Each of these considerations may result in improved yields and reduced DNA damage Library preparation b Library preparation was broadly based on the procedures outlined in [20], using the standard steps for NGS library preparation (i.e., end repair, adaptor ligation and fill-in, and indexing PCR), but with several modifications due to the fragmented nature of the starting material. These modifications included: (1) the omission of a shearing step: (2) the incorporation of Uracil-Specific Excision Reagent (USER) enzyme (#M5508, New England Biolabs, Inc.) in the blunt-end repair step; (3) an ‘on-beads’ McGaughran BMC Genomics (2020) 21:188 Page 8 of 10 Page 8 of 10 place of COT-1 DNA, as COT-1 DNA is not available for H. armigera. place of COT-1 DNA, as COT-1 DNA is not available for H. armigera. place of COT-1 DNA, as COT-1 DNA is not available for H. armigera. clean-up protocol throughout (thus, no elution was per- formed following the majority of bead clean-ups); and (4) the use of a calculation to determine the number of required indexing PCR cycles. Baits were designed by NimbleGen (Roche), with tar- get sites encompassing ~ 1300 loci extracted from the H. armigera annotated genome [65]. Following hybridisa- tion, clean-up, and amplification of the pooled library, qPCR was used to confirm the success of the capture, before sequencing was carried out on an Illumina Next- Seq500 (75 bp PE) at the Biomolecular Resource Facility (Australian National University). The full wet-lab proto- col is provided in Supplementary Material. I omitted the shearing step because sample aliquots (3 μl) run on a 1% agarose gel following DNA extrac- tion showed DNA to predominantly be < 500 bp in length for all samples. USER was included because deamination is a recognised outcome of DNA degrad- ation processes in historical samples and the enzyme functions to excise uracil sites, forming an abasic (apyrimidinic) site while leaving the phosphodiester backbone intact. When followed by T4 DNA poly- merase (#EP0062, ThermoFisher Scientific) treatment (a standard step in library preparation), the result is removal of uracil residues from the DNA, cleavage of the 5′- and 3′- sides of the resulting abasic sites, and removal of the 3′-phosphate groups by T4 poly- nucleotide kinase (PNK; EK0032, ThermoFisher Scien- tific). Thus, USER removes the DNA damage, but the treated molecules are repaired and retained in the li- brary. Bioinformatics pipeline Quality control of raw read data was performed using FastQC v.0.10.1 (http://www.bioinformatics.babraham. ac.uk/projects/fastqc/). Trimmomatic v.0.36 [66] was used to remove adapter sequences, after which trimmed reads were aligned to the H. armigera genome (“Harm_ 1.0”; GenBank assembly accession: GCA_002156985.1), which spans a total length of ~ 337 Mb, using the bwa 0.7.5a-r405 [67] mem algorithm. Duplicate reads were removed from sorted bam files using picard v.2.10.6 (http://broadinstitute.github.io/picard/), and low quality and ambiguous alignments were removed with samtools v.1.5 [68] commands: -q 20 -f 0 × 0002 -F 0 × 0004 -F 0 × 0008. Finally, bam files were indexed with samtools and evaluated with the software mapdamage v.2.0 [42] to quantify DNA damage patterns. Library preparation b The on-beads protocol (SeraMag beads; #45152105050250, GE Healthcare Life Sciences) in- volved carrying beads and attached DNA directly to the subsequent step throughout the protocol, with the relevant solution pipetted gently up and down to re- suspend beads [64]. In contrast to usual methods, which involve elution following every bead clean-up, DNA was only eluted off the beads before and after the indexing PCR – this should go some way towards reducing library loss with each elution step (e.g., a re- cent study of beetles up to 159 years in age found an average DNA loss of 48.7% following bead clean-up during library preparation [17]). Finally, a calculation was used to determine the number of PCR cycles to use instead of applying a blanket number, or using qPCR. This calculation was based off Table 1 in the KAPA Library Amplification Kit Technical Data Sheet (KR0408_V7,17, KAPA BioSystems; available at https://www.kapabiosystems.com/document/kapa-li- Statistical analysis Th b The output bam files, generated above, were analysed with a variety of packages and tools, including samtools (flagstat), picard (CollectWgsMetrics, EstimateLibrary- Complexity) and ngscat v.0.1 [69] to obtain various met- rics of library quality. Statistical analyses (e.g., correlation, t-tests) were performed to examine the rela- tionship between sample age and these metrics using core packages in R v.3.5.1 [70]; the R scripts are pro- vided as Supplementary Material. Supplementary information brary-amplification-kit-tds/?dl=1), with sample con- centrations determined using a Qubit dsDNA HS Assay Kit and Fluorometer (ThermoFisher Scientific). pp y Supplementary information accompanies this paper at https://doi.org/10. 1186/s12864-020-6594-0. Indexing PCRs were performed twice for each sample (12 μl volume of DNA), with a different DNA polymerase in each reaction (#KK2600, KAPA HiFi, Kapa BioSystems; and #M0530, Phusion HiFidelity, New England Biolabs, Inc.) and 9–18 PCR cycles, depending on the sample con- centration following adapter ligation. Following indexing PCRs, samples were quantified and pooled with equimo- larity, then hybridised to baits following a modified ver- sion of the SeqCap EZ Library SR User’s Guide (Roche). The main modification during hybridisation was use of Nimblegen SeqCap EZ Developer Reagent (Roche) in Additional file 1. I wish to thank the various Australian museums and government departments that let me access important museum specimens, including the Australian National Insect Collection (Canberra), the Department of Agriculture and Food (Western Australia), the Department of Agriculture and Fisheries (Queensland), the Agricultural Scientific Collections Trust (New South Wales), and Museum Victoria (Victoria). I would like to thank Rebecca Laver for completing the library preparation for many of the samples in this paper, and Terry Newman for providing statistical advice. Finally, I thank colleagues, past and present, in the Moritz Lab at Australian National University, and at the References 1. 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https://openalex.org/W2895073155	https://europepmc.org/articles/pmc6171174?pdf=render	English	null	Translocated duplication of a targeted chromosomal segment enhances gene expression at the duplicated site and results in phenotypic changes in Aspergillus oryzae	Fungal biology and biotechnology	2,018	cc-by	11,965	Takahashi et al. Fungal Biol Biotechnol (2018) 5:17 https://doi.org/10.1186/s40694-018-0061-6 Takahashi et al. Fungal Biol Biotechnol (2018) 5:17 https://doi.org/10.1186/s40694-018-0061-6 Fungal Biology and Biotechnology Fungal Biology and Biotechnology Open Access Translocated duplication of a targeted chromosomal segment enhances gene expression at the duplicated site and results in phenotypic changes in Aspergillus oryzae Tadashi Takahashi* , Masahiro Ogawa, Atsushi Sato and Yasuji Koyama © The Author(s) 2018. 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: Translocated chromosomal duplications occur spontaneously in many organisms; segmental duplica- tions of large chromosomal regions are expected to result in phenotypic changes because of gene dosage effects. Therefore, experimentally generated segmental duplications in targeted chromosomal regions can be used to study phenotypic changes and determine the functions of unknown genes in these regions. Previously, we performed tandem duplication of a targeted chromosomal segment in Aspergillus oryzae. However, in tandem chromosomal duplication, duplication of chromosomal ends and multiple chromosomal duplication are difficult. In this study, we aimed to generate fungal strains with a translocated duplication or triplication of a targeted chromosomal region via break-induced replication. Results: Double-strand breaks were introduced into chromosomes of parental strains by treating protoplast cells with I-SceI meganuclease. Subsequently, strains were generated by nonreciprocal translocation of a 1.4-Mb dupli- cated region of chromosome 2 to the end of chromosome 4. Another strain, containing a triplicated region of chromosome 2, was generated by translocating a 1.4-Mb region of chromosome 2 onto the ends of chromosomes 4 and 7. Phenotypic analyses of the strains containing segmental duplication or triplication of chromosome 2 showed remarkable increases in protease and amylase activities in solid-state cultures. Protease activity was further increased in strains containing the duplication and triplication after overexpression of the transcriptional activator of proteases prtT. This indicates that the gene-dosage effect and resulting phenotypes of the duplicated chromosomal region were enhanced by multiple duplications, and by the combination of the structural gene and its regulatory genes. Gene expression analysis, conducted using oligonucleotide microarrays, showed increased transcription of a large population of genes located in duplicated or triplicated chromosomal regions. Conclusion: In this study, we performed translocated chromosomal duplications and triplications of a 1.4-Mb targeted region of chromosome 2. Strains containing a duplication of chromosome 2 showed significant increases in protease and amylase activities; these enzymatic activities were further increased in the strain containing a triplication of chromosome 2. This indicates that segmental duplications of chromosomes enhance gene-dosage effects, and that the resulting phenotypes play important phenotypic roles in A. oryzae. Keywords: Targeted chromosomal duplication, Translocated duplication, Break induced replication, Aspergillus oryzae, Phenotypic change, prtT, Screening, Molecular breeding, Biotechnology, Genome engineering, Gross genome editing, Protease © The Author(s) 2018. Background chromosomal deletions [9, 10], have been developed to analyze the nature of A. oryzae and A. sojae. However, in koji molds, the functions of many genes remain unknown. Because of the presence of endogenous multiple ortholo- gous genes, disrupting individual genes in strains of koji molds does not always generate clear phenotypic changes [5]. Overexpression of individual genes via a strong pro- moter can also cause reduced growth phenotypes [11]. However, strains with duplicated chromosomes can show phenotypic changes via increased dosage of unidenti- fied genes [12]. By examining the genome of targeted chromosomal segments and phenotypic changes in the strains, we can determine the role of unknown genes cor- responding to these phenotypic changes. In this study, we generated targeted segmental chromosomal duplications in A. oryzae and examined the phenotypic effects of these duplications. There are two common types of segmen- tal chromosomal duplications: tandem duplication and translocated duplication (Fig. 1). In a tandem duplica- tion, duplicated regions are arranged next to each other on the same chromosome by nonallelic homologous recombination (Fig. 1, top). In translocated duplications, In eukaryotic organisms, duplications, translocations, reversions, and deletions of chromosomal regions occur spontaneously. For example, in Saccharomyces cerevisiae, duplication and translocation of chromosomes frequently occurs via Ty transposable element sequences, which are repeat sequences widely distributed in yeast chro- mosomes [1]. Similarly, previous studies of mammalian cells indicated that duplications, translocations, and dele- tions of chromosomes occur via repeat sequences such as Alu and L1 [2]. Changes in the chromosomal state often induce significant alterations in gene expression and result in phenotypic changes. In humans and other mam- mals, chromosome duplication or translocation events are reported to be among the causes of genetic diseases and cancers [3, 4].h The koji molds Aspergillus oryzae and Aspergillus sojae are filamentous fungi that are used in the fermenta- tion of soy sauce and sake, and in production of indus- trial enzymes. The complete genome sequences of A. oryzae and A. sojae have been determined [5, 6]. Previ- ously, genetic tools such as gene targeting [7, 8] and large Fig. 1 Types of chromosomal duplication. The top part of the figure shows a schematic representation of tandem chromosomal replication by nonallelic homologous recombination, where the duplicated region was arrayed in the same chromosome. Abstract This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Correspondence: ttakahashi@mail.kikkoman.co.jp Noda Institute for Scientific Research, 399 Noda, Noda City, Chiba Pref 278‑0037, Japan Correspondence: ttakahashi@mail.kikkoman.co.jp Noda Institute for Scientific Research, 399 Noda, Noda City, Chiba Pref 278‑0037, Japan Correspondence: ttakahashi@mail.kikkoman.co.jp Noda Institute for Scientific Research, 399 Noda, Noda City, Chiba Pref 278‑0037, Japan © The Author(s) 2018. 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. Takahashi et al. Fungal Biol Biotechnol (2018) 5:17 Page 2 of 17 Page 2 of 17 Translocated duplication of targeted segment of chromosome 2 onto chromosome 4 and a strain bearing translocated triplication We previously constructed strains containing targeted tandem chromosomal duplications [12]. Chromosome 2 of A. oryzae includes genes encoding alkaline protease and alpha-amylase, and their respective regulatory genes prtT and amyR, which are important for fermentation. Accordingly, strains containing a 700-kb tandem chro- mosomal duplication in chromosome 2 showed increased protease and amylase activities under solid-state cul- ture conditions [12]. In the present study, we duplicated a 1.4-Mb region of chromosome 2 and translocated this region onto the end of chromosome 4 (Fig. 2). Success- fully translocated duplications can increase the activities of protease and amylase in solid-state cultures, as shown previously by tandem duplication of chromosome 2 [12]. In subsequent analyses using BLASTN, no I-SceI recog- nition sequence was found in the chromosome of A. ory- zae strain RIB40 [17]. To translocate the 1.4-Mb region of chromosome 2 onto the end of chromosome 4, we placed the 5′ΔpyrG marker (targeted integration of the basic unit of 5′ΔpyrG and subsequent 5-fluoroorotic acid [5-FOA] selection) adjacent to the target donor chromo- somal region. The 3′ΔpyrG marker (targeted integration of the basic unit of 3′ΔpyrG and subsequent 5-FOA selec- tion), which included the I-SceI recognition sequence, was placed adjacent to the acceptor chromosomal region (Fig. 2, top). The resulting parental strain was then used to generate translocated duplication of the targeted region of chromosome 2. For these procedures, proto- plast cells of the parental strain were prepared and gently treated with I-SceI meganuclease and PEG. The treated protoplasts were then incubated at 30 °C on Czapek-Dox minimum medium (CZ) plates containing 1.2 M sorbi- tol. After more than 2 weeks of incubation, we observed a colony that was regenerated from treated protoplasts. The frequency of the number of regenerated colonies was approximately 10−8 per cell. To confirm that translo- cated duplication was achieved in the regenerated strain, genomic DNA of the regenerated strain was extracted The break-induced replication (BIR) mechanism causes translocated chromosomal duplications in yeast [13–15]. However, no previous studies have shown that the same mechanism can be used in filamentous fungi such as A. oryzae. To cause BIR in A. oryzae, it is necessary to arti- ficially introduce double-strand breaks (DSBs) into the chromosomes (Fig. 1, bottom). In most studies on yeast, DSBs are introduced after the expression of genes encod- ing endogenous yeast homothallic switching endonu- clease (HO) or I-SceI endonuclease (SCEI). Background The bottom of the figure shows a schematic representation of translocated duplication by break-induced replication, where the duplicated region was translocated to the other chromosome Fig. 1 Types of chromosomal duplication. The top part of the figure shows a schematic representation of tandem chromosomal replication by nonallelic homologous recombination, where the duplicated region was arrayed in the same chromosome. The bottom of the figure shows a schematic representation of translocated duplication by break-induced replication, where the duplicated region was translocated to the other chromosome Fig. 1 Types of chromosomal duplication. The top part of the figure shows a schematic representation of tandem chromosomal replication by nonallelic homologous recombination, where the duplicated region was arrayed in the same chromosome. The bottom of the figure shows a schematic representation of translocated duplication by break-induced replication, where the duplicated region was translocated to the other chromosome Takahashi et al. Fungal Biol Biotechnol (2018) 5:17 Page 3 of 17 Page 3 of 17 chromosomal duplications are translocated onto another chromosome by break-induced replication (Fig. 1, bot- tom). We previously generated A. oryzae strains contain- ing targeted tandem chromosomal duplications using protoplasts of the parental strain, in which artificially generated consensus sequences (partly deleted selection marker) were introduced into both ends of the duplica- tion target region [12]. If the copy number of the duplica- tion target region is increased, such as in triplication, the phenotypic change in the strain may be further enhanced. However, to maintain stability of a tandem chromosomal duplication, a selection marker must be located at the junction between the two duplicated segments. In A. oryzae, this prevents removal of the duplicated region by recombination events occurring between the duplicated homologous sequences [12]. Therefore, it is technically difficult to increase the copy numbers of genes located in duplicated regions in tandem chromosomal duplica- tions. Moreover, it is difficult to duplicate chromosomal ends in tandem duplications because a selection marker positioned at the chromosomal end is unstable and lost during regeneration (Takahashi et al. unpublished data). In contrast, generating multiple translocated chromo- somal duplications is theoretically possible because the potential for recombination between the two duplicated regions is low in translocated chromosomal duplications, and selective pressures are not required to maintain the translocation. Furthermore, duplication of chromo- somal ends is straightforward in translocated duplication because a selection marker is not required at chromo- somal ends. Background However, no previous studies have gener- ated translocated duplications of targeted chromosomal regions in filamentous fungi such as A. oryzae. Therefore, we generated a translocated duplication of a targeted chromosomal segment in A. oryzae. chromosomal duplications are translocated onto another chromosome by break-induced replication (Fig. 1, bot- tom). We previously generated A. oryzae strains contain- ing targeted tandem chromosomal duplications using protoplasts of the parental strain, in which artificially generated consensus sequences (partly deleted selection marker) were introduced into both ends of the duplica- tion target region [12]. If the copy number of the duplica- tion target region is increased, such as in triplication, the phenotypic change in the strain may be further enhanced. However, to maintain stability of a tandem chromosomal duplication, a selection marker must be located at the junction between the two duplicated segments. In A. oryzae, this prevents removal of the duplicated region by recombination events occurring between the duplicated homologous sequences [12]. Therefore, it is technically difficult to increase the copy numbers of genes located in duplicated regions in tandem chromosomal duplica- tions. Moreover, it is difficult to duplicate chromosomal ends in tandem duplications because a selection marker positioned at the chromosomal end is unstable and lost during regeneration (Takahashi et al. unpublished data). In contrast, generating multiple translocated chromo- somal duplications is theoretically possible because the potential for recombination between the two duplicated regions is low in translocated chromosomal duplications, and selective pressures are not required to maintain the translocation. Furthermore, duplication of chromo- somal ends is straightforward in translocated duplication because a selection marker is not required at chromo- somal ends. However, no previous studies have gener- ated translocated duplications of targeted chromosomal regions in filamentous fungi such as A. oryzae. Therefore, we generated a translocated duplication of a targeted chromosomal segment in A. oryzae. strain containing 1.4-Mb segment of the targeted chro- mosomal region. To the best of our knowledge, this is the first study to generate a targeted translocated chro- mosomal duplication using BIR and analyze the effect of chromosomal duplication on gene expression in fila- mentous fungi. Phenotypes of the strain resulting from this chromosomal duplication show increased activities of protease and amylase, indicating that this method can be used in functional analysis and molecular breeding of Aspergillus strains. Fig. 3 a Confirmation of translocated duplication by PCR. Targeted translocated duplication of a 1.4-Mb region of chromosome 2 to chromosome 4 was confirmed by PCR. Lane 1. B–U, B–L; Lane 2. ct166-U, ct166-L; Lane 3. B–U, ct166-L; Lane 4. ct166-U, B–L (Lanes 1–4. translocated duplication strain J4); Lane 5. B–U, B–L; Lane 6. ct166-U, ct166-L; Lane 7. B–U, ct166-L; Lane 8. ct166-U, B–L (Lanes 5–8. Wild-type strain). b Confirmation of translocated duplication by comparative genome hybridization (CGH) arrays. Vertical bars show ratios of signal intensities for probes of the strain bearing translocated duplication (J4) relative to those of the control strain (RIB40). Ratios of hybridization signals in the 1.4-Mb region of chromosome 2 were twice those of the other region, indicating that the 1380-kb region of chromosome 2, including AO090002001035 to AO090002001558, was duplicated. The ratio of hybridization signals in the 308-kb region of chromosome 4 was nearly zero, indicating that the 308-kb region of chromosome 4, including AO090166000010 to AO090166000123, was deleted (See figure on next page.) Gene expression analysis of translocated chromosomal duplication and triplication strains in solid‑state culture Segmental duplication of large chromosomal regions may upregulate numerous genes in the duplicated chro- mosomal region and cause phenotypic changes via gene dosage effect. To demonstrate the effects of chromo- somal duplication on gene expression, we examined gene expression levels of the strains under conditions of solid-state fermentation. The J4 strain with a translo- cated duplication of a 1.4-Mb region of chromosome 2, I-8 strain with a translocated triplication of the 1.4-Mb region of chromosome 2, and RIB40 strain (control) were cultivated for 65 h at 30 °C in wheat bran medium; then, RNA was extracted and analyzed by gene expression microarrays. The ratios of upregulated genes in dupli- cated chromosomal regions were remarkably higher in strains with duplicated and triplicated chromosomes. As shown in Tables 1 and 2, gene expression was increased by 11% in the whole chromosomal region (1293 vs. 12010). Gene expression was increased by 37 and 64% in the strains with duplicated (166 vs. 446) and triplicated regions (284 vs. 446), respectively. These data indicate that segmental duplication and triplication of target chromosomal regions effectively increased transcription of resident genes. These data were summarized accord- ing to Clusters of Orthologous Groups (COGs) classifi- cation [19]. Per COGs classifications, the influence gene dosage effect on signal transduction mechanisms (T) was low with 25% increases in the J4 (2 vs. 8) and 38% increases in the I-8 (3 vs. 8) strains. The expression of genes categorized into the nucleotide transport and metabolism (F) group, per COGs classification, was sig- nificantly increased (5 vs. 5) in the duplicated region of the strains (Tables 1 and 2). In contrast, gene expression was decreased by 4 and 2.5% in the duplicated (17 vs. 446) and triplicated chromosomal regions, respectively. For the chromosomes in the J4 (duplicated) strain, sites of upregulated genes were identified using probes (Addi- tional file 1: Figure S5), which clearly showed increased gene transcription in the duplicated chromosomal region. Approximately 10% of the genes located outside of the duplicated region (1127 vs. 11,564) were upregu- lated; several of these genes were markedly upregulated in the duplicated strain (Additional file 1: Figure S5), indicating the presence of regulatory factors in the dupli- cated region. The genes upregulated in the J4 strain are summarized in Tables 3, 4, 5 and 6. Translocated duplication of targeted segment of chromosome 2 onto chromosome 4 and a strain bearing translocated triplication vector for introducing 3′ΔpyrG with the I-SceI recogni- tion sequence was integrated near the end of chromo- some 7 (Additional file 1: Figure S3, acceptor region). This was followed by selection using 5-FOA. The resulting triplication-containing parental strain (K1-IF5-2-5FOA) was cultured, and protoplasts were prepared for pheno- typic analyses. Protoplasts were treated with I-SceI and PEG as described above. After incubation for 3 weeks at 30 °C, regenerated colonies were analyzed by PCR. After single-colony isolation (details are described in Addi- tional file 1: Fig. S4), the strain containing homokaryotic nuclei with the translocated chromosomes was thereafter referred to as I-8 strain. vector for introducing 3′ΔpyrG with the I-SceI recogni- tion sequence was integrated near the end of chromo- some 7 (Additional file 1: Figure S3, acceptor region). This was followed by selection using 5-FOA. The resulting triplication-containing parental strain (K1-IF5-2-5FOA) was cultured, and protoplasts were prepared for pheno- typic analyses. Protoplasts were treated with I-SceI and PEG as described above. After incubation for 3 weeks at 30 °C, regenerated colonies were analyzed by PCR. After single-colony isolation (details are described in Addi- tional file 1: Fig. S4), the strain containing homokaryotic nuclei with the translocated chromosomes was thereafter referred to as I-8 strain. Translocated duplication of targeted segment of chromosome 2 onto chromosome 4 and a strain bearing translocated triplication In contrast, chromosome modifications have been performed in A. oryzae using polyethylene glycol (PEG)-mediated intro- duction of enzymes into protoplast cells [12, 16–18]. Therefore, we generated chromosomal duplications by directly treating protoplast cells with I-SceI meganucle- ase and monitoring the resulting chromosomal DSBs. This approach eliminated the need for I-SceI expression and produced a translocated chromosomal duplication Takahashi et al. Fungal Biol Biotechnol (2018) 5:17 Page 4 of 17 Fig. 2 A schematic showing translocated duplication of chromosome 2 in a strain of A. oryzae. A 1.4-Mb target donor region of chromosome 2 was duplicated and translocated to the acceptor region of chromosome 4 using break-induced replication and analyzed by PCR using primers targeting the border of the translocated region (Fig. 3a). occurred in the regenerated strain; this strain was there- after referred to as J4 and used in further analyses. We detected amplification of the 5-kb DNA fragment using combinations of primers targeting the translocated position (primers ct166-U and B-L in lane 4 of Fig. 3a) and original position (primers B-U and B-L in lane 1 of Fig. 3a) in the regenerated strain. In contrast, amplifica- tion of the 3-kb fragment was detected only by using the primer combination corresponding to the original posi- tion (primes B-U and B-L in lane 5 of Fig. 3a, and primers ct166-U and ct166-L in lane 6 of Fig. 3a) in the control strain (RIB40). These analyses confirmed that translo- cated duplication of the targeted chromosomal region As described above, generating multiple translocated chromosomal duplications is theoretically possible. Therefore, we constructed a strain in which the targeted region of chromosome 2 was triplicated, and evaluated the effects of triplication on the phenotype of this strain. Initially, we removed the pyrG marker at the border of the translocated duplication region of chromosome 4 by homologous recombination between the chromo- some and fragment used to remove pyrG (Additional file 1: Figure S3, top). Transformants were then selected on 1.2-M sorbitol-CZ plates containing 5-FOA, and the Page 5 of 17 Takahashi et al. Fungal Biol Biotechnol (2018) 5:17 Takahashi et al. Fungal Biol Biotechnol (2018) 5:17 Page 6 of 17 These results indicate that multiple translocated duplica- tions (such as translocated triplication) of the targeted chromosomal region were achieved in the I-8 strain. These results indicate that multiple translocated duplica- tions (such as translocated triplication) of the targeted chromosomal region were achieved in the I-8 strain. Gene expression analysis of translocated chromosomal duplication and triplication strains in solid‑state culture Twenty-three proteo- lytic genes, 8 amylolytic genes, and 23 xylanolytic genes, which were located outside of the duplicated region, were Confirmation of translocated chromosomal duplications using array comparative genome hybridization (array CGH) CGH was used to confirm the presence of translocated chromosomal duplication in the targeted chromosomal region. Briefly, genomic DNA isolated from RIB40 (con- trol) and J4 strains (Δku70 strain with translocated chromosomal duplication) was labeled with Cy dyes, hybridized on microarray slides at 65 °C for 24 h, and scanned with a laser-scanner. Comparison of the two strains revealed that the signal ratio of the probes in the 1.4-Mb region of chromosome 2 (AO090003001035– AO090003001558) was approximately two, and that of the probes located in the 308-kb region near the terminal end of chromosome 4 (AO090166000010– AO090166000123) was nearly zero (Fig. 3b). These observations confirmed the presence of translocated duplication of the targeted chromosomal region in the J4 strain (Fig. 3b and Additional file 1: Figure S1). The BN1-1 strain (ku70 + strain with translocated chromo- somal duplication) was also analyzed using CGH. Simi- lar results were obtained (Additional file 1: Figure S2), indicating that translocated duplication of the targeted chromosomal region occurred successfully in the J4 and BN1-1 strains. Next, genomic DNA isolated from RIB40 (con- trol) and I-8 strains (Δku70 strain with translocated chromosomal triplication) was labeled with Cy dyes, hybridized on microarray slides, and scanned using a laser-scanner. As shown in Fig. 4, comparisons of triplicate (I-8) and control strains revealed a signal ratio of approximately 3 for the probes located in the 1.4-Mb region of chromosome 2 (AO090003001035– AO090003001558). Moreover, the signal ratios of the probes located in the 308-kb near-telomeric region of chromosome 4 (AO090166000010–AO090166000123) and in the 13-kb near-telomeric region of chromosome 7 (AO090011000001–AO090011000008) were nearly zero, indicating successful translocation of chromosome 2 to the end of chromosomes 4 and 7 in the I-8 strain (Fig. 4). Takahashi et al. Fungal Biol Biotechnol (2018) 5:17 Page 7 of 17 Fig. 4 Confirmation of translocated duplication by array CGH. Results of array CGH indicate that the 1.4-Mb region of chromosome 2 was triplicated and translocated to the ends of chromosomes 4 and 7 Fig. 4 Confirmation of translocated duplication by array CGH. Results of array CGH indicate that the 1.4-Mb region of chromosome 2 was triplicated and translocated to the ends of chromosomes 4 and 7 the duplicated region. Gene expression analysis of translocated chromosomal duplication and triplication strains in solid‑state culture The expression of xlnR was not increased. However, we observed upregulated expres- sion of genes related to xylanase, most of which were positively regulated by xlnR [20]. This indicate that xlnR2 (AO090003001292) affected the expression of these genes. The expression of prtT, amyR, and xlnR2 was upregulated. Increased expression of proteolytic genes, located outside of the duplicated region, was observed in the J4 strain, suggesting that gene dosage effect exerted by prtT occurred in the duplicated region. Similarly, the upregulated expression of amylolytic genes indicated that gene dosage effect, exerted by amyR, occurred in Takahashi et al. Gene expression analysis of translocated chromosomal duplication and triplication strains in solid‑state culture Fungal Biol Biotechnol (2018) 5:17 Page 8 of 17 Table 1 Percent increase in the expression of upregulated genes in the duplicated chromosomal region of the J4 strain p < 0.02 and fold change > 1 indicate upregulated genes % increase shows the percent ratio of upregulated genes to total genes in the whole genome or that of the genes in the duplicated region COG description Total Up-regulated genes % Duplicated region Up-regulated genes % [A] RNA processing and modification 176 3 2 8 1 13 [B] Chromatin structure and dynamics 38 2 5 4 2 50 [C] Energy production and conversion 371 56 15 9 3 33 [D] Cell cycle control, cell division, chromosome partitioning 101 10 10 5 2 40 [E] Amino acid transport and metabolism 453 65 14 18 9 50 [F] Nucleotide transport and metabolism 97 15 15 5 5 100 [G] Carbohydrate transport and metabolism 500 103 21 27 15 56 [H] Coenzyme transport and metabolism 133 13 10 4 3 75 [I] Lipid transport and metabolism 314 37 12 12 7 58 [J] Translation, ribosomal structure and biogenesis 277 5 2 6 3 50 [K] Transcription 180 4 2 9 3 33 [L] Replication, recombination and repair 172 11 6 7 2 29 [M] Cell wall/membrane/envelope biogenesis 98 11 11 5 1 20 [N] Cell motility 2 0 0 0 0 0 [O] Posttranslational modification, protein turnover, chaperones 390 19 5 12 6 50 [P] Inorganic ion transport and metabolism 195 21 11 9 2 22 [Q] Secondary metabolites biosynthesis, transport and catabolism 447 68 15 9 4 44 [R] General function prediction only 1370 186 14 51 18 35 [S] Function unknown 333 27 8 16 7 44 [T] Signal transduction mechanisms 310 26 8 8 2 25 [U] Intracellular trafficking, secretion, and vesicular transport 241 5 2 9 1 11 [V] Defense mechanisms 50 11 22 0 0 0 [W] Extracellular structures 3 0 0 0 0 0 [Y] Nuclear structure 6 0 0 0 0 0 [Z] Cytoskeleton 94 10 11 2 0 0 Unannotated 5659 585 10 211 70 33 Total 12,010 1293 11 446 166 37 Table 1 Percent increase in the expression of upregulated genes in the duplicated chromosomal region of the J4 strain xpression of upregulated genes in the duplicated chromosomal region of the J4 strain p < 0.02 and fold change > 1 indicate upregulated genes p g g ent ratio of upregulated genes to total genes in the whole genome or that of the genes in the duplicated region duplication strains under solid-state culture conditions (Fig. Gene expression analysis of translocated chromosomal duplication and triplication strains in solid‑state culture 5a). Both translocated (J4) and tandem (700k-dup) chromosomal duplications led to increased activity of protease and amylase. Moreover, the activity of acid carboxypeptidase in the translocated duplication strain showed slightly higher levels than that in the wild-type strain (RIB40 control), but lower levels than that in the tandem duplication strain. A list of genes located in the overlapped region between J4 and 700k-dup is provided in Additional file 1: Table S2. We then examined enzy- matic activity in the triplication strain I-8 under solid- state culture conditions. Protease activity was increased by more than fourfold in the triplication strain (I-8) com- pared to that in the control strain (RIB40), and was higher than those in the translocated and tandem duplication strains (J4 and 700k-dup) (Fig. 5a). The activity of amyl- ase was also higher in the triplication strain (I-8) than in the duplication strains (J4 and 700k-dup), indicating upregulated in the duplicated region (Table 6). In con- trast, 1252 genes located outside of the duplicated region were downregulated in the J4 strain, indicating that genes involved in negative regulation were also present in the duplicated region. duplication and triplication strains in solid‑state culture To examine how chromosomal duplication and tripli- cation affect phenotypes, we assessed enzymatic activ- ity in translocated chromosomal duplication strains under solid-state conditions. We have previously shown increased activity of protease, amylase, and acid carboxypeptidase in strains with the 700-kb region (AO090003001003–AO090003001258) of the tandem duplication of chromosome 2 under conditions of solid- state cultivation on wheat bran media [12]. Therefore, we measured the activities of these enzymes in translocated Takahashi et al. duplication and triplication strains in solid‑state culture Fungal Biol Biotechnol (2018) 5:17 Page 9 of 17 Table 2 Percent increase in upregulated genes in the triplicated chromosomal region of the I-8 strain p < 0.02 and fold change > 1 indicate upregulated genes % increase shows the percent ratio of upregulated genes to total genes in the whole genome or that of the genes in the duplicated region COG description Total Up-regulated genes % Duplicated region Up-regulated genes % [A] RNA processing and modification 176 9 5 8 7 88 [B] Chromatin structure and dynamics 38 3 8 4 3 75 [C] Energy production and conversion 371 42 11 9 5 56 [D] Cell cycle control, cell division, chromosome partitioning 101 13 13 5 5 100 [E] Amino acid transport and metabolism 453 60 13 18 14 78 [F] Nucleotide transport and metabolism 97 11 11 5 5 100 [G] Carbohydrate transport and metabolism 500 70 14 27 24 89 [H] Coenzyme transport and metabolism 133 13 10 4 3 75 [I] Lipid transport and metabolism 314 39 12 12 9 75 [J] Translation, ribosomal structure and biogenesis 277 8 3 6 6 100 [K] Transcription 180 10 6 9 8 89 [L] Replication, recombination and repair 172 14 8 7 5 71 [M] Cell wall/membrane/envelope biogenesis 98 10 10 5 3 60 [N] Cell motility 2 0 0 0 0 0 [O] Posttranslational modification, protein turnover, chaperones 390 21 5 12 10 83 [P] Inorganic ion transport and metabolism 195 18 9 9 4 44 [Q] Secondary metabolites biosynthesis, transport and catabolism 447 54 12 9 5 56 [R] General function prediction only 1370 138 10 51 24 47 [S] Function unknown 333 29 9 16 14 88 [T] Signal transduction mechanisms 310 21 7 8 3 38 [U] Intracellular trafficking, secretion, and vesicular transport 241 10 4 9 7 78 [V] Defense mechanisms 50 11 22 0 0 0 [W] Extracellular structures 3 0 0 0 0 0 [Y] Nuclear structure 6 0 0 0 0 0 [Z] Cytoskeleton 94 12 13 2 2 100 Unannotated 5659 537 9 211 118 56 Total 12,010 1153 10 446 284 64 ble 2 Percent increase in upregulated genes in the triplicated chromosomal region of the I-8 strain n upregulated genes in the triplicated chromosomal region of the I-8 strain the triplicated chromosomal region of the I-8 strain p < 0.02 and fold change > 1 indicate upregulated genes of the prtT gene, and a pyrG marker. duplication and triplication strains in solid‑state culture pAPRT in circular state was used to transform the RP1 strain (pyrG deletion strain), del 1546K4 strain (pyrG deletion strain derived from the J4 strain), and TLTAs11B strain (pyrG deletion strain derived from the I-8 strain). Single transformants showing clear large halos on casein plates, which indi- cates high protease activity [12], were selected for each prtT overexpression strain; overexpression of prtT was confirmed by real-time PCR (Table 8). that gene dosage showed increased phenotypic effects after multiple duplication of the chromosomal region. The BP-B3 strain, a tandem duplication strain bearing a 9-kb region (AO090003001033-AO090003001036) that included alp (gene encoding alkaline protease, AO090003001036) [21], showed 1.7-fold increase in protease activity compared to that of the control strain (Fig. 5a). The expression of alp and prtT in the duplica- tion strains were examined by real-time PCR (Table 7). y We then overexpressed the transcription factor prtT and examined its effects in duplicated and triplicated chromosomal translocation strains. PrtT regulates pro- teolytic enzymes in A. oryzae [22]. Our gene expression microarrays revealed moderate fivefold increases in prtT expression in duplication strains. These data suggest that prtT is rate-limiting, indicating increased protease activity in prtT over-expressing strains. The prtT over- expression vector pAPRT contains an amyB promoter and terminator, connected to the open reading frame i The expression of prtT was increased more than 10-fold in the APRT, 1546 K-APRT, and TLTA-APRT strains compared to that in the control strain (Table 8). Accordingly, the activity of protease in solid-state cul- tures (Fig. 5a) was approximately threefold higher in the APRT-transformed strain than in the RIB40 strain (wild type). This indicates that a single copy of alp was rate- limiting for protease activity under condition of prtT overexpression. Moreover, protease activity was similar in the 1546 K-APRT and TLTA-APRT strains and nearly Takahashi et al. Fungal Biol Biotechnol (2018) 5:17 Page 10 of 17 Table 3 Upregulation of proteolytic genes located outside of the duplicated chromosomal region in the J4 strain a Relative gene expression in the J4 strain compared to that in the RIB40 strain was measured by gene expression array. duplication and triplication strains in solid‑state culture p < 0.02 Systematic name Relative genea expression Description AO090001000135 2.7 Mep20-metalloproteinase AO090003000354 3.2 LapII, transferrin receptor and related proteins AO090009000148 2.1 OpsA;aspartyl protease AO090009000171 3.5 SPRT-like metalloprotease AO090009000593 4.4 Metal-dependent amidase/aminoacylase/carboxypeptidase AO090010000493 4.5 NpII-neutral protease II AO090010000534 4.4 Serine carboxypeptidases (lysosomal cathepsin A) AO090010000540 3.9 Aminoacylase ACY1 and related metalloexopeptidases AO090011000036 9.7 Np I-neutral protease I AO090011000052 4.7 LapI-leucine aminopeptidase AO090011000235 3.0 TppA-tripeptidyl peptidase A AO090012000022 2.9 Metal-dependent amidase/aminoacylase/carboxypeptidase AO090012000080 2.4 Metal-dependent amidase/aminoacylase/carboxypeptidase AO090012000706 4.0 Carboxypeptidase C (cathepsin A) AO090020000288 3.1 Aminoacylase ACY1 and related metalloexopeptidases AO090020000351 4.0 Serine carboxypeptidases (lysosomal cathepsin A) AO090023000382 2.6 Carboxypeptidase C (cathepsin A) AO090026000083 4.5 AorO-aorsin AO090026000680 3.8 Serine carboxypeptidases (lysosomal cathepsin A) AO090103000264 3.9 Predicted molecular chaperone distantly related to HSP70-fold metalloproteases AO090138000101 4.0 Putative intracellular protease/amidase AO090138000114 7.1 Meltrins, fertilins and related Zn-dependent metalloproteinases of the ADAMs family AO090701000220 2.3 Carboxypeptidase C (cathepsin A) Table 3 Upregulation of proteolytic genes located outside of the duplicated chromosomal region in the J4 strain Systematic name Relative genea expression Description egulation of proteolytic genes located outside of the duplicated chromosomal region in the J4 strain a Relative gene expression in the J4 strain compared to that in the RIB40 strain was measured by gene expression array. p < 0.02 Table 4 Upregulation of amylolytic genes located outside of the duplicated region in the J4 strain a Relative gene expression in the J4 strain compared to that in the RIB40 strain was measured by gene expression array. p < 0.02 Systematic name Relative genea expression Description AO090001000259 2.8 Beta-galactosidase AO090001000492 2.6 Arabinogalactan endo- 1,4-beta-galactosidase AO090012000389 8.3 Beta-galactosidase/beta- glucuronidase AO090023000944 2.5 Alpha-amylase AO090038000471 2.5 Maltase glucoamylase and related hydrolases, glyco- syl hydrolase family 31 AO090103000378 2.9 Alpha-amylase AO090120000158 6.4 Beta-galactosidase AO090701000558 2.2 Alpha-glucosidases, family 31 of glycosyl hydrolases The APRT, 1546 K-APRT, and TLTA-APRT strains showed similarly increases in the activity of acid carboxy- peptidase compared to that of the RIB40 strain. This sug- gests that acid carboxypeptidase genes are transactivated by prtT, but are not located in the duplicated chromo- somal region. Single-copy integration of pAPRT in the APRT and TLTA-APRT strains, and two-copy integration of pAPRT in the 1546 K-APRT strain, were confirmed by quantitative PCR (Additional file 1: Figure S7). duplication and triplication strains in solid‑state culture Table 4 Upregulation of amylolytic genes located outside of the duplicated region in the J4 strain q (i g ) The growth phenotypes of translocated duplication strains on CZ plates are presented in Fig. 5b. The strains were cultured on 1.2 M sorbitol-CZ plates at 30 °C for 7 days. The J4 strain showed a slight delay in growth compared to the growth of the RIB40 strain. The I-8 strain showed a slight delay in growth compared to that of the J4 strain. The growth rate of the strains gradually decreased as the copy number of the duplicated region from chromosome 2 increased. The deletion of a 308-kb region from chromosome 4, resulting from translocation in the J4 and I-8 strains, may have caused the delay in growth. The growth rates of the APRT and 1546 K-APRT strains were similar to those of the RIB40 and J4 strains, respectively. However, the TLTA-APRT strain showed a a Relative gene expression in the J4 strain compared to that in the RIB40 strain was measured by gene expression array. p < 0.02 sixfold higher than that in the RIB40 strain; this indicates that two copies of alp were sufficient for protease activity when prtT was overexpressed. Takahashi et al. Fungal Biol Biotechnol (2018) 5:17 Page 11 of 17 Table 5 Upregulation of xylanolytic genes located outside of the duplicated region in the J4 strain Positively regulated by XlnR [20] a Relative gene expression in the J4 strain compared to that in the RIB40 strain was measured by gene expression array. duplication and triplication strains in solid‑state culture p < 0.02 Systematic name Relative genea expression Description AO090001000164 2.3 Protein with predicted nucleotide binding activity* AO090001000383 6.9 Extracellular catechol oxidase* AO090005000476 2.8 Beta-xylosidase AO090005000531 3.6 Endoglucanase B; predicted secretion signal peptide* AO090005000698 3.7 Beta-xylosidase* AO090010000063 5.5 Sugar transporter (MFS family); transcriptionally induced by growth on xylose* AO090011000141 3.6 Putative exoarabinase* AO090023000001 6.7 AbfB-alpha-L-arabinofuranosi- dase B AO090023000401 3.3 PglB-polygalacturonase B* AO090038000426 4.1 Dehydrogenases related to short-chain alcohol dehy- drogenases* AO090038000631 3.5 xdhA1-Xylitol dehydroge- nase* AO090103000087 3.0 Putative endoglucanase precursor* AO090103000268 6.2 Beta-xylosidase* AO090103000326 2.5 Beta-1,4-xylanase AO090103000423 2.8 XynF1, beta-1,4-xylanase* AO090103000426 4.0 Shikimate 5-dehydrogenase* AO090124000023 3.1 AbfA,;alpha-L-arabinofura- nosidase AO090701000885 3.1 As Abf-alpha-L-arabinofura- nosidase* AO090001000649 3.4 Alpha-glucosidases, family 31 of glycosyl hydrolases* AO090011000140 3.6 Beta-glucosidase-related glycosidases* AO090011000715 2.7 Ak eglA-Endoglucanase AO090023000056 2.1 Endoglucanase B AO090038000175 2.5 Endoglucanase Table 6 Upregulation of proteolytic, amylolytic, and xylanolytic genes, and expression of their respective regulatory genes, located in the duplicated region of the J4 strain a Relative gene expression in the J4 strain compared to that in the RIB40 strain was measured by gene expression array. p < 0.02 Systematic name Relative genea expression Description AO090003001036 3.2 Alp, subtilisin-related protease AO090003001208 2.0 AmyR AO090003001209 2.5 AgdA, maltase glucoamylase and related hydrolases, glycosyl hydrolase family 31 AO090003001211 5.1 prtT AO090003001258 2.4 Peptidase family M48 AO090003001292 3.0 XlnR2 AO090003001305 2.7 Alpha-D-galactosidase (melibiase) AO090003001341 8.3 Endoglucanase AO090003001497 2.1 Glycosidases AO090003001511 2.3 Beta-glucosidase-related glycosi- dases Table 5 Upregulation of xylanolytic genes located outside of the duplicated region in the J4 strain Table 6 Upregulation of proteolytic, amylolytic, and xylanolytic genes, and expression of their respective regulatory genes, located in the duplicated region of the J4 strain a Relative gene expression in the J4 strain compared to that in the RIB40 strain was measured by gene expression array. p < 0.02 b Data are expressed as mean ± SD, and all experiments were conducted in triplicate Discussion In this study, we generated strains of A. oryzae in which a targeted chromosomal region was translocated and duplicated. CGH and PCR were used to confirm that targeted translocation indeed occurred. Because CGH is commonly used to detect chromosomal rearrangement in yeast. It was confirmed that the original genome of A. oryzae did not contain an I-SceI recognition sequence [17], indicating a low possibility of unexpected chromo- somal rearrangement. g The strain with the translocated duplication of the targeted chromosomal region was obtained by treat- ing protoplast cells with the I-SceI enzyme. No strains containing translocated duplication were generated in the absence of I-SceI, indicating that translocated chro- mosomal duplication in A. oryzae requires artificially introduced DSBs and depends on the break-induced replication mechanism. We previously obtained tandem chromosomal duplications from protoplasted cells with- out I-SceI-derived chromosomal DSBs in A. oryzae [12] and hypothesized that the tandem chromosomal duplica- tions were generated by a nonallelic homologous recom- bination mechanism instead of classical unequal sister chromatid exchange in diploid budding yeast [23]. In addition, the time required for regenerating cells and fre- quency of regenerated colonies differed between tandem and translocated chromosomal duplications in A. oryzae. Specifically, tandem chromosomal duplications were pro- duced after incubating protoplast cells for 5–7 days on regeneration plates, and the frequency of regenerated a Relative gene expression in the J4 strain compared to that in the RIB40 strain was measured by gene expression array. p < 0.02 a Relative gene expression in the J4 strain compared to that in the RIB40 strain was measured by gene expression array. p < 0.02 severe delay in growth compared to that of the I-8 strain. As shown in Table 8, the expression of alp was extremely high in the TLTA-APRT strain compared to that in the APRT or 1546 K-APRT strains. The expression of alp was originally high in the control strain (RIB40), suggesting that increased expression of alp caused growth delays in the TLTA-APRT strain. severe delay in growth compared to that of the I-8 strain. As shown in Table 8, the expression of alp was extremely high in the TLTA-APRT strain compared to that in the APRT or 1546 K-APRT strains. The expression of alp was originally high in the control strain (RIB40), suggesting that increased expression of alp caused growth delays in the TLTA-APRT strain. Takahashi et al. a Relative quantity of gene expression was measured by real-time PCR ean ± SD, and all experiments were conducted in triplicate a Relative quantity of gene expression was measured by real-time PCR Discussion Fungal Biol Biotechnol (2018) 5:17 Page 12 of 17 Fig. 5 Phenotypes of translocated duplication strains. a Enzyme activity in strains bearing chromosomal duplication under solid-state culture conditions. Total protease, alpha-amylase, and acid carboxypeptidase activity in solid-state cultures; RIB40 (control), J4 (the strain bearing translocated duplication of chromosome 2), I-8 (the strain bearing translocated triplication of chromosome 2), APRT (prtT overexpression strain), 1546K-APRT (the strain bearing translocated duplication of chromosome 2 with prtT overexpression), TLTA-APRT (the strain bearing translocated triplication of chromosome 2 with prtT overexpression), Ao-700k-dup (the strain bearing tandem duplication of a 700-kb region of chromosome 2), BP-B3 (the strain bearing tandem duplication of a 9-kb region of chromosome 2). b Growth phenotypes of strains bearing chromosomal duplication cultured on CZ plates. The strains were inoculated onto 1.2 M sorbitol CZ plates and incubated at 30 °C for 7 days Fig. 5 Phenotypes of translocated duplication strains. a Enzyme activity in strains bearing chromosomal duplication under solid-state culture conditions. Total protease, alpha-amylase, and acid carboxypeptidase activity in solid-state cultures; RIB40 (control), J4 (the strain bearing translocated duplication of chromosome 2), I-8 (the strain bearing translocated triplication of chromosome 2), APRT (prtT overexpression strain), 1546K-APRT (the strain bearing translocated duplication of chromosome 2 with prtT overexpression), TLTA-APRT (the strain bearing translocated triplication of chromosome 2 with prtT overexpression), Ao-700k-dup (the strain bearing tandem duplication of a 700-kb region of chromosome 2), BP-B3 (the strain bearing tandem duplication of a 9-kb region of chromosome 2). b Growth phenotypes of strains bearing chromosomal duplication cultured on CZ plates. The strains were inoculated onto 1.2 M sorbitol CZ plates and incubated at 30 °C for 7 days Table 7 Quantitative expression of alp and prtT in strains bearing chromosomal duplication Strain alp prtT Relativea quantity (dR) Upperb error bars Lowerb error bars Relativea quantity (dR) Upperb error bars Lowerb error bars RIB40 1 0.58 0.37 1 0.26 0.21 J4 3.11 0.58 0.49 4.14 0.61 0.53 I-8 7.10 2.18 1.98 5.81 1.32 1.54 D2 2.84 1.16 0.71 4.28 1.75 1.08 BP-B3 1.84 1.47 0.70 2.19 1.67 0.92 Table 7 Quantitative expression of alp and prtT in strains bearing chromosomal duplication Table 7 Quantitative expression of alp and prtT in strains bearing chromosomal duplication Takahashi et al. Discussion Fungal Biol Biotechnol (2018) 5:17 Page 13 of 17 Table 8 Quantitative expression of alp and prtT in prtT overexpression strains a Relative quantity of gene expression was measured by real-time PCR b Data are expressed as mean ± SD, and all experiments were conducted in triplicate Strain alp prtT Relativea quantity (dR) Upperb error bars Lowerb error bars Relativea quantity (dR) Upperb error bars Lowerb error bars RIB40 1 0.08 0.07 1 0.15 0.13 APRT 5.26 1.19 1.00 15.69 2.45 2.14 1546 K APRT 8.07 1.10 1.03 15.34 2.27 2.03 TLTA-APRT 17.31 1.52 1.35 18.33 2.34 2.05 Table 8 Quantitative expression of alp and prtT in prtT overexpression strains Relative quantity of gene expression was measured by real-time PCR b Data are expressed as mean ± SD, and all experiments were conducted in triplicate in the strains containing duplicated and triplicated chromosome regions as compared to that of the control strain (Tables 1 and 2). Thus, the transcription levels of genes in the duplicated chromosomal regions were pre- dominantly affected, and the resulting phenotypes were enhanced as the copy number increased. This suggests that segmental chromosomal duplication can be used to identify unknown gene functions based on phenotypic observations.i colonies from the protoplast cells was approximately 10−7 [12]. In contrast, strains with translocated chromo- somal duplications were generated only after protoplast cell regeneration was conducted for more than 2 weeks, and the frequency of regenerated colonies from the pro- toplasted cell was approximately 10−8. To obtain trans- located chromosomal triplications in regenerated state, nearly 3 weeks were necessary to regenerate the colonies, and most regenerated cells were heterokaryons con- taining both the original chromosome and translocated chromosome (Additional file 1: Figure S4). Hence, in the present study, single-colony isolation was necessary to obtain a pure translocated chromosomal triplication strain, indicating that more time is required for recom- bination to form translocated chromosomal regions in A. oryzae. These results indicate that tandem duplication of a targeted chromosomal region achieved in our pre- vious study [12], and translocated duplication of a tar- geted chromosomal region achieved in the present study, depend on completely different recombination mecha- nisms and techniques. Protease activity was used to confirm the duplication stability of chromosome 2. Genome stability of A. oryzae strains in which translocated duplication was generated by mutagenesis was described in a United States patent US8900647B2. Discussion The genomes of the strains bearing trans- located duplication were stable after 10 generation of subculture. Additionally, in our previous report [12], we showed that targeted tandem duplication of chromosome 2 remained stable after 5 rounds of subculture. Translo- cated duplication is more stable than tandem duplication because the possibility of losing the duplicated region via recombination is low. Translocated duplication strains were generated from both ku70 + and ku70- strains; the BN1-1 strain was derived from ku70 + and J4 strain was derived from ku70− (Table 9). This suggests that non-homologous end-joining pathways involving Ku70/80 heterodimers are not important in the mechanism of translocated duplication. As shown previously, undesirable deletions or illegitimate recombinations were not observed in the combination of the Δku70 strain and 5FOA counter selection [17]. This indicates that the Δku70 strain of A. oryzae is suitable for pyrG-mediated targeted chromo- somal duplication used previously [12] and in our pre- sent study. Therefore, it is possible to identify interesting phenotypes by constructing a chromosome duplication strain library that covers the whole genome and to detect unknown regulatory genes by screening and analyzing the library in A. oryzae. In the present study, we generated duplication and trip- lication strains of A. oryzae chromosome 2 including alp and prtT. The strains bearing duplicated and triplicated chromosome 2 showed significant increases in the activ- ity of protease under solid-state culture conditions. However, protease activity in the BP-B3 strain (gen- erated using tandem duplication of the 9-kb region, which included alp but not prtT) was less than those of the J4 and 700k-dup strains (generated using duplica- tion of a chromosomal region containing both alp and prtT) (Fig. 5a). This indicates that duplication of both alp and prtT, or a structural gene and its regulatory gene, enhances the activity of protease. The duplicated region of chromosome 2 included the alpha-amylase gene and amyR, suggesting that amylase activity in the strains was enhanced via similar mechanism. Oligonucleotide microarrays revealed increased gene expression with increasing amounts of genetic material Page 14 of 17 Takahashi et al. Fungal Biol Biotechnol (2018) 5:17 Table 9 Strains used in the present study A. Conclusion In this study, we achieved translocated chromosomal duplication and triplication of a 1.4-Mb targeted chro- mosomal region by directly introducing I-SceI mega- nuclease into A. oryzae protoplast cells. Strains with duplication and triplication of chromosome 2 showed substantial increases in the activity of protease and amyl- ase. Gene dosage effects were enhanced by combining the structural gene and its regulatory gene, indicating that segmental duplications of chromosomes play impor- tant phenotypic roles in koji mold strains. Discussion oryzae RIB40 strain Genotype Source or reference RIB40 Wild type Ku70RC7-2 Δku70, ΔpyrG [10] RP-1 ΔpyrG [7, 8] Ao-700k-dup Tandem duplication of 700kb region of chromosome 2 [12] B1036p5′ΔCTp3′Δ 5′ΔpyrG, 3′ΔpyrG (parental strain of BN1-1) Present study ku70-B1036p5′ΔCTp3′Δ Δku70, 5′ΔpyrG, 3′ΔpyrG (parental strain of J4) Present study BN1-1 Translocated duplication of 1.4 Mb region of chromosome 2 to chromosome 4 Present study J4 Δku70, translocated duplication of 1.4 Mb region of chromosome 2 to chromosome 4 Present study del1546K4 Δku70, translocated duplication of 1.4 Mb of region chromosome 2 to chromosome 4, ΔpyrG Present study K1-IF5-2-5FOA Δku70, translocated duplication of 1.4 Mb region of chromosome 2 to chromosome 4, 5′ΔpyrG, 3′ΔpyrG (parental strain of I-8) Present study I-8 Δku70, translocated duplication of 1.4 Mb region of chromosome 2 to chromosome 4 and chromo- some 7 Present study TLTAs11B Δku70, translocated duplication of 1.4 Mb region of chromosome 2 to chromosome 4 and chromo- some 7, ΔpyrG Present study APRT pAPRT Present study 1546 K-APRT Δku70, translocated duplication of 1.4 Mb region of chromosome 2 to chromosome 4, pAPRT Present study TLTA-APRT Δku70, translocated duplication of 1.4 Mb region of chromosome 2 to chromosome 4 and chromo- some 7, pAPRT Present study BP-B3 Tandem duplication of 9 kb region of chromosome 2 Present study Table 9 Strains used in the present study Source or reference 30 min in a solution containing 0.05 mL of 1.2 M sorbitol, 0.02 mL of PEG, and 50 U of I-SceI endonuclease (New England Biolabs, Ipswich, MA, USA). After the addition of 0.07 mL PEG, the cells were incubated at room tem- perature for 1 h. Then, the cells were spread on regenera- tion plates containing 1.2 M sorbitol-CZ and incubated at 30 °C for 2–3 weeks. Regenerated colonies on regenera- tion plates were transferred to CZ plates for further anal- ysis. Information on the genome of Aspergillus oryzae was collected from the genome database at the National Institute of Technology and Evaluation (http://www.bio. nite.g.o.jp/dogan/Top) and AspGD (http://www.asper gillusgenome.org). All oligonucleotide primers used in this study are listed in Additional file 1: Table S1. Strains, media, and transformation Strains, media, and transformation Host strains included A. oryzae RIB40 (ATCC 42149), A. oryzae RP-1(ΔpyrG) [7, 8], and A. oryzae Ku70RC7-2 (Δku70, ΔpyrG) [10]. Positive selection of pyrG-defi- cient strains was performed on Czapek-Dox minimum medium (CZ) plates containing 20 mM uridine and 1.5 mg/mL 5-fluoroorotic acid (5-FOA). Solid-state cul- tivation was performed using medium containing 20% wheat bran and 80% water (wt/wt). Transformation of Aspergillus strains using protoplasts and PEG was con- ducted as previously described [17, 24]. Strains bearing translocated chromosomal duplication were obtained as follows. Approximately 1 × 108 protoplasts were pre- pared from the host strain and maintained on ice for DNA techniques and expression analysis The resulting fragments were treated with DpnI and ligated into the vector using an In-Fusion Cloning Kits (TaKaRa) to obtain pCT166pyr3ʹd. The fragment for removing pyrG from the J4 strain was constructed as follows: a 2-kb amplification fragment and a 2.3-kb fragment were amplified from RIB40 genomic DNA using the primer pairs TL-B5626L and TL-B3616U-IF, and TL-ct3685L- IF and TL-ct1388U, respectively. Fragments were ligated using In-fusion cloning kits (TaKaRa) to obtain the vec- tor for removing pyrG from the J4 strain. To construct the vector for integrating 3′ΔpyrG at the S11 locus of chromosome 7, a 1.3-kb fragment of genomic DNA from the RIB40 strain was amplified using the primers S11-U and S11-L. This fragment was cloned using a TA clon- ing kit (TOYOBO) to prepare pS11T. pS11T was ampli- fied via PCR using the primers S11-IFU and S11-IFL. The basic unit of 3′ΔpyrG, BP1130-I, was amplified using the primers P363U and P3297L. The resulting fragments were treated with DpnI and ligated into the vector using In-Fusion Cloning Kits (TaKaRa) to obtain pS11pyr3ʹd. The fragment for removing pyrG from the I-8 strain was constructed as follows: a 2.8-kb fragment was ampli- fied using the primers TL-B3616U-IF-S11 and B6217L, and a 2.7-kb fragment was amplified using the primers TL-S11-14389U-IF-B and S11-17060L. The two PCR products were then ligated using In-Fusion Cloning Kits (TaKaRa) to obtain the vector for removing pyrG from the I-8 strain. Details of pyrG removal using homologous fragments were described previously [10]. Construction of vector used for partial deletion of pyrG Construction of vectors used to create the 5′ΔpyrG and 3′ΔpyrG constructs was carried out in a manner similar to a method described previously [12]. The schematic of vector construction is shown in Additional file 1: Fig- ure S6. The 5′ΔpyrG construct was pyrG-truncated by removing 631 bp from the 5′ end of pyrG, which included the promoter region. The 3′ΔpyrG construct was pyrG- truncated by removing 1236 bp from the 3′ end of pyrG, which included the terminator region. A 462-bp consen- sus region was present between 5′ΔpyrG and 3′ΔpyrG, and 3′ΔpyrG included an I-SceI site in the consensus region. To construct the 5′ΔpyrG vector, a 5′ΔpyrG vec- tor backbone was constructed using the primers P499L, P509U, P1130U-IF5, and P1592L-IF5. DNA techniques and expression analysis The resulting fragments were treated with DpnI and ligated into the vector using In-Fusion Cloning Kits (TaKaRa) to obtain pS11pyr3ʹd. The fragment for removing pyrG from the I-8 strain was constructed as follows: a 2.8-kb fragment was ampli- fied using the primers TL-B3616U-IF-S11 and B6217L, and a 2.7-kb fragment was amplified using the primers TL-S11-14389U-IF-B and S11-17060L. The two PCR products were then ligated using In-Fusion Cloning Kits (TaKaRa) to obtain the vector for removing pyrG from the I-8 strain. Details of pyrG removal using homologous fragments were described previously [10]. Construction of parental strain for strains bearing translocated chromosomal duplication PCR amplification was performed using TaKaRa Ex Taq DNA polymerase (TaKaRa, Shiga, Japan) in a T100 thermal cycler (Bio-Rad, Hercules, CA, USA). Quantita- tive RT-PCR was performed in a MxPro3000P (Agilent Technologies, Santa Clara, CA, USA) using PrimeScript™ RT reagent with gDNA Eraser (Perfect Real Time) and SYBR® Premix Ex Taq™ II (Tli RNase H Plus) (TaKaRa). The expression of alp and prtT were analyzed by com- parative quantification using MxPro software version 4.10 (Agilent Technologies). AO090005000807 (ortholog of transcription factor TFIID) was used as normalizer, and primers are listed in Table S2. Oligonucleotide arrays were purchased from Agilent Technologies; the experi- mental protocol was detailed in a previous study [17, 25]. The CGH and gene expression array data obtained in the present study have been deposited into the NCBI Gene Expression Omnibus [26] and are accessible using GEO Series accession number GSE120604. Then, a 5.8 kb fragment was amplified from the pPB9 plasmid [12] using the primer pair P2788L/P2828U, and a 0.5 kb-fragment was amplified from the pBP9-sceI (a pBP9-based vector bearing pyrG, including the I-SceI recognition site) [12] using the primer pair P1130U-IF3/ P1592-IF3. The fragments were then ligated using an In- Fusion cloning kit (TaKaRa), and the basic construct for 3′ΔpyrG BP1130-I was generated. To construct the vec- tor for integrating 3′ΔpyrG at the CT166 locus of chro- mosome 4, a 3-kb fragment of genomic DNA from the RIB40 strain was amplified using the primers ct166-U and ct166-L. This fragment was cloned using a TA clon- ing kit (TOYOBO) to prepare pCT166T. pCT166T was amplified via PCR using the primers ct166-IFU and ct166-IFL. The basic unit of 3′ΔpyrG, BP1130-I, was amplified using the primers P363U and P3297L. DNA techniques and expression analysis DNA techniques and expression analysis Genomic DNA from Aspergillus strains was extracted as described previously [24]. RNA samples were pre- pared from mycelia that were inoculated on wheat bran medium and cultivated at 30 °C for 65 h. After cultiva- tion, the mycelia were harvested and ground in liquid nitrogen using a mortar and pestle; RNA was extracted using Isogen reagent (Nippon Gene, Toyama, Japan). Fur- ther purification was performed using RNeasy Mini kits (Qiagen, Hilden, Germany) according to the manufac- turer’s instructions. RNA was used in quantitative real- time PCR (RT-PCR) and gene expression microarrays. Takahashi et al. Fungal Biol Biotechnol (2018) 5:17 Page 15 of 17 Then, a 5.8-kb fragment was amplified from the pPB9 plasmid [12] using the primer pair P2788L/P2828U, and a 0.5 kb-fragment was amplified from the pBP9-sceI (a pBP9-based vector bearing pyrG, including the I-SceI recognition site) [12] using the primer pair P1130U-IF3/ P1592-IF3. The fragments were then ligated using an In- Fusion cloning kit (TaKaRa), and the basic construct for 3′ΔpyrG BP1130-I was generated. To construct the vec- tor for integrating 3′ΔpyrG at the CT166 locus of chro- mosome 4, a 3-kb fragment of genomic DNA from the RIB40 strain was amplified using the primers ct166-U and ct166-L. This fragment was cloned using a TA clon- ing kit (TOYOBO) to prepare pCT166T. pCT166T was amplified via PCR using the primers ct166-IFU and ct166-IFL. The basic unit of 3′ΔpyrG, BP1130-I, was amplified using the primers P363U and P3297L. The resulting fragments were treated with DpnI and ligated into the vector using an In-Fusion Cloning Kits (TaKaRa) to obtain pCT166pyr3ʹd. The fragment for removing pyrG from the J4 strain was constructed as follows: a 2-kb amplification fragment and a 2.3-kb fragment were amplified from RIB40 genomic DNA using the primer pairs TL-B5626L and TL-B3616U-IF, and TL-ct3685L- IF and TL-ct1388U, respectively. Fragments were ligated using In-fusion cloning kits (TaKaRa) to obtain the vec- tor for removing pyrG from the J4 strain. To construct the vector for integrating 3′ΔpyrG at the S11 locus of chromosome 7, a 1.3-kb fragment of genomic DNA from the RIB40 strain was amplified using the primers S11-U and S11-L. This fragment was cloned using a TA clon- ing kit (TOYOBO) to prepare pS11T. pS11T was ampli- fied via PCR using the primers S11-IFU and S11-IFL. The basic unit of 3′ΔpyrG, BP1130-I, was amplified using the primers P363U and P3297L. DNA techniques and expression analysis Then, 5.8- and 0.5-kb fragments were amplified via PCR from the pPB9 plasmid (a pUC-based plasmid bearing a 3.0-kb fragment containing pyrG) [12] using the primer pairs P499L/ P509U and P1130U-IF5/P1592L-IF5. The fragments were then ligated using an In-Fusion cloning kit (TaKaRa), and the basic construct for 5′ΔpyrG BP1130 was generated. To construct a vector for integrating 5′ΔpyrG at the B1036 site of chromosome 2, a 3-kb fragment was PCR-ampli- fied from the genome of the RIB40 strain using primers B1036-U and B1036-L. Cloning of this fragment using a TA cloning kit (TOYOBO, Osaka, Japan) produced pB1036T, and pB1036T and BP1130 were PCR-amplified using the primers B1036-IFU and B1036IFL, and P363U and P3297L, respectively. The resulting two fragments were treated with DpnI and ligated using In-Fusion Clon- ing Kits (TaKaRa) to generate the pB1036pyr5′d vector. To construct the 3′ΔpyrG vector including the I-SceI site, a 3′ΔpyrG vector backbone was constructed using the primers P2788L, P2828U, P1130U-IF3, and P1592L-IF3. Construction of parental strain for strains bearing translocated chromosomal duplication To construct parental strains for generating strains with translocated chromosomal duplication, RP-1 (ΔpyrG) and ku70RC7-2 (Δku70, ΔpyrG) were transformed with pB1036pyr5′d. After confirming vector integration at target sites, transformants were subjected to selection Takahashi et al. Fungal Biol Biotechnol (2018) 5:17 Page 16 of 17 Page 16 of 17 Solid‑state cultivation and measurements of enzyme activity Solid‑state cultivation and measurements of enzyme activity Solid‑state cultivation and measurements of enzyme activity using 5-FOA, and strains with 5′ΔpyrG at the target site of chromosome 2 were isolated (Additional file 1: Figure S6B). The strains B1036pyrG5′Δ and ku70-B1036pyrG5′Δ were transformed with pCT166pyr3′d. Strains with pCT166pyr3′d integrated at the target site were selected using 5-FOA to obtain parental strains for generating the strains B1036p5′ΔCTp3′Δ and ku70-B1036p5′ΔCTp3′Δ containing translocated chromosomal duplication (Addi- tional file 1: Figure S6C). Construction of parental strain for translocated triplication was conducted as follows. The del1546 K strain, obtained by removing pyrG from chromosome 4 of the J4 strain (Additional file 1: Figure S4), was transformed with pS11pyr3ʹd. After confirming vector integration of chromosome 7, transformants were subjected to selection using 5-FOA. The strains with 3′ΔpyrG at the target site of chromosome 7 (K1-IF5-2- 5FOA) were isolated (Additional file 1: Figure S4). Solid-state cultures were generated by inoculating 1 × 108 conidiospores into 5 g of wheat bran medium in a 150-mL Erlenmeyer flask and incubating at 30 °C for 4 days. Water (50 mL) was then added to the flask and extracted after shaking. The liquid fraction was filtered through filter paper and used as sample extract. Pro- tease activity was determined by mixing the substrate and 2% milk casein (pH 7) with sample extracts and incubating at 30 °C for 20 min. Reactions were stopped using trichloroacetic acid, and quantities of liberated amino acids were measured at 660 nm using a tyrosine standard. One unit (U) was defined as the amount of enzyme yielding 1 µg of tyrosine per min at 30 °C and at pH 7. The activity of alpha-amylase was determined using Alpha-Amylase Activity kits (Kikkoman Bio- chemifa, Minato-ku, Japan) according to the manufac- turer’s instructions. Briefly, reaction buffer containing 100 mM acetate (pH 5), 2 mM 2-chloro-4 nitrophenyl 65-azido-65-deoxy-beta-maltopentaocid, 50 U/mL glu- coamylase, 6 U/mL beta-glucosidase, 50 mM NaCl, and 2 mM CaCl2 was mixed with 300 volumes of diluted sample extract and incubated for 5 min at 37 °C. The reactions were stopped using trichloroacetic acid, and liberated 2-chloro-4 nitrophenol contents were deter- mined at 400 nm. One unit (U) was defined as the amount of enzyme yielding 1 µmol of 2-chloro-4 nitro- phenol per min. Construction of prtT overexpression strainsh Construction of prtT overexpression strains The prtT overexpression vector pAPRT was constructed as follows. A 3.7 kb amyB fragment, amplified from RIB40 genome DNA using the primers amyU and amyl, was cloned by a TA cloning kit (TOYOBO) to generate pAmyTA. A 6.7-kb fragment, amplified from pAmyTA using the primer pair Amy118UIF/TA816UIF, and a 2.9-kb pyrG fragment amplified from pBP9 [12] using the primer pair P363U/P3229L, were fused using an In- Fusion-Cloning Kit (TaKaRa) to obtain pAmyPYR. A 7.6-kb fragment amplified from pAmyPYR, and a 2.1 kb fragment containing prtT ORF and amplified from RIB40 genomic DNA using the primer pair prtTstU/prtTteL, were fused using an In-Fusion-Cloning Kit (TaKaRa) to obtain the prtT overexpression vector pAPRT. The RP1, del1546K4, and TLTAS11B strains were transformed with pAPRT in a circular state to obtain the APRT, 1546 K-APRT, and TLTA-APRT strains, respectively. The copy numbers of the pAPRT vector integrated in the strains were estimated by quantitative PCR (Addi- tional file 1: Figure S7). The APRT and TLTA-APRT strains contained one copy of APRT. The 1546 K-APRT strain contained two copies of pAPRT. Determining the copy number of pAPRT in the strains via Southern blot was difficult. This is because A. oryzae strains originally contain three copies of amylase genes, and the duplicated chromosomal region contained prtT and amylase genes. The results of PCR suggested that one copy of pAPRT in the 1546 K-APRT strain was integrated at one of the amylase loci (data not shown). The other copy of pAPRT appeared to be randomly integrated in the strains. The activity of acid carboxypeptidase was determined using Acid Carboxypeptidase Assay kits (Kikkoman Bio- chemifa) according to the manufacturer’s instructions. The reaction buffer containing 50 mM acetate (pH 3), 0.5 mM Cbz-Tyr-Ala (carboxybenzoxy-L-tyrosyl-l-ala- nine), and 5 mM NAD was mixed with 30 volumes of diluted sample extract and incubated for 10 min at 37 °C. The reaction was stopped using 0.5 M Tris–HCl buffer (pH 8.5) containing 5 mM WST-8 and 13 U of alanine dehydrogenase, and the mixture was incubated at 37 °C for 20 min. Subsequently, 0.5 mM 1-methoxy-5-methyl- phenazinium methylsulfate was added to reaction mix- ture and incubated at 37 °C for 10 min. Quantities of liberated NADH were measured at 460 nm. 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https://openalex.org/W4223497809	https://arodes.hes-so.ch/record/10228/files/Monteiro_2022_ability_to_care.pdf	English	null	Ability to Care for an Ill Loved One During the First COVID-19 Lockdown: Mediators of Informal Caregivers’ Stress in Europe	Frontiers in psychiatry	2,022	cc-by	8,252	Edited by: Chung-Ying Lin, National Cheng Kung University, Taiwan 1 La Source, School of Nursing Sciences, HES-SO University of Applied Sciences and Arts Western Switzerland, Lausanne, Switzerland, 2 Aix Marseille Université, LPCPP, Aix-en-Provence, France, 3 AVASAD, Association Vaudoise d’Aide et de Soins à Domicile Route de Chavannes, Lausanne, Lausanne, Switzerland, 4 HESAV, School of Health Sciences, HES-SO University of Applied Sciences Western Switzerland, Switzerland, 5 Service de Psychiatrie Générale, Psychiatry Department, CHUV, Lausanne, Switzerland, 6 Pôle Centre Rive Gauche et Centre Ressource de Réhabilitation Psychosociale et de Remédiation Cognitive, Centre Hospitalier Le Vinatier, UMR 5229, CNRS, Université Claude Bernard Lyon 1, Lyon, France, 7 Département des Sciences de la Santé Publique et de la Motricité, Haute Ecole de la Province de Namur, Namur, Belgium, 8 Centre Neuro Psychiatrique St. Martin, Dave Namur, Belgium, 9 Community Psychiatry Service, Department of Psychiatry, Lausanne University Hospital and University of Lausanne, Lausanne, Switzerland Reviewed by: Chia-Wei Fan, AdventHealth University, United States Emma Berry, Queen’s University Belfast, United Kingdom Informal caregivers are overlooked, healthcare actors. They are at particular risk of distress and suffer from poor mental health. This study aimed to investigate the perceived stress and modulating factors during the ﬁrst COVID-19 lockdown in Europe, regardless of the illness that care recipients suffer from. Sociodemographic data, coping resources, and perceived stress level using the Perceived Stress Scale (PSS-10) questionnaire were assessed using a web-based survey in Switzerland, France, and Belgium with 232 informal caregivers. Mediation analyses were used to identify the factors that modulate stress. Higher perceived stress among informal caregivers was associated with a younger age for the care recipient, family relationship with the care recipient, cohabitation, and female sex of the informal caregiver. These associations were partially mediated by the fear of getting ill (age, cohabitation), the conviction that lockdowns had a negative impact on health (age, kinship), and the perceived deterioration of the care recipient’s health (gender). The fear of losing the ability to cope with caregiving tasks due to an illness (COVID-19 and/or other) and the negative impact of the lockdown on care recipients’ health, particularly on the mental health of young care recipients, increased the stress of informal caregivers. Our results emphasize the importance of informal caregiving support to prevent heightened stress in lockdown conditions, regardless of care recipient illness or kinship. Informal caregivers are overlooked, healthcare actors. Ability to Care for an Ill Loved One During the First COVID-19 Lockdown: Mediators of Informal Caregivers’ Stress in Europe Shadya Monteiro1†, Margot Fournier1†, Jérôme Favrod1, Anne-Laure Drainville1, Léa Plessis2, Sylvie Freudiger3, Krzysztof Skuza4, Charlene Tripalo5, Nicolas Franck6, Marie-Clotilde Lebas7, Jocelyn Deloyer8, Hélène Wilquin2, Philippe Golay9 and Shyhrete Rexhaj1* Keywords: informal caregiver, lockdown, COVID-19, perceived stress, mental health They are at particular risk of distress and suffer from poor mental health. This study aimed to investigate the perceived stress and modulating factors during the ﬁrst COVID-19 lockdown in Europe, regardless of the illness that care recipients suffer from. Sociodemographic data, coping resources, and perceived stress level using the Perceived Stress Scale (PSS-10) questionnaire were assessed using a web-based survey in Switzerland, France, and Belgium with 232 informal caregivers. Mediation analyses were used to identify the factors that modulate stress. Higher perceived stress among informal caregivers was associated with a younger age for the care recipient, family relationship with the care recipient, cohabitation, and female sex of the informal caregiver. These associations were partially mediated by the fear of getting ill (age, cohabitation), the conviction that lockdowns had a negative impact on health (age, kinship), and the perceived deterioration of the care recipient’s health (gender). The fear of losing the ability to cope with caregiving tasks due to an illness (COVID-19 and/or other) and the negative impact of the lockdown on care recipients’ health, particularly on the mental health of young care recipients, increased the stress of informal caregivers. Our results emphasize the importance of informal caregiving support to prevent heightened stress in lockdown conditions, regardless of care recipient illness or kinship. Correspondence: Shyhrete Rexhaj s.rexhaj@ecolelasource.ch †These authors share ﬁrst authorship Specialty section: This article was submitted to Public Mental Health, a section of the journal Frontiers in Psychiatry Received: 11 January 2022 Accepted: 08 March 2022 Published: 12 April 2022 Citation: Monteiro S, Fournier M, Favrod J, Drainville A-L, Plessis L, Freudiger S, Skuza K, Tripalo C, Franck N, Lebas M-C, Deloyer J, Wilquin H, Golay P and Rexhaj S (2022) Ability to Care for an Ill Loved One During the First COVID-19 Lockdown: Mediators of Informal Caregivers’ Stress in Europe. Front. Psychiatry 13:852712. doi: 10.3389/fpsyt.2022.852712 Specialty section: This article was submitted to Public Mental Health, a section of the journal Frontiers in Psychiatry Specialty section: This article was submitted to Public Mental Health, a section of the journal Frontiers in Psychiatry Received: 11 January 2022 Accepted: 08 March 2022 Published: 12 April 2022 Received: 11 January 2022 Accepted: 08 March 2022 Published: 12 April 2022 ORIGINAL RESEARCH published: 12 April 2022 doi: 10.3389/fpsyt.2022.852712 ORIGINAL RESEARCH published: 12 April 2022 doi: 10.3389/fpsyt.2022.852712 ORIGINAL RESEARCH published: 12 April 2022 doi: 10.3389/fpsyt.2022.852712 Design and Recruitment g This cohort study was an observational retrospective study targeting informal caregivers to assess their perceived stress, attitudes, and resources during the ﬁrst COVID-19 lockdown. Convenience sampling was used to collect data, and a link to the online survey was sent by the researchers to informal caregivers in the French-speaking part of Switzerland, France, and Belgium through various communication channels (e.g., social networks, family support associations, and clinical networks). Questionnaires about the lockdown were completed retrospectively at the end of the ﬁrst wave, between May and October 2020, and the survey was closed to focus on the early phase of the pandemic. To be included, participants had to be over 18 years old; live in Switzerland, France, or Belgium; and be an informal caregiver of at least one person. Self-identiﬁcation as an informal caregiver was chosen to make it possible for all informal caregivers to participate without attempting to control their involvement in direct care or support to the care recipient in the particular context of the pandemic. To be considered informal caregivers for the purpose of data analysis, participants were to provide valid information about at least three of the following four items concerning the care recipient: gender, age, illness duration, and diagnosis. No inclusion or exclusion criteria were applied to care recipients. Studies have reported the general eﬀects of pandemics on informal caregivers. Several studies have noted such psychological consequences as worry, stress, nervousness, and anxiety (19–23). The major concerns and stressors for informal caregivers are their own health and job loss. Being ill would impact greatly aﬀects care and the possibility of transmitting COVID-19 (24). The fear of infecting their loved ones and the anticipated guilt should this happen have even led to greater avoidance of healthcare facilities among the informal caregiver population, be it for medical appointments or emergency care for themselves or their relatives (19). Moreover, informal caregivers were burdened by a new need to make decisions related to the health of their ill loved ones, on issues, such as symptom management or palliative care, to name but a few (21, 25). Some studies have also shown that informal caregivers can suﬀer as a result of their socioeconomic situations (18). The risk of contracting the virus at work or losing income could aﬀect the level of care the ill person receives (18, 21, 26). INTRODUCTION factors (28). Perceived stress refers to an individual’s feelings or thoughts about the levels of stress that they experience at a given time or over a given period of time (29). The Perceived Stress Scale (PSS) developed by Cohen and Williamson evaluates whether the person feels able to cope with the event (28) and how the person perceives his or her control of the event (30). This study aimed to report the perceived stress of informal caregivers and the factors mediating this stress during the ﬁrst COVID-19 lockdowns in Switzerland, France, and Belgium. We reported sociodemographic factors associated with perceived stress and identify mediators that modulate these associations, thus providing actionable points to alleviate stress among informal caregivers. In March 2020, the World Health Organization (WHO) declared the new coronavirus (COVID-19) outbreak to be a pandemic and called on countries to take action for population health and health services (1, 2). To reduce the risk of contagion, many European countries imposed containment measures during the coronavirus pandemic. Care consultations, day hospitals, and community facilities have quickly implemented strategies to protect patients from infection while providing routine care, such as teleconsultations (3, 4). However, restrictions on liberty, quarantine, and isolation have substantial, long-lasting, negative psychological impacts (5), potentially aﬀecting more severely vulnerable populations. Informal caregivers help family members, friends, or neighbors to cope with disabilities or long- term illnesses and usually play an essential role in patient support. On the basis of studies in this domain, the term “informal caregiver” is applied to anyone who believes they have a caregiver role (6). Studies have shown that this role aﬀects the quality of life, well-being, and mental health (7–11). The detrimental eﬀect of poor informal caregiver health is twofold, as it also aﬀects care recipients’ health (12, 13). During the ﬁrst wave of the COVID-19 pandemic, informal caregivers were more often requested to (14– 16) provide support without being able to rely on their speciﬁc usual support (17) due to a deterioration in the condition of their loved ones or because of the cancelation of certain healthcare services (18). Design and Recruitment The various consequences studied in the current scientiﬁc literature do not specify the perceived stress associated with how informal caregivers experience lockdown. However, it is important to clarify this link under high-pressure conditions to promote eﬀective coping strategies for informal caregivers. Frontiers in Psychiatry \| www.frontiersin.org Citation: Monteiro S, Fournier M, Favrod J, Drainville A-L, Plessis L, Freudiger S, Skuza K, Tripalo C, Franck N, Lebas M-C, Deloyer J, Wilquin H, Golay P and Rexhaj S (2022) Ability to Care for an Ill Loved One During the First COVID-19 Lockdown: Mediators of Informal Caregivers’ Stress in Europe. April 2022 \| Volume 13 \| Article 852712 Frontiers in Psychiatry \| www.frontiersin.org 1 Mediators of Informal Caregivers’ Stress Monteiro et al. Perceived Stress Scale The survey assessed the informal caregivers’ experience of the lockdowns, their attitudes toward the care recipient, their resources to cope with containment, including social support, their economic situation, their health, and their perceptions of information provided on the virus (refer to Table 2 and The PSS-10 comprises 10 items to evaluate the frequency of stress during a deﬁned period using a scale ranging from “never” to “very often” (28). We used the validated French version of the PSS-10 (33, 34). Wording was adjusted to refer to COVID-19 lockdowns as stress events and to assess TABLE 1 \| Sociodemographic variables and their association with perceived stress. Instruments Online Survey The web-based survey is a self-report questionnaire collecting three types of data, i.e., (i) sociodemographic data; (ii) level of perceived stress during the COVID-19 lockdown; and (iii) attitudes and resources during the ﬁrst containment, which corresponds to factors that may modulate perceived stress. The survey was adapted from a study assessing the impact of containment measures in the general French population (31, 32). It was adjusted by the researchers to match the socio- health speciﬁcities of the three targeted countries: Switzerland, France, and Belgium. In this study, lockdowns are considered stress-triggering events as deﬁned by Lazarus and Folkman (27): the person ﬁrst interprets whether the stressor is a threat; should that be the case, the person evaluates the available strategies to cope with the event (27). The impact of a particular event on one’s health is not solely determined by the event’s inherent intensity; it depends on perception and personal and contextual The REDCap web application was used to build the survey and collect data anonymously. April 2022 \| Volume 13 \| Article 852712 2 Mediators of Informal Caregivers’ Stress Monteiro et al. Sociodemographic Data perceived stress during the previous month. The PSS-10 score was calculated as previously described (33). Participants with more than one missing item were removed from the PSS-10 questionnaire. Remaining missing data were imputed using the corresponding median value. Three categories of sociodemographic data can be distinguished (Table 1), i.e., (i) speciﬁc information related to the informal caregiver, (ii) housing conditions, and (iii) speciﬁc information related to the care recipient as provided by the informal caregiver. Values of the corresponding statistical test (degree of freedom) are displayed with the p-values. Signiﬁcant tests are indicated in bold. p-value < 0.05; *p-value < 0.01. test (degree of freedom) are displayed with the p-values. Signiﬁcant tests are indicated in bold. p-value < 0.05; *p-value < 0.01. Perceived Stress Scale TABLE 2 \| List of attitudes and resources of informal caregivers and their association with PSS-10 scores. TABLE 2 \| List of attitudes and resources of informal caregivers and their association with PSS-10 scores. Support to care recipient (during containment) Frequency of face-to-face interaction σ = 0.163 p = 0.014 Frequency of phone calls σ = −0.060 p = 0.417 Frequency of interaction by text σ = 0.072 p = 0.342 Disruption of therapeutic follow-up t(227) = −0.932 p = 0.352 *Perceived deterioration of care recipient’s health t(199) = −3.875 p = 1.45 × 10−4 Resources to cope with containment Support available t(190) = −0.109 p = 0.913 Words of relatives and friends t(230) = –0.632 p = 0.528 Belief in a positive outcome t(230) = 2.517 p = 0.013 Knowledge and scientiﬁc progress t(151) = 2.129 p = 0.035 Experience, ability to face difﬁculties t(220) = −0.783 p = 0.434 Community actions and support t(230) = 0.726 p = 0.469 Possible positive impact on the planet t(230) = 2.99 p = 0.003 Possible positive impact at the individual level t(230) = 4.293 p = 2.6 × 10−5 Personal economic situation (during containment) Negative impact of containment on budget σ = 0.201 p = 0.002 Health Fear of falling ill F(3,228) = 10.520 p = 2 × 10−6 Concerns about the access to masks, gel, etc. σ = 0.179 p = 0.006 *Negative impact of containment on health σ = 0.622 p = 4,5 × 10−26 Clarity of COVID-19-related information r = −0.032 p = 0.625 Values of the corresponding statistical test (degree of freedom) are displayed with the p-values. Signiﬁcant tests are indicated in bold. p-value < 0.05; *p-value < 0.005. Values of the corresponding statistical test (degree of freedom) are displayed with the p-values. Signiﬁcant tests are indicated in bold. p-value < 0.05; p-value < 0.005. In the second step, mediation analyses for PSS-10 scores were performed to explore whether the relationship between perceived stress and the previously identiﬁed signiﬁcant sociodemographic variables was mediated by other variables. Models were estimated separately for each previously identiﬁed potential mediator; for each model, the dependent variable was the PSS-10 score, and the independent variable was one of the sociodemographic variables. Perceived Stress Scale For the nominal variables “link to the care recipient” and “fear of getting ill,” we dichotomized the variables according to the best multinomial model using the exact likelihood with a uniform prior on all parameters (35). Supplementary Material). Responses were measured using a 4- or 5-point Likert scale. These factors potentially modulate perceived stress, and we tested whether they mediated the association between stress and sociodemographic variables. Statistical Analyses The database (n = 250 entries) was processed as follows: (i) removal of cases not providing information on care recipients (n = 10); (ii) removal of duplicates (n = 4); (iii) exclusion of persons living neither in Switzerland, France, nor Belgium (n = 3); and (iv) removal of participants with > 1 missing item in the PSS-10 questionnaire (n = 1). Remaining missing data were imputed using the corresponding median value. For categorical sociodemographic variables, categories corresponding to less than 4% of the cohort were dropped (i.e., “other” for sex) or pooled (i.e., marital status, educational level, employment status, link to care recipient, and class of disorder). For potential mediators, we selected the clinically most meaningful in relation to stress and the cohort (e.g., more than 20% of the subjects answered at least two diﬀerent items). Complete mediation was present when the path between PSS- 10 scores and sociodemographic variables (i.e., the direct eﬀect) was no longer signiﬁcant after introduction of the mediator and the indirect eﬀect was signiﬁcant; partial mediation was present when the direct and indirect eﬀects were statistically signiﬁcant. Conﬁdence intervals were estimated using bias- corrected bootstrapping with 1,000 draws. Mediation analyses were performed using Jamovi version 1.6.15 (36, 37). Ethical Considerations In the ﬁrst step, the associations between perceived stress (PSS-10 score) and sociodemographic variables, on the one hand (Table 1), and potential mediators (Table 2), on the other, were assessed. For continuous variables (e.g., age), we tested the association with the PSS-10 score using Pearson’s correlation; for ordinal variables (e.g., education level), we tested the association with PSS-10 score using Spearman’s correlation; for categorical variables, we compared PSS-10 scores between groups using either Student’s t-test in the case of two groups (e.g., sex) or one-way ANOVA in the case of three groups or more (e.g., marital status). The tests were performed using SPSS version 25. The research protocol was approved by the local ethics committee of Switzerland (Commission cantonale d’éthique de la recherche sur l’être humain [CER-VD]), a member of Swissethics. Frontiers in Psychiatry \| www.frontiersin.org Perceived Stress Scale Variable name Categories Mean ± sd [range]or n (%) PSS-10 score Test (d.f) p-value Informal caregivers, n = 232 Age 54.0 ± 13.9 [19–87] r = −0.178 p = 0.007 *Sex female 184 (79%) 18.8 ± 0.5 t(228) = 2.995 p = 0.003 male 46 (20%) 15.2 ± 1.1 other 2 (1%) Marital status married, partnership 138 (59%) 17.9 ± 0.6 F(2,231) = 1.011 p = 0.366 single 49 (21%) 19.5 ± 1.2 separated, widowed 45 (19%) 17.7 ± 1.1 Country Switzerland 129 (56%) 18.0 ± 0.7 F(2,231) = 0.192 p = 0.826 France 73 (31%) 18.6 ± 0.9 Belgium 30 (13%) 17.8 ± 1.4 Education level Compulsory school 9 (4%) 19.7 ± 7.0 σ = 0.005 p = 0.940 Secondary level 50 (22%) 17.7 ± 7.0 Tertiary level 173 (75%) 18.2 ± 7.7 Employment status employed 122 (53%) 19.0 ± 0.7 F(2,222) = 3.148 p = 0.045 retired, inactive 88 (38%) 16.6 ± 0.8 other 22 (9%) 19.8 ± 1.6 Occupational rate 81 ± 23 [15–100%] r = −0.041 p = 0.653 Link to the care recipient parent 71 (31%) 19.8 ± 0.8 F(5,231) = 3.361 p = 0.006 spouse 49 (21%) 17.6 ± 1.0 offspring 47 (20%) 18.6 ± 1.1 sibling 40 (17%) 18.2 ± 1.2 relative 13 (6%) 16.3 ± 2.4 other 12 (5%) 10.9 ± 1.8 Housing conditions N of cohabitants r = 0.101 p = 0.126 Living with the care recipient no 138 (59%) 16.7 ± 0.6 t(230) = –3.831 p < 0.001 yes 94 (41%) 20.4 ± 0.7 Care-recipient *Age 54.0 ± 25.1 [7–99] r = –0.212 p = 0.001 Sex female 94 (41%) 17.7 ± 0.8 t(227) = –0.691 p = 0.490 male 135 (58%) 18.4 ± 0.6 other 3 (1%) Illness duration 11.0 ± 9.2 [1–49] r = 0.042 p = 0.536 Class of disorder psychiatric 104 (45%) 18.6 ± 0.7 F(2,231) = 0.822 p = 0.512 neurological 12 (5%) 19.0 ± 2.1 physiological 28 (12%) 18.7 ± 1.5 other cases 31 (13%) 15.9 ± 1.5 >1 condition 57 (25%) 18.3 ± 1.0 Values of the corresponding statistical test (degree of freedom) are displayed with the p-values. Signiﬁcant tests are indicated in bold. p-value < 0.05; *p-value < 0.01. TABLE 1 \| Sociodemographic variables and their association with perceived stress. Frontiers in Psychiatry \| www.frontiersin.org April 2022 \| Volume 13 \| Article 852712 3 Mediators of Informal Caregivers’ Stress Monteiro et al. Association Between Perceived Stress and Sociodemographic Variables In summary, six variables, such as perception of care recipient’s health deterioration, possible positive impact on the planet, possible positive impact at the individual level, fear of falling ill (dichotomized), negative impact of containment on one’s health, and negative impact of containment on one’s budget, which were related to resources or attitude were retained for subsequent analyses. For the 14 sociodemographic variables (Table 1), we assessed the association with the level of perceived stress using the PSS-10. Six variables were robustly associated with the PSS-10, such as age of informal caregiver, sex of informal caregiver, employment status of informal caregiver, relationship with the care recipient, living with the care recipient, and age of care recipient. Informal caregivers had higher PSS-10 scores when their care recipients were younger, when they were relatives of the care recipient, or when they lived with him or her. Retired or inactive participants had lower PSS-10 scores than other occupational groups. Demographics Study participants were informal caregivers (n = 232) who were recruited in Switzerland (56%), France (31%), and Belgium (13%) (Table 1). They were mainly women (79%), with a mean age of April 2022 \| Volume 13 \| Article 852712 Frontiers in Psychiatry \| www.frontiersin.org 4 Monteiro et al. Mediators of Informal Caregivers’ Stress 54.0 ± 13.9 years. Most participants were relatives of their care recipients (parents, 31%; spouses, 21%; oﬀspring, 20%; siblings, 17%; or other family relatives, 6%) and were living with the care recipients (59%). PSS-10 scores among informal caregivers were higher when they feared getting ill with COVID-19 and/or another disease, when they reported that the lockdown would likely have a negative impact on their health or budget, and when they felt that the health of their care recipient was deteriorating. In contrast, PSS-10 scores were lower when informal caregivers considered that a lockdown would have a positive impact at the individual level or for the planet. The nominal variable “fear of falling ill” was dichotomized to allow subsequent mediation analyses: the two categories retained were “yes” (fear of COVID-19 and/or another disease) and “no,” according to the best multinomial model. Care recipients were mainly men (58%) with a mean age of 54 ± 25.1 years and diverse illnesses (e.g., psychiatric disorders, 45%; physiological pathologies, such as cancer, 12%; more than one diagnosis, 25%). The mean illness duration was 11 years (range, 1–49 years). Identiﬁcation of Mediators for the Association Between Perceived Stress and Sociodemographic Variables We further analyzed only the ﬁve most robust sociodemographic variables (p < 0.01) and tested whether some of them were interdependent; mediating models were computed to assess whether some of the variables concerning the care recipient (i.e., link to care recipient, living with care recipient, and age of care recipient) fully mediated the association between PSS-10 levels and the sociodemographic variables directly related to the informal caregiver (i.e., age and sex). The nominal variable “relation to care recipient” needed to be dichotomized for subsequent analyses, and the two categories retained were “family member” and “other relative,” according to the best multinomial model. Six models were tested, indicating that the age of the care recipient fully mediated the association between PSS-10 and age of the informal caregiver (p = 0.027, Supplementary Table 1). Therefore, the ages of the informal caregivers were not analyzed further. g p For each of the four sociodemographic variables associated with PSS-10 scores, we tested six previously selected potential mediators (Figure 1A). As previously detailed, two nominal variables were dichotomized (i.e., link to care recipient and fear of falling ill); hence, mediations speciﬁc to some family members (e.g., parents vs. spouses) or to a particular illness were not assessed. Partial mediations were detected in ﬁve cases (Figure 1B). The relationship between younger age of the care recipient and higher PSS-10 score for the informal caregiver was partially mediated, on the one hand, by the conviction that lockdowns had a negative impact on health (Mediation 1), and on the other hand, by the fear of the informal caregiver of falling ill (Mediation 2). A higher PSS-10 mean among women than among men is partially mediated by an increase in the perceived deterioration of the care recipient’s health (Mediation 3). Increased perceived stress in informal caregivers who are family members of the care recipient vs. other relationships was partially mediated by the conviction that lockdowns have negative impacts on health (Mediation 4). Living together with the care recipient is associated with higher perceived stress of the informal caregiver, a relationship partially mediated by the fear of illness (Mediation 5). In summary, four sociodemographic variables were retained for subsequent analyses, such as sex of informal caregiver, relationship with the care recipient, living with the care recipient, and age of care recipient. Selection of Variables Potentially Mediating the Perceived Stress Participants rated their attitudes and resources during the ﬁrst lockdown of the pandemic toward caregiving, resources, economic status, and health (Table 2). Statistical analyses indicated that 10 of the 18 corresponding items were associated with PSS-10. We applied a stringent cutoﬀto further analyze the most robust ones (p < 0.005). Six variables, such as perception of care recipient’s health deterioration, possible positive impact of containment on the planet, possible positive impact of containment at the individual level, negative impact of containment on one’s budget, fear of falling ill, and negative impact of containment on one’s health, were considered. DISCUSSION This European study focused on informal caregivers to assess their perceived stress following the COVID-19 lockdown and identify the mediating factors. Perceived stress among informal caregivers was robustly associated with four sociodemographic factors (i.e., age of care recipient, family link to the care recipient, gender of the informal caregiver, and cohabitation), and these associations were partially mediated by three distinct attitudes of the informal caregivers. The relationship between increased Frontiers in Psychiatry \| www.frontiersin.org April 2022 \| Volume 13 \| Article 852712 5 Mediators of Informal Caregivers’ Stress Monteiro et al. FIGURE 1 \| (A). The standardized effect size (β) for indirect mediation of all the mediation models tested. Bold highlights p-value < 0.05. In all cases, direct effects are signiﬁcant. (B). Diagram illustrating the signiﬁcant mediations and reporting the standardized effect size (β) of the corresponding effects. p < 0.05. FIGURE 1 \| (A). The standardized effect size (β) for indirect mediation of all the mediation models tested. Bold highlights p-value < 0.05. In all cases, direct effects are signiﬁcant. (B). Diagram illustrating the signiﬁcant mediations and reporting the standardized effect size (β) of the corresponding effects. p < 0.05. FIGURE 1 \| (A). The standardized effect size (β) for indirect mediation of all the mediation models tested. Bold highlights p-value are signiﬁcant. (B). Diagram illustrating the signiﬁcant mediations and reporting the standardized effect size (β) of the correspond ed effect size (β) for indirect mediation of all the mediation models tested. Bold highlights p-value < 0.05. In all cases, direct effects trating the signiﬁcant mediations and reporting the standardized effect size (β) of the corresponding effects. p < 0.05. FIGURE 1 \| (A). The standardized effect size (β) for indirect mediation of all the mediation models tested. Bold highlights p-value < 0.05. In all cases, direct effects are signiﬁcant. (B). Diagram illustrating the signiﬁcant mediations and reporting the standardized effect size (β) of the corresponding effects. *p < 0.05. of the measures implemented and governmental communication strategies, we found no diﬀerences in perceived stress among informal caregivers in the three countries. A second strength is a focus on informal caregivers, regardless of the care recipient condition. Although the study participants faced a range of illnesses, we did not detect any eﬀect of diagnosis on their perceived stress. Our results support the idea that guidance for informal caregivers should be provided independently of the patient’s diagnosis. Frontiers in Psychiatry \| www.frontiersin.org DISCUSSION perceived stress of the informal caregiver and lower age of the care recipient was partially mediated by informal caregivers’ fear of falling ill, on the one hand, and by the conviction that lockdowns have a negative impact on health, on the other. Compared with other relationships, increased perceived stress among informal caregivers who are family members of the care recipient was also partially mediated by the conviction that lockdowns have negative impacts on health. Higher perceived stress levels among women were partially mediated by an increase in the perceived deterioration of the care recipient’s health. The higher perceived stress level among caregivers living with the care recipient was partially mediated by fear of illness. The sociodemographic factors associated with perceived stress among informal caregivers in this study are consistent with several reports: female sex, close kinship, cohabitation, and younger age are associated with burden, depressed mood, and health problems among informal caregivers (9, 38, 39). The ﬁrst strength of this study is its international design, which covers Switzerland, France, and Belgium. Despite the diversity April 2022 \| Volume 13 \| Article 852712 6 Mediators of Informal Caregivers’ Stress Monteiro et al. Furthermore, our data conﬁrmed that these known risk factors were speciﬁcally modulated by lockdowns. Thus, the ﬁndings of a recent Italian study (40) can be extended to the three European countries included in this study. The analyses further clariﬁed which attitudes and beliefs mediated these associations, thus providing actionable points to alleviate stress and burden among informal caregivers. Furthermore, our data conﬁrmed that these known risk factors were speciﬁcally modulated by lockdowns. Thus, the ﬁndings of a recent Italian study (40) can be extended to the three European countries included in this study. The analyses further clariﬁed which attitudes and beliefs mediated these associations, thus providing actionable points to alleviate stress and burden among informal caregivers. Cohabitation is another major source of stress and burden for informal caregivers (40, 48–50). In the present cohort, 59% of the informal caregivers were living with their care recipients, and the fear of illness partially mediated their perceived stress. It is interesting to note that the fear of illness includes causes other than COVID-19 and, therefore, may involve the fear of not being able to manage their caregiving duties. Thus, the fear of contagion does not predominate when informal caregivers are considered globally (vs. DISCUSSION when focusing on informal caregivers in contact with at-risk persons, such as the elderly) (40), whereas the fear of not coping with caregiving tasks is a shared concern. A Japanese study speciﬁcally indicated that 73% of caregivers of persons with schizophrenia worried about who would care for their patient if they became infected with COVID-19 (50). The link between cohabitation and stress, and the concern about not being able to meet the care recipient’s needs, may also be prevalent outside the pandemic context. This continuous strain places informal caregivers under pressure and is detrimental to their health. Interestingly, while Zucca et al. concluded that younger age of the informal caregiver of patients with dementia is a risk factor for higher stress in the context of lockdowns (40), our results show that this association is mediated by the younger age of the care recipient when various diseases are considered. Therefore, young age of the informal caregiver per se is not the only risk factor for increased stress. It might seem counterintuitive that the age of the care recipient is negatively associated with the level of perceived stress among informal caregivers, that is, that stress is higher when the care recipient is younger. In fact, it is not the at-risk population for COVID-19 complications (e.g., the elderly) that informal caregivers are most worried about, possibly because the health measures that are taken to control the pandemic also protected vulnerable persons. The mediation analyses highlighted two explanations for higher perceived stress results among informal caregivers with young care recipients: (i) informal caregivers’ fear of falling ill, possibly as she or he will be unable to cope with the caregiving tasks and (ii) the conviction that lockdowns have a negative impact on health, possibly as they perceived this problem early in the mental health of younger persons. However, several reports have warned about the negative impact of COVID-19 on the mental health of young people (i.e., children, adolescents, and students) in the early stages of the pandemic (41–43). Therefore, informal caregivers’ concern about the negative impact of lockdown on health for younger care recipients is likely to underlie mental health issues, but this point could not be disentangled based on the questionnaire. DISCUSSION Interestingly, the conviction that lockdown had a negative impact on health also mediated the association of increased perceived stress in informal caregivers who were family members and those with other types of relationships. Family members might be more aware of the importance of mental resources for their care recipients than more distant relatives. g p The cross-sectional design of this study is a limitation that does not allow us to determine whether the impact of cohabitation was exacerbated by lockdowns. Another limitation linked to the study design is the retrospective collection of data; the level of perceived stress may change signiﬁcantly as the situation evolves. The time window for the survey was limited to the end of the ﬁrst wave to prevent bias as much as possible. The shortcoming of this pragmatic choice is the small cohort, which precluded more reﬁned statistical analyses. Furthermore, the population targeted by the survey limits the generalizability of the results. Indeed, the channels used to share the survey (e.g., online social networks) speciﬁcally targeted informal caregivers in contact with informal caregivers’ associations or with health professionals. Study participants were better educated than is usually reported (51), suggesting that some groups were missed, possibly due to digital poverty or lack of health literacy skills. Moreover, the time spent providing care or support to the care recipient and the frequency of contact between the informal caregiver and the care recipient were not used as eligibility criteria because the pandemic context may have aﬀected them drastically. We showed that among informal caregivers, those who are relatives of the care recipient have reached similarly high levels of perceived stress regardless of the type of kinship (e.g., parents, siblings, or oﬀspring). Moreover, we did not detect any eﬀect of diagnosis on perceived stress, which is in agreement with other studies (40, 52). Overall, these ﬁndings underscore the importance of informal caregivers’ oriented support in the context of sanitary restrictions. With regard to Lazarus and Folkman, this study conﬁrms the link between personal resources and the low level of stress perceived in this pandemic situation (27). For example, belief in a positive outcome is associated with lower levels of perceived stress, supporting the view that an optimistic attitude can improve the eﬀectiveness of coping strategies (51). In practice, this result is favorable for tailored interventions for informal caregivers (53). Frontiers in Psychiatry \| www.frontiersin.org REFERENCES medical illnesses: a systematic review. Int J Environ Res Public Health. (2020) 17:5888. doi: 10.3390/ijerph17165888 1. World Health Organization. Coronavirus Disease 2019 (COVID-19): Situation Report, 51. Geneva: World Health Organization (2020). 1. World Health Organization. Coronavirus Disease 2019 (COVID-19): Situation Report, 51. Geneva: World Health Organization (2020). 12. Elliott TR, Pezent GD. Family caregivers of older persons in rehabilitation. NeuroRehabilitation. (2008) 23:439–46. Report, 51. Geneva: World Health Organization (2020). 13. Magliano L, Fiorillo A, Malangone C, De Rosa C, Maj M. Special Section: a memorial tribute: patient functioning and family burden in a controlled, real- world trial of family psychoeducation for schizophrenia. Psychiatr Serv. (2006) 57:1784–91. doi: 10.1176/ps.2006.57.12.1784 2. Thome J, Deloyer J, Coogan AN, Bailey-Rodriguez D, da Cruz ESOAB, Faltraco F, et al. The impact of the early phase of the COVID-19 pandemic on mental-health services in Europe. World J Biol Psychiatry. (2021) 22:516–25. doi: 10.1080/15622975.2020.1844290 3. Capozzo R, Zoccolella S, Frisullo ME, Barone R, Dell’Abate MT, Barulli MR, et al. 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(2020) 395:912–20. doi: 10.1016/S0140-6736(20)3 0460-8 16. Manacorda T, Bandiera P, Terzuoli F, Ponzio M, Brichetto G, Zaratin P, et al. Impact of the COVID-19 pandemic on persons with multiple sclerosis: early ﬁndings from a survey on disruptions in care and self-reported outcomes. J Health Serv Res Policy. (2021) 26:189–97. doi: 10.1177/13558196209 75069 6. Rexhaj S, Leclerc C, Bonsack C, Golay P, Favrod J. Feasibility and accessibility of a tailored intervention for informal caregivers of people with severe psychiatric disorders: a pilot study. Front Psychiatry. ACKNOWLEDGMENTS We are very grateful to Anne Leroy for her support in preparing the survey and Mercedes Puteo for sharing it with her network. We thank the informal caregivers for their participation in this survey. DISCUSSION Our ﬁndings suggest that supporting caregivers’ health and addressing the negative impact of the lockdown on mental health among young care recipients should be routinely included in intervention strategies to prevent heightened stress among informal caregivers. We reported that perceived stress in informal caregivers is higher for women than for men, which is consistent with reports covering the lockdown period (40, 44). In the general population, perceived stress due to the lockdown was also higher among women than among men, indicating that sex-speciﬁc factors were at stake (41, 42, 45). However, it is noteworthy that women were overrepresented in studies on informal care, arguably because they were more likely to self-identify as informal caregivers and engage in higher levels of care (9, 38, 46). In the context of the pandemic, the burden on informal caregivers was more likely to increase when it was initially high, and women were more likely than men to have an increased caregiving burden due to COVID-19 (47). This analysis further indicates that women’s perceived stress is partially mediated by a subjective perception that care recipients’ health is deteriorating, which may reﬂect their greater physical and emotional involvement in caregiving than what would be common among men. April 2022 \| Volume 13 \| Article 852712 7 Mediators of Informal Caregivers’ Stress Monteiro et al. DATA AVAILABILITY STATEMENT manuscript. All authors critically reviewed the ﬁnal manuscript and approved the ﬁnal version. The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. SUPPLEMENTARY MATERIAL SM, JF, and SR conceived the study. SM, JF, SR, and MF designed the study. SM, SR, JF, NF, HW, LP, M-CL, and JD developed the survey. SM, JF, LP, SF, KS, CT, NF, M-CL, and JD recruited the participants. MF, SR, and PG analyzed and interpreted the data. SM, MF, A-LD, and SR wrote the The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpsyt. 2022.852712/full#supplementary-material ETHICS STATEMENT This work was supported by the Swiss National Science Foundation under grant number 10001C_185422. This work was supported by the Swiss National Science Foundation under grant number 10001C_185422. The studies involving human participants were reviewed and approved by the Commission Cantonale d’éthique de la Recherche sur l’être Humain (CER-VD). Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements. REFERENCES Mohler-Kuo M, Dzemaili S, Foster S, Werlen L, Walitza S. 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Available online at: https://www.jamovi.org Conﬂict of Interest: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. 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. 37. Core Team R. R: A Language and Environment for Statistical Computing. (Version 4.0). Vienna: R Foundation for Statistical Computing (2020). 38. Cohen SA, Sabik NJ, Cook SK, Azzoli AB, Mendez-Luck CA. Diﬀerences within diﬀerences: gender inequalities in caregiving intensity vary by race and ethnicity in informal caregivers. J Cross Cult Gerontol. (2019) 34:245–63. doi: 10.1007/s10823-019-09381-9 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. 39. Etters L, Goodall D, Harrison BE. Caregiver burden among dementia patient caregivers: a review of the literature. J Am Acad Nurse Pract. (2008) 20:423–8. doi: 10.1111/j.1745-7599.2008.00342.x 40. Zucca M, Isella V, Lorenzo RD, Marra C, Cagnin A, Cupidi C, et al. Being the family caregiver of a patient with dementia during the coronavirus disease 2019 lockdown. Front Aging Neurosci. (2021) 13:653533. doi: 10.3389/fnagi. 2021.653533 Copyright © 2022 Monteiro, Fournier, Favrod, Drainville, Plessis, Freudiger, Skuza, Tripalo, Franck, Lebas, Deloyer, Wilquin, Golay and Rexhaj. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. REFERENCES No use, distribution or reproduction is permitted which does not comply with these terms. 41. Debowska A, Horeczy B, Boduszek D, Dolinski D. A repeated cross-sectional survey assessing university students’ stress, depression, anxiety, and suicidality in the early stages of the COVID-19 pandemic in Poland. Psychol Med. (2020):1–4. doi: 10.1017/S003329172000392X [Epub ahead of print]. April 2022 \| Volume 13 \| Article 852712 Frontiers in Psychiatry \| www.frontiersin.org 9
https://openalex.org/W2902656146	https://zenodo.org/records/4062935/files/62%2025Oct18%2014jan18%2011034%20How%20Software%20Size%20ed%20iqbal.pdf	English	null	How software size influence productivity and project duration	International Journal of Electrical and Computer Engineering	2,019	cc-by	7,076	Corresponding Author: Mridul Bhardwaj, Amity School of Engineering and Technology, Amity University, Noida Up, India. Email: mridul2707@gmail.com How software size influence productivity and project duration Mridul Bhardwaj1, Ajay Rana2, Neeraj Kumar Sharma3 1,2Amity School of Engineering and Technology, Amity University, Noida Up, India 3HIMT, Greater NOIDA, UP, India Mridul Bhardwaj1, Ajay Rana2, Neeraj Kumar Sharma3 1,2Amity School of Engineering and Technology, Amity University, Noida Up, India 3HIMT, Greater NOIDA, UP, India Article Info Article history: Received Jan 14, 2018 Revised Oct 25, 2018 Accepted Nov 20, 2018 Keywords: Effort Productivity Project duration Software size International Journal of Electrical and Computer Engineering (IJECE) Vol. 9, No. 3, June 2019, pp. 2006~2017 ISSN: 2088-8708, DOI: 10.11591/ijece.v9i3.pp2006-2017 International Journal of Electrical and Computer Engineering (IJECE) Vol. 9, No. 3, June 2019, pp. 2006~2017 ISSN: 2088-8708, DOI: 10.11591/ijece.v9i3.pp2006-2017 International Journal of Electrical and Computer Engineering (IJECE) Vol. 9, No. 3, June 2019, pp. 2006~2017 ISSN: 2088-8708, DOI: 10.11591/ijece.v9i3.pp2006-2017  2006 Copyright © 2019 Institute of Advanced Engineering and Science. All rights reserved. ABSTRACT To make a perfect project plan, the software size of the order from the customer is the most important factor. The biggest challenge for the project manager is to estimate the project end date in the beginning of the project i.e. in project planning phase with realistic accuracy. Apart from other major inputs to estimate the project end date, expected team capability (productivity) and estimated software size are the major inputs that may influence the project end date. Software size is one of the most significant independent metric available in the planning phase and project manager has to estimate the other metrics based on the initial estimated software size. There is no direct relationship available between software size and project duration or software size and team productivity, however, there are industry data published by Quantitative Software Management and ISBSG that shows how these metrics influence each other. In this paper, using the data published by ISBSG and Quantitative Software Management, we try to statistically establish how productivity and project duration are influenced by software size. We have done linear regression analysis by generating the secondary data based on the data published by ISBSG and Quantitative Software Management. Linear regression equation validated with the actual project data and experimental results suggest that that productivity is significantly dependent on software size, however, project duration does not significantly depend on software size but may also be dependent on other metrics like team size, apart from software size. Keywords: Effort Productivity Project duration Software size Copyright © 2019 Institute of Advanced Engineering and Science. All rights reserved. Journal homepage: http://iaescore.com/journals/index.php/IJECE Corresponding Author: Mridul Bhardwaj, Amity School of Engineering and Technology, Amity University, Noida Up, India. Email: mridul2707@gmail.com 1. INTRODUCTION All Software and Information Technology Organizations are Project Based organizations. The Organization is an integration of Project Teams working in tandem. Each Manager whether vertically or horizontally aligned has to work and deliver projects with varied scope and size. Estimation on the project end date at the beginning of the project, not only ensures a smooth execution but also helps the organization on optimum utilization of available resources and an enhanced dollar value for future projects. Software size is one of the most significant metric available in planning phase and project manager has to estimate the other metrics based on the initial estimated software size. The importance of software size is explained in “Estimate Software Functional Size before Requirement phase of Development Life Cycle” [1]. “Imagine you are driving on an important trip to a distant place where you have not been before. You will not feel comfortable to start such a journey without knowing at least the general direction of the destination, e.g. distance, the available routes, road conditions etc. Armed with this information and a good map one can feel more comfortable about taking the trip. These are essential; however your comfort may be seriously compromised during the journey. Road works or diversions can also hit your journey plan, but at least you have a good chance of having an early warning, if you check it before starting the journey. Car breakdowns or wheel punctures can be less predictable, but they can happen. Managing a software project is much harder Journal homepage: http://iaescore.com/journals/index.php/IJECE Int J Elec & Comp Eng 2007  ISSN: 2088-8708 than planning a trip. The biggest difference is that no matter how hard you try, the specifications are not static and there are challenges in predicting team productivity and project duration.” than planning a trip. The biggest difference is that no matter how hard you try, the specifications are not static and there are challenges in predicting team productivity and project duration.” Each software development project is unique and possess different challenges to control and monitor project execution. The project manager faces major challenge to identify the important metrics to control and monitor the project execution. As per Putnam, Lawrence H., and Ware [2], these metrics are software size, effort, project duration and productivity. 1. INTRODUCTION These metrics tells project manager about what to deliver (size), how it was delivered in past (productivity) and how long will it take to deliver with current team capability (project duration). It is always a challenge before the project manager to estimate these metrics because there is no direct relationship among these metrics, however, there are industry data published by Quantitative Software Management and ISBSG that shows how these metrics influence each other. In this paper, using the data published by ISBSG and Quantitative Software Management, we try to statistically establish how productivity and project duration is influenced by software size. f d h i i l l i hi h f ll i a. Liner regression analysis for software size (independent varia a. Liner regression analysis for software size (independent variable) and productivity (dependent variable) b Liner regression analysis for software size (independent variable) and project duration (dependent a. Liner regression analysis for software size (independent variable) and productivity (dependent variable) b. Liner regression analysis for software size (independent variable) and project duration (dependent variable) a. Liner regression analysis for software size (independent variable) and productivity (dependent variable) a. Liner regression analysis for software size (independent variable) and productivity (dependent variab b. Liner regression analysis for software size (independent variable) and project duration (depe variable) c. Verification of liner regression equations with actual data points 2. PROBLEM STATEMENT Most of the Software development projects are conceptualized to implement the requirements provided by the business users. Users could be an end user, a group of users or a company. Usually in the age of B2B Commerce the software development projects are essentially taken up by software industry companies and there are many local and external stakeholders to the project. The stakeholders are varied between technical and non-technical people. Generally, the marketing team and customer interfacing teams of the client order software. The scope of a software development project may be by the virtue of a new development of software or enhancement of existing software depending upon the user base and their requirements. The business user wants these requirements (most of these are functional in nature) to be implemented as soon as possible, however, they may or may not be aware about the technical challenges that would require to overcome before it can actually be delivered. The Project Manager, who is accountable for successful completion of project, needs to estimate the project duration and the total efforts. In the planning phase, project manager faces the challenge to estimate the project duration to complete the project. Only metrics available is the functional size that can be calculated based on the requirements provided by the user, however, these requirements are also expected to be changed. To estimate the project duration, Project manager must understand his team capability and composition. Team composition and capability can be measured in terms of productivity i.e. number of function points delivered in 1 month, therefore, the project manager needs to understand how he can estimate productivity and project duration using the software size. Unfortunately, most of the time the project manager does not have any method or metrics that help him in estimating the project duration and productivity with the help of software size. A project manager ponders whether there is any measurable relation which exists between software size and duration or software size and productivity. 3. STUDY OBJECTIVE During project execution, the project manager has to monitor and control the following metrics to analyze the impact on estimated project duration and the team productivity. They are: y p p j p a. Software size (It is expected to increase as the project progresses) y p p j p a. Software size (It is expected to increase as the project progresses) b. Team Size (stable team will improve the overall team productivity) b. Team Size (stable team will improve the overall team productivity) Software Size is the most critical input for planning and rollout of a project. The software size is measurable and it is considered as an input to form the development team i.e. team size which in turn decides the project duration and the delivery date. A mathematical model will not only help the project manager to establish a correlation between software size and the project duration but also will help project managers to deliver the project on time. Accuracy of the calculation will trim down project losses and will enable organizations to deliver software within agreed timelines. Is there a mathematical correlation between software size and productivity and the project duration? If yes, how it can be calculated and measured accurately? This is problem which the authors address in this particular study. The objective of the study is to perform data analysis to develop in-depth understanding of software size influence on productivity and project duration. How software size influence productivity and project duration (Mridul Bhardwaj)  2008 2008 ISSN: 2088-8708  a. Is there any co-relation between productivity and software size? b. Can we assume higher team productivity for big projects? c. Is there any co-relation between project duration and software size? Key Software development metrics like efforts, project duration, project cost etc. are dependent on software size and productivity. Our study is an attempt to find the answer of above mention questions. We aim to analyze data published by industry of various projects and arrive at mathematical solution to establish the influence of software size on the productivity of the project as well as how it impact the project duration. This study will help the software professionals and IT Project Managers worldwide in proactivity planning and mitigate the risk if any. Technical Challenges Major challenge to achieve the research objective was the availability of actual primary dataset for statistical analysis. 4.1. Software size Software Size is the measure of functionality that will be delivered by software and that can be validated by end user of the software. It is a numerical measure of functional and non-functional requirements. Software size is an independent metric and all other metrics can be viewed as function of the software size. Software Size only depends on what to deliver rather on how to deliver. If software size of two projects are same then it does not imply that both the projects are same or they deliver the same requirement but it only indicates that both software are of same size. Software Size is the important metric to estimate other metrics e.g. total effort and project duration. It always remains a challenge to estimate the software size in the beginning of the project because only high level requirements are available during the pre-requirement phase of the project, however, in size-based project estimation, details of software requirement are not important but relative size of the project is sufficient for initial estimate of the metrics. For example we may not be able to estimate the software size of proposed project as detail software requirements are not available, but we can compare the complexity of the proposed project with the already delivered projects. This will help the project manager to establish initial baseline of key metrics based on the past performance of the similar projects. There are many software sizing methods viz. function points, use case points, story points (for agile projects), object based count [3]-[6] etc. but IFPUG function point is the most widely used and acceptable software sizing method. Int J Elec & Comp Eng, Vol. 9, No. 3, June 2019 : 2006 - 2017 3. STUDY OBJECTIVE We have used secondary data that was generated using the statistics (mean and standard deviation) published by ISBSG. Data was generated using the Mini tab tool and data was assumed as normally distributed. 4.2. Productivity or rate of delivery Effort required to deliver unit software size is defined as Productivity or Rate of delivery. Productivity is require to estimate the overall project effort to deliver the project. Productivity depends on the various factor like team experience or capability to work in similar technology or domain, the team will be more productive if they have earlier worked on similar technology or domain. It also depends on the business process understanding as it will help to translate the business requirements to technical requirements. If the same team had earlier worked together then it will help in improving the productivity as it will reduce the time required to resolve the collaboration issues. It is challenging to define team productivity and to give productivity a number but it an essential metric that not only need to be estimated in the beginning but also need to be consistently monitored during the project life cycle. The project manager can use the historical data of similar projects to estimation the productivity of the team. As most of the productivity numbers are published in range so project manager should carefully chose the productivity number within that range. Figure 1 [7] explain how productivity can be used to drive the overall project efforts. If there is uncertainty to establish the team productivity then Iterative development model is more preferable to waterfall model. In waterfall model actual productivity of the team will only be known after the construction phase so project manage will not find any opportunity to re-define the productivity number and hence the other key metrics (effort and time), however, iterative development model provide project manager the opportunity to re-define these key metrics. Mridul Bhardwaj and Ajay Rana [8]-[11] explains how effort, schedule and project duration should be re-define using iterative model as shown in Figure 2. Int J Elec & Comp Eng, Vol. 9, No. 3, June 2019 : 2006 - 2017  2009 2009  Int J Elec & Comp Eng ISSN: 2088-8708 Figure 1. Driving efforts using size and productivity Figure 2. Iterative model to re-define effort and time during project life cycle Figure 1. Driving efforts using size and productivity Figure 1. Driving efforts using size and productivity Figure 1. Driving efforts using size and productivity Figure 2. Iterative model to re-define effort and time during project life cycle Figure 2. Iterative model to re-define effort and time during project life cycle 4.3. Project duration Project calendar duration represents time required to complete the project. Project duration is the estimation of project end date for given project start date and estimate the date when we can delivery the project. It must be understood that project duration and total project effort are not interchangeable metric. There is no direct relationship which exists between time and effort. It must be understood that by adding more team members we can only reduce the overall project duration to a certain level but adding more resources may result in longer project duration and increase cost. e.g if total estimated project effort is 12 person months and there is a project team of 4 people. In this case estimated project duration will be 3 months provided there is no planned idle time. We can’t deliver this project in one month with 12 member team because adding more member will not only add new communication channels but also increase the integration effort. This relationship must be understood and it is better to have an optimal team size. Figure 3 [7] describe the relationship between Team size and Project duration, increase in team size will help in reducing the overall project duration only till point of reflection but increase in team size beyond the point of reflection will increase the project duration. Project managers must understand the point of reflection as it will help him to commit timelines with project stakeholders. It is not easy to identify the point of reflection but work break down structure or Delphi techniques can help project manager in identifying the optimal team size. Figure 3. How increase in team size will impact project duration? Figure 3. How increase in team size will impact project duration? 5. RELATED WORK 5.1. Data published by quantitative software management p y q g Quantitative Software Management (QSM) [12] has published the data based on the study of 2,231 closed projects. Projects are classified as new development, Major/Minor enhancement, Conversion or Maintenance project based on the ratio of new functionality to be added versus total functionality (added, modified and deleted). Table 1 shows the median project metrics for various project type. a. New Development (more than 75% new functionality) b. Major Enhancement (new functionality between 25%-75%) c. Minor Enhancement (new functionality between 5%-25%) d. Conversion (less than 5% functionality) s e. Maintenance (No new functionality) Quantitative Software Management (QSM) [12] has published the data based on the study of 2,231 closed projects. Projects are classified as new development, Major/Minor enhancement, Conversion or Maintenance project based on the ratio of new functionality to be added versus total functionality (added, modified and deleted). Table 1 shows the median project metrics for various project type. N D l ( h 75% f i li ) Quantitative Software Management (QSM) [12] has published the data based on the study of 2,231 closed projects. Projects are classified as new development, Major/Minor enhancement, Conversion or Maintenance project based on the ratio of new functionality to be added versus total functionality (added, modified and deleted). Table 1 shows the median project metrics for various project type. N D l t ( th 75% f ti lit ) Table 1. Median Project Metrics for Various Project Type Project Type Median Project Metrics New Development Major Enhancement Minor Enhancement Conversion Maintenance % of Projects 16% 61% 14% 7% 2% Median Size (FP) 291.00 119.00 153.00 109.00 68.00 Median Effort Months 29.70 19.30 28.10 23.40 18.60 Median % Functional Efforts 12% 11% 12% 10% 19% Median Productivity FP/PM 9.16 5.79 5.19 5.06 2.7 Median Duration 7.57 7.23 6.42 6.43 4.73 Median Defects 37 16 38.5 35 16 Table 1. Median Project Metrics for Various Project Type Median Project Metrics Data shows, Rate of delivery (productivity) is higher for new development projects compared to maintenance and enhancement projects. Productivity (rate of delivery) for new development projects is 3 times to the productivity (rate of delivery) of maintenance projects and 50% more than productivity of enhancement projects, however, there are more defects injected in development projects compare to maintenance projects. 5. RELATED WORK How software size influence productivity and project duration (Mridul Bhardwaj) 5. RELATED WORK As per Putnam, Lawrence H., and Ware [2], software size, effort, project duration and productivity are the key metrics for monitoring and control of software development project. There is no direct  2010 2010   ISSN: 2088-8708 relationship among these metrics, however, there are industry data available that shows how these metrics influence each other. relationship among these metrics, however, there are industry data available that shows how these metrics influence each other. 5.1. Data published by quantitative software management Data shows Figure 4, Figure 5 and Figure 6 that median number of defects for new development projects are more than double for number of defects for maintenance projects. Figure 7 shows how productivity (rate of delivery) changes against software size. It shows that productivity improves significantly as project size improve. Data in Table 1 shows that median project size for new development project is 291 function points and median project size for maintenance project is 68 function points. New development projects are bigger in size and rate of delivery is significantly higher than maintenance projects. Figure 4. Median productivity for various project type Figure 5. Median defects for various project type Figure 5. Median defects for various project type Figure 4. Median productivity for various project type Figure 5. Median defects for various project type Figure 4. Median productivity for various project type Int J Elec & Comp Eng, Vol. 9, No. 3, June 2019 : 2006 - 2017 ISSN: 2088-8708  2011 Int J Elec & Comp Eng  2011 2011 Figure 6. Median productivity against project size Figure 7. Total efforts and productivity Figure 6. Median productivity against project size Figure 7. Total efforts and productivity Figure 6. Median productivity against project size Figure 7. Total efforts and productivity Figure 8 shows that rate of delivery decreases sharply as we spend more efforts. It shows that if we increase the team size it may impact negatively on overall team productivity. When we distribute the overall efforts to the various project phases, it is important to know how effort distribution to various phases would impact the key metrics like schedule, productivity and defects etc. Study of Quantitative Software Management (QSM) [12] shows that overall productivity improves to 28% if more than 20% efforts spends during analysis and design. Table 2 shows how productivity, schedule and defects improves if we spend more than 20% efforts during analysis and design phases. Overall duration of the project can be reduced by 14% and also overall project effort can be reduced by 10% if more than 20% efforts spend during the analysis and design phase. Table 2. 5.2.2. Total project efforts as function of software size and maximum team size Dependent Variable=Efforts (Project Efforts in Person Hours) Independent Variable 1=Size (Software Size in Function Points) Independent Variable 2=Maximum Team Size (Full Time Equivalent of Maximum Team Size) 5.2.2. Total project efforts as function of software size and maximum team size Dependent Variable=Efforts (Project Efforts in Person Hours) Independent Variable 1=Size (Software Size in Function Points) Independent Variable 2=Maximum Team Size (Full Time Equivalent of Maximum Team Size) Efforts = 37.48 * Size (.504) * Maximum Team Size (0.759) Above equation is derived based on 203 new development projects in ISBSG project repository and having R2(adj) value as 0.61 which means 61% variability is explained by the above equation. Median MRE value is 0.37 which implies that there could be 37% variance in actual value and value obtain using the derived equation. 5.2.4. Project duration as function of software size Dependent Variable=Project Duration (Total active elapsed time in mon Independent Variable 1=Size (Software Size in Function Points) Dependent Variable=Project Duration (Total active elapsed time in mo Independent Variable 1=Size (Software Size in Function Points) Project duration = 0.543 * Size (.408) Above equation is derived, based on 494 new development projects in ISBSG project repository and having R2(adj) value as 0.30 which means 30% variability is explained by the above equation. Median MRE value is 0.39 which implies that there could be 39% variance in actual value and value obtain using the derived equation. 5.2.3. Total project efforts as function of software size Dependent Variable=Efforts (Project Efforts in Person Hours) Independent Variable 1=Size (Software Size in Function Points) 5.2.3. Total project efforts as function of software size Dependent Variable=Efforts (Project Efforts in Person Hours) Independent Variable 1=Size (Software Size in Function Points) Efforts = 23.25 * Size (.814) Efforts = 23.25 * Size (.814) Above equation is derived, based on 534 new development projects in ISBSG project repository and having R2(adj) value as 0.45 which means 45% variability is explained by the above equation. Median MRE value is 0.55 which implies that there could be 55% variance in actual value and value obtain using the derived equation. 5.2.1. Productivity as function of software size and maximum team size Dependent Variable=Productivity (Project Delivery Rate in Hours per Function Point) Independent Variable 1=Size (Software Size in Function Points) Independent Variable 2=MaxTeamSize (Full Time Equivalent of Maximum Team Size) Productivity = 37.48 * Size (-0.496) * MaxTeamSize (0.759) Productivity = 37.48 * Size (-0.496) * MaxTeamSize (0.759) Above equation is derived based on 203 new development projects in ISBSG project repository and having R2(adj) value as 0.32 which means 32% variability is explained by the above equation. Median MRE value is 0.37 which implies that there could be 37% variance in actual value and value obtain using the derived equation. 5.1. Data published by quantitative software management Impact of Effort Spend in Analysis and Design on Key Metrics Impact of Effort Spend in Analysis and Design Effort Spend >20% Effort Spend <20% Impact Productivity FP/PM 7.93 6.2 28% Median Duration (Months) 6.2 7.23 -14% Median Efforts (PM) 20.29 22.59 -10% Median Defects 19.5 20 -3% Figure 8. Impact of effort spend in analysis and design 7.93 6.2 20.29 19.5 6.2 7.23 22.59 20 28% -14% -10% -3% -20% -10% 0% 10% 20% 30% 40% 0 5 10 15 20 25 Productivity FP / PM Median Duration (Months) Median Efforts (PM) Median Defects Impact of Effort spend in Analysis and Design Effort spend >20% Effort spend<20% Impact Figure 8. Impact of effort spend in analysis and design 5.2. Data published by international software benchmarking standard group (ISBSG) 5.2. Data published by international software benchmarking standard group (ISBSG) International Software Benchmarking Standard Group (ISBSG) in its Benchmarking release published the relationship for software size, efforts, project duration, productivity and maximum team size. These relationship is based on 4,106 software projects from around the world, and made available on Release 10 of Estimating, Benchmarking & Research Suite CD [13]. This relationship have been derived after the How software size influence productivity and project duration (Mridul Bhardwaj)  2012  2012 ISSN: 2088-8708 statistical analysis of projects in the ISBSG repository. ISBSG study [14] showed that software size and maximum team size are the key metrics for estimating project duration and efforts. statistical analysis of projects in the ISBSG repository. ISBSG study [14] showed that software size and maximum team size are the key metrics for estimating project duration and efforts. 6. APPROACH ISBSG data does not represent any meaningful regression equation between productivity and software size which is one of the most significant metric known in the beginning of the project, however, it does represent the regression equation of productivity with software size and maximum team size (explain in 5.2.1) but most of the time maximum team size is not known in the beginning of the project. Following sections explain the step by step approach taken to establish the regression equation. 5.2.5. Project duration as function of effort Dependent Variable=Project Duration (Total active elapsed time in months Independent Variable 1=Effort (Total Planned effort in person hours) Project duration=0.370 * Effort (.328) Above equation is derived, based on 1681 new development projects in ISBSG project repository and having R2(adj) value as 0.35 which means 35% variability is explained by the above equation. Median MRE value is 0.36 which implies that there could be 36% variance in actual value and value obtain using the derived equation. Int J Elec & Comp Eng, Vol. 9, No. 3, June 2019 : 2006 - 2017 2013  20  Int J Elec & Comp Eng ISSN: 2088-8708 6.1. Steps to obtain the linear regression equation In both the equations software size is taken as independent variables, while productivity and project duration is taken as dependent variables p y p j p e. Verify the linear equations obtain in step 5 with actual data for 10 closed projects. p y p j p e. Verify the linear equations obtain in step 5 with actual data for 10 closed projects. 6.1. Steps to obtain the linear regression equation Using the model published by ISBSG and discussed in section 6.2, we simulate the productivity and project duration for different software size in the range 50 to 2000 function points, assuming software size it is normally distributed, we followed the following steps to establish the linear regression equation for productivity and project duration with software size. Table in annexure shows the complete dataset. a. For all projects, efforts were derived using the regression equation explain in section 6.2.3. Figure 9 shows the graph for the size and corresponding efforts. Figure 9. Total effort v/s size Figure 9. Total effort v/s size b. Productivity for each project was calculate using the effort and size calculated in step 1 (by dividing software size and effort). Figure 10 shows the graph for software size and productivity. b. Productivity for each project was calculate using the effort and size calculated in step 1 (by dividing software size and effort). Figure 10 shows the graph for software size and productivity. Figure 10. Productivity v/s size Figure 10. Productivity v/s size c. Project duration in person month was estimated using the regression equation describe in 6.2.5. Figure 11 shows the graph for project duration and software size. c. Project duration in person month was estimated using the regression equation describe in 6.2.5. Figure 11 shows the graph for project duration and software size. c. Project duration in person month was estimated using the regression equation describe in 6.2.5. Figure 11 shows the graph for project duration and software size. How software size influence productivity and project duration (Mridul Bhardwaj) How software size influence productivity and project duration (Mridul Bhardwaj)  2014  14 ISSN: 2088-8708 Figure 11. Project duration v/s size Figure 11. Project duration v/s size d. Using the statistical tools, obtain the linear regression equations for productivity and project duration depending on software size. In both the equations software size is taken as independent variables, while productivity and project duration is taken as dependent variables d. Using the statistical tools, obtain the linear regression equations for productivity and project duration depending on software size. In both the equations software size is taken as independent variables, while productivity and project duration is taken as dependent variables d. Using the statistical tools, obtain the linear regression equations for productivity and project duration depending on software size. 7. EXPERIMENT RESULTS Using the statistical software Minitab version 16, following linear regression equation for productivity and size is obtained. R2(Adj) for this equation is 93% that means linear equation explain 92% of variability in dataset. Figure 12. to Figure 15. shows the output of regression analysis of size versus productivity and size versus project time line. Following linear regression equation for project duration and size is also obtained. R2(Adj) for this equation is 92% that means linear equation explain 92% of variability in dataset. Productivity = 4.378+0.001923Size Figure 12. Regression analysis for productivity and size Figure 13. Fitted line plot for regression analysis for productivity and size Project duration = 4.378+0.001923Size Figure 13. Fitted line plot for regression analysis for productivity and size Figure 13. Fitted line plot for regression analysis for productivity and size 8. VERIFICATION OF EXPERIMENT RESULTS Linear regression equation for productivity and project duration was verified for actual project metrics of 10 closed projects. Actual project size of these projects varies from 175 function points to 1468 function points. For all these projects, actual size, total effort and project duration was collected. We calculated the actual productivity using total effort and size. Productivity and project duration was also calculated based on the regression equation obtained in section 7. Variation in the actual metric and calculated metric is calculated. Variation in productivity (Actual v/s Calculated) was between -2.6% to 20.4%, except one data point. All the projects is having a positive variation which means the actual productivity was more than the productivity calculated using the regression equation. We can conclude that productivity is significantly dependent on software size. Productivity and software size linear regression relationship provides productivity estimates with 20% variation thus regression equation can be a good estimate of productivity in the initial phases of the project. Table 3. Verification Results Actual Project Data of Closed Projects As Per Regression Equcation % Variation S.N Actual Size Total Efforts Productivity (FP/PH) Project Duration in Months Productivity (FP/PH) Project Duration In Months In Productivity Project Duration in Months 1 175 1400 0.125 4.465 0.1236 4.714525 1.1% -5.3% 2 228 1583 0.144 4.974 0.125296 4.816444 14.9% 3.3% 3 258 1985 0.130 5.232 0.126256 4.874134 3.0% 7.3% 4 325 2600 0.125 5.749 0.1284 5.002975 -2.6% 14.9% 5 339 2568 0.132 5.848 0.128848 5.029897 2.4% 16.3% 6 467 3199 0.146 6.665 0.132944 5.276041 9.8% 26.3% 7 567 3611 0.157 7.214 0.136144 5.468341 15.3% 31.9% 8 775 4506 0.172 8.195 0.1428 5.868325 20.4% 39.6% 9 1245 6803 0.183 9.944 0.15784 6.772135 15.9% 46.8% 10 1468 7567 0.194 10.635 0.164976 7.200964 17.6% 47.7% Table 3. Verification Results Variation of project duration (Actual v/s Calculated) is increasing with project size and this increment is not linear, thus we can conclude that linear equation does not explain the relationship for project duration and software size. We can conclude that project duration does not significantly depend on software size only but may also depend on another metric apart from software size. This other metric could be the team size, this leaves scope to further analysis the project duration with software size and team size as depended variables. Figure 13. Fitted line plot for regression analysis for productivity and size Int J Elec & Comp Eng, Vol. 9, No. 3, June 2019 : 2006 - 2017  2015  2015 ISSN: 2088-8708 2015 Int J Elec & Comp Eng Figure 14. Regression analysis for project duration and size Figure 15. Fitted line plot of regression analysis for project duration and size Figure 14. Regression analysis for project duration and size Figure 15. Fitted line plot of regression analysis for project duration and size 9. RESULT AND DISSCUSION Linear regression analysis of data published by ISBSG [13] provides linear regression equation for productivity and project duration with software size. Verification of linear regression with actual data of 10 projects conclude that productivity is significantly dependent on software size. Productivity and software size How software size influence productivity and project duration (Mridul Bhardwaj) 2016  2016  ISSN: 2088-8708 linear regression relationship provides productivity estimates with 20% variation thus regression equation can be the good estimate of productivity in the initial phases of the project. linear regression relationship provides productivity estimates with 20% variation thus regression equation can be the good estimate of productivity in the initial phases of the project. We also conclude that project duration does not significantly depend on software size but may also depend on other metric apart from software size. This other metric could be the team size, this leaves a scope to further analyze the project duration with software size and team size as depended variables. It can be inferred that software size is the key metrics to manage software development project effectively. QSM study suggests that rate of delivery (productivity) is higher for new development projects compared to maintenance and enhancement projects. Productivity for new development projects are 3 times to the rate of delivery for maintenance projects and 50% more than the productivity for enhancement projects, however, there are more defects injected in development projects compared to maintenance projects. QSM data also suggest that productivity improves significantly as the project size increases, however, it decreases with the increase in efforts, and hence, the increasing team size may have negative impact on productivity, cost and increase the risk of delivering the high cost but low quality projects. Our study also concluded that distribution of efforts over various project phases also impacts the productivity and schedule. If we spend more than 20% effort during analysis and design phase then we can improve productivity and project duration, by 28%, 14% respectively. Hence, if a Project Manager applies estimation and techniques prescribed in this study to determine the productivity of a project, the Project Manager should take the software size as the most important input factor. Also, factors like the team size are subjective factors, it may or may not have a direct correlation on the productivity of any software project. REFERENCES [1] Mridul Bhardwaj and Ajay Rana, “Estimate Software Functional Size before Requirement phase of Development Life Cycle,” International Journal of Innovations & Advancement in Computer Science, vol. 3 Issue 4 June-2014, pp 79-83, 2014. pp [2] Putnam, Lawrence H., and Ware Myers, “Five Core Metrics-The Intelligence behind Successful So Management,” New York: Dorset House Publishing Company, Inc., 2002. [3] S. M. Satapathy, B. P. Acharya and S. K. Rath, "Early Stage Software Effort Estimation Using Random Forest Technique Based on Use Case Points," in IET Software, vol. 10, no. 1, pp. 10-17, 2 2016. doi: 10.1049/iet- sen.2014.0122 [4] M. Rizwan and J. Qureshi, "Agile Software Development Methodology for Medium and Large Projects," in IET Software, vol. 6, no. 4, pp. 358-363, August 2012. doi: 10.1049/iet-sen.2011.0110 [5] J. Popovic, D. Bojic and N. Korolija, "Analysis of Task Effort Estimation Accuracy Based on Use Case Point Size," in IET Software, vol. 9, no. 6, pp. 166-173, 12 2015. doi: 10.1049/iet-sen.2014.0254 [6] E. Khatibi and V. Khatibi Bardsiri, "Model to Estimate the Software Development Effort Based on In-Depth Analysis of Project Attributes," in IET Software, vol. 9, no. 4, pp. 109-118, 8 2015. doi: 10.1049/iet-sen.2014.0169 [7] Mridul Bhardwaj and Ajay Rana, “Key Software Metrics and its Impact on each other for Software Development Projects,” International Journal of Electrical and Computer Engineering (IJECE) Vol. 6, No. 1, February 2016, pp. 242~248, 2015. [6] E. Khatibi and V. Khatibi Bardsiri, "Model to Estimate the Software Development Effort Based on In-Depth Analysis of Project Attributes," in IET Software, vol. 9, no. 4, pp. 109-118, 8 2015. doi: 10.1049/iet-sen.2014.0169 [6] E. Khatibi and V. Khatibi Bardsiri, "Model to Estimate the Software Development Effort Based on In-Depth Analysis of Project Attributes," in IET Software, vol. 9, no. 4, pp. 109-118, 8 2015. doi: 10.1049/iet-sen.2014.0169 [7] Mridul Bhardwaj and Ajay Rana, “Key Software Metrics and its Impact on each other for Software Development Analysis of Project Attributes, in IET Software, vol. 9, no. 4, pp. 109 118, 8 2015. doi: 10.1049/iet sen.2014.0169 [7] Mridul Bhardwaj and Ajay Rana, “Key Software Metrics and its Impact on each other for Software Development Projects,” International Journal of Electrical and Computer Engineering (IJECE) Vol. 6, No. 1, February 2016, pp. 242~248, 2015. [8] Mridul Bhardwaj and Ajay Rana, “Impact of Size and Productivity on Testing and Rework Efforts for Web-based Development Projects,” ACM SIGSOFT Software Engineering Notes, vol. 9. RESULT AND DISSCUSION Team size depends upon the number of team resources and the productivity of the team resources is a subjective matter which cannot be implied empirically or mathematically. 10. FUTURE SCOPE The Current study concludes that software size has a significant impact on team productivity, however, the study is unable to establish any significant impact of software size on project duration on measurable terms. Project duration has a positive correlation with software size, however, we need to study further to analyze y a. How project duration influence by software size and team size. y a. How project duration influence by software size and team size. a. How project duration influence by software size and team size. p j y b. As software size is a key metrics so we need to establish how we can effectively estimate the software size in the early stage of project life cycle when only high level requirements are available. b. As software size is a key metrics so we need to establish how we can effectively estimate the software size in the early stage of project life cycle when only high level requirements are available. The results of the future scope of study will benefit Software Organizations to plan and execute projects on a mathematical grid accuracy which in turn could become a standardized operating procedure as further studies may establish mathematical relationship between software size and project duration. The results of the future scope of study will benefit Software Organizations to plan and execute projects on a mathematical grid accuracy which in turn could become a standardized operating procedure as further studies may establish mathematical relationship between software size and project duration. [14] Book, “Practical Software Project Estimation,” by International Software Benchmarking Standards Group, Page no 246-248 [10] Bhardwaj, Mridul; Rana, Ajay. “Estimation of Testing and Rework Efforts for Software Development Projects,” Asian Journal of Computer Science and Information Technology, [S.l.], v. 5, n. 5, p. 33-37, May 2015. ISSN 2249- 5126. How software size influence productivity and project duration (Mridul Bhardwaj) [11] Bhardwaj, Mridul; Rana, Ajay. “Impact of Size and Productivity on Testing and Rework Efforts for Non-Web- based Development Projects,” International Journal of Advanced Technology in Engineering and Science, vol 3, spl issue 2, March-15 REFERENCES 40 Number 2, March-2015, pp 1-4, 2015. [9] Mridul Bhardwaj and Ajay Rana, “Estimation of Testing and Rework Efforts for non-Web-based Software Development Projects,” IEEE international conference on “Futuristic Trends in Computational Analysis and Knowledge Management” at Greater NOIDA, 25-27, February 2015. Int J Elec & Comp Eng, Vol. 9, No. 3, June 2019 : 2006 - 2017  2017 2017  Int J Elec & Comp Eng ISSN: 2088-8708 [10] Bhardwaj, Mridul; Rana, Ajay. “Estimation of Testing and Rework Efforts for Software Development Projects,” Asian Journal of Computer Science and Information Technology, [S.l.], v. 5, n. 5, p. 33-37, May 2015. ISSN 2249- 5126 [10] Bhardwaj, Mridul; Rana, Ajay. “Estimation of Testing and Rework Efforts for Software Development Projects,” Asian Journal of Computer Science and Information Technology, [S.l.], v. 5, n. 5, p. 33-37, May 2015. ISSN 2249- 5126. [11] Bhardwaj, Mridul; Rana, Ajay. “Impact of Size and Productivity on Testing and Rework Efforts for Non-Web- based Development Projects,” International Journal of Advanced Technology in Engineering and Science, vol 3, spl issue 2, March-15 [11] Bhardwaj, Mridul; Rana, Ajay. “Impact of Size and Productivity on Testing and Rework Efforts for Non-Web- based Development Projects,” International Journal of Advanced Technology in Engineering and Science, vol 3, spl issue 2, March-15 p [12] “QSM Software Almanac, Application Development Series,” Research Edition, 2014. [13] ISBSG “The Benchmark data for Software estimation,” Release 10, 2011. [14] Book, “Practical Software Project Estimation,” by International Software Benchmarking Standards Group, Page no 246-248
https://openalex.org/W3048579654	https://www.int-arch-photogramm-remote-sens-spatial-inf-sci.net/XLIII-B2-2020/369/2020/isprs-archives-XLIII-B2-2020-369-2020.pdf	English	null	INDOOR 3D POINT CLOUDS SEMANTIC SEGMENTATION BASES ON MODIFIED POINTNET NETWORK	The international archives of the photogrammetry, remote sensing and spatial information sciences/International archives of the photogrammetry, remote sensing and spatial information sciences	2,020	cc-by	4,358	1. INTRODUCTION enhance the automation and intelligence of point cloud segmentation, the segmentation methods for point cloud base on deep learning has became a latest research hot spot. Deep learning is a kind of novel technology that automatically extract the high level features of the input data by the structure of deep neural network. Currently,the segmentation methods base on deep learning are mainly divided into 3 types: ①Convert point cloud into multi-view images then input the images into 2DCNN to realize the segmentation of 3D elements. (Su et al., 2015) put forward MVCNN network, which utilized 2DCNN network structure. However,converting point cloud to image will lose the spatial information of point cloud, which will affect the precision of segmentation; ②The neural networks that use voxel as input. Daniel and Sebastian (2015) put forward VoxNet network model base on point cloud voxelization and supervised 3DCNN. This network preprocess the point cloud into voxel, then use 3D convolutional kernel to carry out convolution operation, which is the original 3DCNN network but along with the disadvantages, such as: additional computation,“dimension explosion” etc. ③The deep neural network that directly use points as input. Qi et al. (2016), from Stanford, put forward PointNet network, which utilize multi- layer perception (MLP) to extract the global feature of point cloud and use maximum symmetric function to solve the problem of irregular format to achieve a good segmentation precision. However this network only pay attention to the global INDOOR 3D POINT CLOUDS SEMANTIC SEGMENTATION BASES ON MODIFIED POINTNET NETWORK J. Zhao1,2,3,X. Zhang1,3, Y. Wang1,3,* School of Geomatics and Urban Information, Beijing University of Civil Engineering and Architecture, 102616 Beij zhaojiangh@bucea.edu.cn 1 School of Geomatics and Urban Information, Beijing University of Civil Engineering and Architecture, 102616 Beijing ,China - zhaojiangh@bucea.edu.cn Key laboratory of Modern Urban Surveying and Mapping. National Administration of Surveying , Mapping and Geo 102616 Beijing, China 2 Key laboratory of Modern Urban Surveying and Mapping. National Administration of Surveying , Mapping and Geoinformation, 102616 Beijing, China 3 Beijing Key Laboratory For Architectural Heritage Fine Reconstruction & Health Monitoring 102616 Beijing China 3 Beijing Key Laboratory For Architectural Heritage Fine Reconstruction & Health Monitoring, 102616 Beijing, (zhangxiaoguang,2108521519005)@stu.bucea.edu.cn KEY WORDS: 3D LiDAR Point Cloud; Point Cloud Segmentation; Semantic Segmentation; Deep Learning; Indoor Structural Elements; PointNet ABSTRACT: Indoor 3D point clouds semantics segmentation is one of the key technologies of constructing 3D indoor models,which play an important role on domains like indoor navigation and positioning,intelligent city, intelligent robot etc. The deep-learning-based methods for point cloud segmentation take on higher degree of automation and intelligence. PointNet,the first deep neural network which manipulate point cloud directly, mainly extracts the global features but lacks of learning and extracting local features,which causes the poor ability of segmenting the local details of architecture and affects the precision of structural elements segmentation . Focusing on the problems above,this paper put forward an automatic end-to-end segmentation method base on the modified PointNet. According to the characteristic that the intensity of different indoor structural elements differ a lot, we input the point cloud information of 3D coordinate, color and intensity into the feature space of points. Also,a MaxPooling is added into the original PointNet network to improve the ability of attracting and learning local features. In addition, replace the 1×1 convolution kernel of original PointNet with 3×3 convolution kernel in the process of attracting features to improve the segmentation precision of indoor point cloud. The result shows that this method improves the automation and precision of indoor point cloud segmentation for the precision achieves over 80% to segment the structural elements like wall,door and so on ,and the average segmentation precision of every structural elements achieves 66%. * Corresponding author This contribution has been peer-reviewed. https://doi.org/10.5194/isprs-archives-XLIII-B2-2020-369-2020 \| © Authors 2020. CC BY 4.0 License. The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLIII-B2-2020, 2020 XXIV ISPRS Congress (2020 edition) The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLIII-B2-2020, 2020 XXIV ISPRS Congress (2020 edition) 1.1 General Instructions 3D LiDAR technology has gradually became a important method of understanding the 3D indoor scene because of this advantages that it can acquire massive point cloud data with high speed,low cost and high precision. However, the semantic segmentation has became a hot spot of research fields like: 3D indoor modeling, indoor navigation, robot pattern etc. The traditional segmentation methods for point cloud has developed for a period of time. So there are amounts of classic segmentation algorithms, such as: segmentation methods base on edge(Himmelsbach et al.,2009), segmentation methods base on surface (Li et al., 2011; Zhang et al., 2015; Hu et al., 2012) segmentation methods base on clustering (Chen et al., 2012; Sun et al., 2006; Lin et al., 2016), segmentation methods base on machine learning (Rusu et al., 2008; Rusu et al., 2009; Aldoma et al., 2011). With the improvement form lots of researchers, the traditional segmentation methods for point cloud has been enhanced constantly. However the traditional segmentation methods for point cloud require manually designed feature descriptors, which demand the designers possess empiric knowledge. There are lots of thresholds needed to be selected for traditional segmentation methods during the process of point cloud segmentation which is complicated, only suitable for specified tasks and poor in generalization. To 369 The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLIII-B2-2020, 2020 XXIV ISPRS Congress (2020 edition) The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLIII-B2-2020, 2020 XXIV ISPRS Congress (2020 edition) The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLIII-B2-2020, 2020 XXIV ISPRS Congress (2020 edition) Figure 1. Dataset main scenarios （Mainly divided into two parts :data set with reflection intensity information and data set without reflection intensity information ） feature and ignore the local feature. So this network has poor capacity of details segmentation. feature and ignore the local feature. So this network has poor capacity of details segmentation. This paper used the modified PointNet to segment the point cloud of indoor structural elements. The main works are as follow: (1)Currently, there are few indoor point cloud dataset which contain intensity information. 1.1 General Instructions So this paper constructed a indoor point cloud data set contains 8 types of indoor structural elements with intensity information for the experiment; (2)Focusing on the indoor structural elements (door, wall, window etc.) with different intensity information, this paper input intensity information,coordinate information and color information into the neural network as tensor to improve the segmentation precision of PointNet to segment the indoor structural elements. Figure 1. Dataset main scenarios （Mainly divided into two parts :data set with reflection intensity information and data set without reflection intensity information ） (3)Focusing the problem that the original PointNet only pay attention to extract the global features of point cloud but ignore local features, this paper modified the structure of PointNet network to let it has better ability to extract local features and improve the segmentation precision of structural elements. 2. CONSTRUCTING THE INDOOR POINT CLOUD DATA SET Faro Focus 3D X130 scanner and main technical indicators Data Set Year Format Feature of Point Cloud Producer S3DIS (Armeni et al., 2017) 2017 Point Cloud RGB-D (X,Y,Z,R,G,B) Stanford Scannet (Dai et al., 2017) 2017 Point Cloud RGB-D (X,Y,Z,R,G,B) Stanford Table 1. Current open large indoor point cloud datasets Configuration Faro Focus3D X130 Ranging/m 0.5～130 Distance Accuracy Index 0.6mm/10m Scanning View 360°×120° Scanning precision 0.1mm/50m Speed 1,200,000points/s Table 2. Faro Focus 3D X130 scanner and main technical indicators Data Set Year Format Feature of Point Cloud Producer S3DIS (Armeni et al., 2017) 2017 Point Cloud RGB-D (X,Y,Z,R,G,B) Stanford Scannet (Dai et al., 2017) 2017 Point Cloud RGB-D (X,Y,Z,R,G,B) Stanford Figure 2. Structure of PointNet This contribution has been peer-reviewed. https://doi org/10 5194/isprs-archives-XLIII-B2-2020-369-2020 \| © Authors 2020 CC BY 4 0 License 2. CONSTRUCTING THE INDOOR POINT CLOUD DATA SET The structure of PointNet network is showed in Figure 2. It utilized transformation network T-net for rigid transformation and use maximum symmetric function to handle the problem of disorder. The algorithm can be divided into 3 flowing steps: (1)Utilize T-Net to learn a transformation matrix and then multiply the matrix with point cloud to make sure that the rigid transformation steadiness of point cloud. The structure of PointNet network is showed in Figure 2. It utilized transformation network T-net for rigid transformation and use maximum symmetric function to handle the problem of disorder. The algorithm can be divided into 3 flowing steps: Currently,there are only 2 public large-scale indoor 3D point cloud data set which are showed in table 1. But these 2 public data set contain no reflection intensity information. Hence,we created a data set contains 4 areas, 8 semantic elements and 70,000,000 labeled points for better segmentation result of indoor structural elements. The point cloud of this data set contains not only spatial coordinate information (X,Y,Z), color information (R,G,B), but also reflection intensity information. We mainly utilized Faro Focus3D X130 scanner to acquire point cloud and the technical index of it are showed in Table 2. After acquiring the original point cloud, preprocessed the original point cloud like: registering, denoising, getting sparse. After prepocessing, labeled the point cloud manually and divided the data set into training set and testing set which is showed in Figure 1 . g g p (1)Utilize T-Net to learn a transformation matrix and then multiply the matrix with point cloud to make sure that the rigid transformation steadiness of point cloud. (2)Utilize MLP to extract high dimensional feature from the point cloud which has been transformed by T-Net and use maximum symmetric to manipulate the disordered point cloud to extract global feature. (3)Fuse the global and local features and then utilize MLP to downsample the features for the probability score to measure what type the points might belong to. Figure 2. Structure of PointNet Table 1. Current open large indoor point cloud datasets Configuration Faro Focus3D X130 Ranging/m 0.5～130 Distance Accuracy Index 0.6mm/10m Scanning View 360°×120° Scanning precision 0.1mm/50m Speed 1,200,000points/s Table 2. 3.2.2 Local feature extraction 1 pool pool values axis concat tf concat    Figure 5. Segmentation results of indoor dataset 4. RESULT ANALYSIS Figure 6. Comparison of Reflective including intensity and without intensity Figure 6. Comparison of Reflective including intensity and without intensity After modifying the PointNet network, we utilized this network to segment the S3DIS data set and the indoor data set we created. We Selected the Area1, Area2, Area3, Area4, Area6 as training set, and chose Area5 as testing set. Besides, we chose Area1, Area2, Area3 as training set and Area4 as testing set. To validate the effectiveness of the modified PointNet, we compared the segmentation result of using the modified network and original PointNet on S3DIS data set. We also compare the result of using the data set we constructed with reflection intensity and without reflection intensity. In addition, compare the result of using different kernel size of 3×3 and 1×1. We used Tensorflow deep learning framework, adam optimizer to train the network. The learning rate is 0.001,batch siez is 16, epoch is 100 and the training consumed 21hours. The hardware and software we used to train the network are showed in Table3 and Table 4. Figure 6. Comparison of Reflective including intensity and without intensity 3.2.1 The input Feature space Because of the difference of distance between points and the sparse structure of point cloud, the point cloud need to be converted to the format that can be understand by neural network. The 3D coordinate （X,Y,Z ）represents the spatial information and the color (R,G,B) represents the texture information. Also, the coordinates of original point cloud are normalized before input. The point cloud are divided according to the room they belongs to and then normalized base on the coordinate of the room. After acquiring the coordinate (X0,Y0,Z0), every room are divided into the area of 1m×1m. In addition, the reflection intensity information is added, because different structural elements contain different information of reflection intensity. Finally,every points in the point cloud are input as   0 0 0 , , , , , , , , Z Y X B G R Z Y X , the feature space of 10 dimension, into the neural network. This contribution has been peer-reviewed. https://doi.org/10.5194/isprs-archives-XLIII-B2-2020-369-2020 \| © Authors 2020. CC BY 4.0 License. 4.1 Result The segmentation results of using modified PointNet network to segment S3DIS data set are showed in Fig 4, and the results of segmenting the data set we constructed are showed in Figure5. The upper half part of the result figure shows input data, and the lower half part shows the segmentation result. The data set we constructed consist with the part contains reflection intensity and the part without that and Figure 6.shows the comparing result. The upper half part shows the result of using the intensity information, and the lower half part shows the result without using intensity information. The objects in the red pane are important objects needed to be compared Figure 6. shows that the result of upper half part is better than the lower one. 3.2.2 Local feature extraction During the process of optimizing the original PointNet network, we found that adding MLP with 4 layers on the original MLP helps to improve the precision of segmentation. Hence,we add 2 more layers of MLP behind the MLP-2 layer of the original network as the extracting layer for local features. Also,Adding a MLP layer behind the original MLP-5 and parallel with the extracting layer for local features as the extracting layer for global features. Compared with the original PointNet, our structure use more MLP layers to extracting the local feature, which have better ability to extract the correlation features between points. When optimizing the index of MLP layer network used to extract the high dimensional features, we found that the convolutional kernel in bigger size will get better segmentation precision during the semantic segmentation of indoor scene. So,we replace some of the 1×1 convolutional kernel of original PointNet network with 3×3 convolutional kernel to improve the segmentation precision of the network to segment the indoor structural elements. In addition, we add a MaxPooling layer behind the MLP-4 layer to extract the local feature for better ability to input the local feature of point cloud. Besides, let every point contain not only local feature but also global feature by fusing the local feature extracted by the first MaxPooling layer and the global feature extracted by the second MaxPooling layer. Utilizing the Concat operation of tensorflow framework to achieve the fusion of features. The algorithm of the Concat operation is showed in (1).   Figure 4. Segmentation Results of Area5 in S3DIS Dataset Figure 4. Segmentation Results of Area5 in S3DIS Dataset Figure 5. Segmentation results of indoor dataset Figure 6. Comparison of Reflective including intensity and without intensity Figure 4. Segmentation Results of Area5 in S3DIS Dataset Figure 5. Segmentation results of indoor dataset   ], 1, 4096 [,4 2 max_ . 1 VALID padding conv d pool tf pool   )' ' ], 1, 4096 [,8 ( 2 max_ . 2 VALID padding conv d pool tf pool   (1) ]) 2 ,1 [ ,3 ( . 1 pool pool values axis concat tf concat      ] [ _ p g p f p )' ' ], 1, 4096 [,8 ( 2 max_ . 2 VALID padding conv d pool tf pool   (1) ]) 2 ,1 [ ,3 ( . 3.2 Modify PointNet We modified the original PointNet network. First, add reflection intensity information of the point cloud into the feature space. Then we add a layer to extract local feature and then fuse the global feature and local feature. The modified network structure is showed in Figure 3. g Figure 3. The Structure of Modified PointNet indicators Figure 3. The Structure of Modified PointNet This contribution has been peer-reviewed. 370 The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLIII-B2-2020, 2020 XXIV ISPRS Congress (2020 edition) The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLIII-B2-2020, 2020 XXIV ISPRS Congress (2020 edition) The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLIII-B2-2020, 2020 XXIV ISPRS Congress (2020 edition) The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLIII-B2-2020, 2020 XXIV ISPRS Congress (2020 edition) 4.3 Result analysis Figure 5. shows the mIoU of every categories of using modified PointNet algorithm and original PoingNet algorithm to segment the Area5 in S3DIS. We found that the modified network got higher precision than PointNet except chair, which showed in Table 5. Consequently, the modification for PointNet of this paper is effective. 4.2 Precision norms According to the analysis, we think there are two main reasons: ①The ability of the modified network base on the PointNet is weak to learn the local details of wall and board, so the segmentation results are poor. ②The number of different semantic elements are not well-distributed, so the segmentation precision of the elements which contain small quantity, like lamp, will be lower. Hence, the following research could follow these two following aspects: (1)Keep on improving the structure of network for a better ability to learn the local features of point cloud and improve the segmentation precision. (2)Enlarge the size of data set and make sure the uniformity of the number of every categories. 5. CONCLUSION Network mIoU ceiling floor window door board lamp Publicity cabinet others PointNet (with I) 65.46 92.06 95.89 79.29 82.53 40.06 25.36 62.42 50.09 ointNet (without I) 64.97 91.27 94.76 77.21 81.39 38.52 24.79 61.63 50.16 Ours（with I） 68.24 92.93 96.23 80.68 85.37 42.63 26.43 63.56 50.58 Ours (without I) 66.78 92.13 95.53 78.36 83.12 41.73 25.63 62.26 50.45 Table 7 The Segmentation Accuracy of our Dataset Network mIoU ceiling floor window door board lamp Publicity cabinet others PointNet (with I) 65.46 92.06 95.89 79.29 82.53 40.06 25.36 62.42 50.09 PointNet (without I) 64.97 91.27 94.76 77.21 81.39 38.52 24.79 61.63 50.16 Ours（with I） 68.24 92.93 96.23 80.68 85.37 42.63 26.43 63.56 50.58 Ours (without I) 66.78 92.13 95.53 78.36 83.12 41.73 25.63 62.26 50.45 FN FP TP TP IoU    (2)     k i i IoU k mIoU 0 1 1 (3)       k i k j ij k i ii p p OA 0 0 0 (4) is (2) (3) (4) 5. CONCLUSION Focusing on the problem that PointNet network has poor ability to extract local feature of point cloud,this paper put forward a modified end-to-end deep neural network base on PointNet for semantic segmentation of indoor 3D LiDAR point cloud. Base on the structure of PointNet, we replac the original 1×1 convolutional kernel to 3×3 convolutional kernel. And add several layers of MLP and MaxPooling layer to improve the ability of extracting local feature. Also add a layer to fuse features for the segmentation task of indoor structural elements. At last we compare the results of using S3DIS and the data set Focusing on the problem that PointNet network has poor ability to extract local feature of point cloud,this paper put forward a modified end-to-end deep neural network base on PointNet for semantic segmentation of indoor 3D LiDAR point cloud. Base on the structure of PointNet, we replac the original 1×1 convolutional kernel to 3×3 convolutional kernel. And add several layers of MLP and MaxPooling layer to improve the ability of extracting local feature. Also add a layer to fuse features for the segmentation task of indoor structural elements. At last,we compare the results of using S3DIS and the data set we constructed and the original PointNet. The modified network base on PointNet acquires higher precision compared with the original PointNet according to the result which proves that the modification are effective Network PointNet （IoU） Ours （IoU） mIoU 53.87 56.84 ceiling 90.80 91.09 floor 95.33 96.13 wall 70.56 73.35 beam 10.25 0.15 columu 13.92 26.51 window 66.26 67.04 door 78.62 80.36 table 55.32 62.86 chair 54.75 54.53 sofa 15.32 29.26 bookcase 50.76 51.14 board 45.52 52.35 clutter 52.86 54.17 Table 5. IoU of Area5 in S3DIS Dataset Size of convolutiona l kernel in PointNet MIoU(%) OA(%) 1×1（original PointNet） 54.12% 80.59 3×3 54.96% 81.03 Table 6. Segmentation Results of PointNet with Different Convolution Kernels on S3DIS At last,we compare the results of using S3DIS and the data set we constructed and the original PointNet. The modified network base on PointNet acquires higher precision compared with the original PointNet according to the result, which proves that the modification are effective. This contribution has been peer-reviewed. 4.2 Precision norms The Intersection over (IoU), mean Intersection over Union (mIoU), overall accuracy (OA) are frequently-used norms in 371 The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLIII-B2-2020, 2020 XXIV ISPRS Congress (2020 edition) The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLIII-B2-2020, 2020 XXIV ISPRS Congress (2020 edition) The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLIII-B2-2020, 2020 XXIV ISPRS Congress (2020 edition) Table 7. The Segmentation Accuracy of our Dataset omestic and foreign countries to evaluate segmentation on of every semantic elements. Therefore, this paper use IoU and OA as norms to evaluate the precision. IoU is o of the area of segmentation result to the area of ground mIoU is the average of IoU, OA is the ratio of the number points accurately segmented to the number of total points. gorithm of IoU is showed in (2),TP means true positive, ns false positive,FN means false negative. The algorithm U is showed in (3), the algorithm of OA is showed in (4), means there are K+1 types, P represent every point and resents the number of the points are correctly segmented, esents the points belong to type i but segmented as type j, resents the points belong to type j but segmented as type FN FP TP TP IoU    (2)     k i i IoU k mIoU 0 1 1 (3)       k i k j ij k i ii p p OA 0 0 0 (4) esult analysis 5. shows the mIoU of every categories of using modified Table 7. shows the comparison of the results of using both modified network and original PointNet to segment the data set with reflection intensity and without intensity. Table 6. shows that adding reflection intensity information into the point cloud helps to improve the segmentation precision of segmenting door, window, board and so on no matter we use the modified network or the original PointNet. However,the improvement is not obvious for ceiling, floor and others. The segmentation results of using modified network and the data set we constructed are better than using original PointNet on segmenting ceiling, floor, window, door and so on. However the segmentation results of segmenting board and lamp are not good enough, which the precision only achieve 45%. REFERENCES HIMMELSBACH M, Luettel T, Wuensche H., 2009. Real-time object classification in 3D point clouds using point feature histograms. International Conference on IEEE: 994-1000. LI Na, MA Yi-wei, YANG Yang et al., 2011. Segmentation of Building Facade Point Clouds using RANSAC. Science of Surveying and Mapping, 36(5):144-145 ZHANG Fan, GAO Yunlong, HUANG Xianfeng et al.,2015. Spherical Projection Based Straight Line Segment Extraction for Single Station Terrestrial Laster Point Cloud. Acta Geodaetica et Cartographica Sinica, 44 (6):655-662. ZHANG Fan, GAO Yunlong, HUANG Xianfeng et al.,2015. Spherical Projection Based Straight Line Segment Extraction for Single Station Terrestrial Laster Point Cloud. Acta Geodaetica et Cartographica Sinica, 44 (6):655-662. 372 The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLIII-B2-2020, 2020 XXIV ISPRS Congress (2020 edition) The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLIII-B2-2020, 2020 XXIV ISPRS Congress (2020 edition) HU Wei, LU Xiaoping, LI Cheng et al.,2012. Extended RANSAC Algorithm for Building Roof Segmentation from Li DAR Data. Bulletin of Surveying and Mapping,11:31-34 CHEN Xiangyang, YANG Yang, XIANG Yunfei, 2012. Measurement of Point Cloud Data Segmentation Based on Euclidean Clustering Algorithm. Bulletin of Surveying and Mapping, (11): 31-34+39. SUN Hongyan, SUN Xiaopeng, LI Hua, 2006. 3D Point Cloud Model Segmentation Based on K-means Cluser Analysis. Computer Engineering and Applications, (10): 42-45 LIN Xiangguo, NING Xiaogang, XIA Shaobo, 2016. A method For powerline LiDAR point cloud segmentation using K- meaning clustering of a feature space. Science of Surveying and Mapping, 41(5):60-63 RUSU R B, Blodow N, Marton Z C et al., 2008. Aligning point clouds views using persistent feature histograms. IEEE/RSJ International Conference on Intelligent Robots and Systems, 3384-3391. RUSU R B, Blodow N, Beetz M., 2009. Fast point feature histograms (FPFH) for 3D registration. IEEE International Conference on Robotics & Automation. ALDOMA A, Vincze M, Blodow N et al., 2011. CAD model recognition and 6 DOF pose estimation using 3D cues. IEEE International Conference on Computer Vision Workshops, ICCV 2011 Workshops, Barcelona, Spain, November:6-13. Su H, Maji S, Kalogerakis E et al., 2015. Multi-view Convolutional Neural Networks for 3D Shape Recognition. IEEE International Conference on Computer Vision, 945-953. Daniel M and Sebastian S., 2015. VoxNet:A 3D convolutional neural network for real-time object recognition. Proceedings of 2015 IEEE/RSJ International Conference on Intelligent Robots and Systems, Congress Center Hamburg, Sept 28-Oct 2. Hamburg, Germany, 922-928. C.R.Qi, H.Su, K.Mo et al., 2016. REFERENCES Pointnet: Deep learning on point sets for 3d classification and segmentation. Computer Vision and pattern Recognition, 217-223. Armeni I, Sax S, Zamir A R et al.,2017. Joint 2D-3D-Semantic Data for Indoor Scene Understanding. Computer Vision and pattern Recognition, 113-118. Dai A, Chang A X, Savva M et al., 2017. ScanNet: Richly- annotated 3D Reconstructions of Indoor Scenes. Computer Vision and pattern Recognition, 261-268 This contribution has been peer-reviewed. https://doi.org/10.5194/isprs-archives-XLIII-B2-2020-369-2020 \| © Authors 2020. CC BY 4.0 License. APPENDIX ( Foundation Support: the National Natural Science Foundation of China,No.41601409; National Natural Science Foundation of China,No.41501495 Foundation Support: the National Natural Science Foundation of China,No.41601409; National Natural Science Foundation of China,No.41501495 373
https://openalex.org/W2995503356	https://res.mdpi.com/d_attachment/remotesensing/remotesensing-11-02942/article_deploy/remotesensing-11-02942-v2.pdf	English	null	Spectral Diversity Successfully Estimates the α-Diversity of Biocrust-Forming Lichens	Remote sensing	2,019	cc-by	11,985	Received: 18 September 2019; Accepted: 6 December 2019; Published: 9 December 2019 Abstract: Biocrusts, topsoil communities formed by mosses, lichens, liverworts, algae, and cyanobacteria, are a key biotic component of dryland ecosystems worldwide. Experiments carried out with lichen- and moss-dominated biocrusts indicate that climate change may dramatically reduce their cover and diversity. Therefore, the development of reproducible methods to monitor changes in biocrust diversity and abundance across multiple spatio-temporal scales is key for evaluating how climate change may impact biocrust communities and the myriad of ecosystem functions and services that rely on them. In this study, we collected lichen-dominated biocrust samples from a semi-arid ecosystem in central Spain. Their α-diversity was then evaluated using very high spatial resolution hyperspectral images (pixel size of 0.091 mm) measured in laboratory under controlled conditions. Support vector machines were used to map the biocrust composition. Traditional α-diversity metrics (i.e., species richness, Shannon’s, Simpson’s, and Pielou’s indices) were calculated using lichen fractional cover data derived from their classiﬁcations in the hyperspectral imagery. Spectral diversity was calculated at diﬀerent wavelength ranges as the coeﬃcient of variation of diﬀerent regions of the reﬂectance spectra of lichens and as the standard deviation of the continuum removal algorithm (SD_CR). The accuracy of the classiﬁcations of the images obtained was close to 100%. The results showed the best coeﬃcient of determination (r2 = 0.47) between SD_CR calculated at 680 nm and the α-diversity calculated as the Simpson’s index, which includes species richness and their evenness. These ﬁndings indicate that this spectral diversity index could be used to track spatio-temporal changes in lichen-dominated biocrust communities. Thus, they are the ﬁrst step to monitor α-diversity of biocrust-forming lichens at the ecosystem and regional levels, a key task for any program aiming to evaluate changes in biodiversity and associated ecosystem services in drylands. Keywords: biocrusts; biological soil crust; spectral diversity; chlorophyll; continuum removal; biodiversity; α-diversity; support vector machine; remote sensing Spectral Diversity Successfully Estimates the α-Diversity of Biocrust-Forming Lichens Javier Blanco-Sacristán 1,* , Cinzia Panigada 1, Giulia Tagliabue 1, Rodolfo Gentili 1 , Roberto Colombo 1, Mónica Ladrón de Guevara 2,3, Fernando T. Maestre 4 and Micol Rossini 1 1 Remote Sensing of Environmental Dynamics Lab, University of Milano – Bicocca, 20126 Milan, Italy; cinzia.panigada@unimib.it (C.P.); giulia.tagliabue@unimib.it (G.T.); rodolfo.gentili@unimib.it (R.G.); roberto.colombo@unimib.it (R.C.); micol.rossini@unimib.it (M.R.) ( ); ( ) 2 Universidad Rey Juan Carlos, 28933 Móstoles, Spain; monigue28@yahoo.es 3 Centre for Ecological Research and Forestry Applications, CREAF CSIC UAB, 08193 Barcelona, Spain 4 Department of Ecology, University of Alicante, 03690 San Vicente del Raspeig, Alicante, Spain; ft.maestre@gmail.com * Correspondence: javier.blanco@unimib.it; Tel.: +34-610-681-243 remote sensing remote sensing remote sensing 1. Introduction Biocrusts are communities formed by photoautotrophic (algae, lichens, cyanobacteria, liverworts, and bryophytes) and heterotrophic (bacteria, fungi, protozoa, and nematodes) organisms that live on the soil surface and cover a large part of the non-vegetated surface in drylands worldwide [1]. These communities inﬂuence fundamental ecosystem processes in drylands, including—but not limited Remote Sens. 2019, 11, 2942; doi:10.3390/rs11242942 www.mdpi.com/journal/remotesensing 2 of 16 Remote Sens. 2019, 11, 2942 to—nutrient cycling, soil respiration, and runon-runoﬀdynamics [2], and are critical for maintaining the multiplicity of ecosystem services they provide [3]. Attributes of biocrust communities, such as their cover, composition, and diversity, largely modulate their impacts on multiple ecosystem functions simultaneously [4,5], and thus have been suggested as indicators of ecosystem functioning in drylands [6,7]. Multiple lines of evidence suggest that ongoing climate change can dramatically aﬀect biocrust communities, reducing their cover and diversity [8]. Reference [9] estimated a global reduction of their cover by 40% globally within the next 65 years, and quick reductions in their cover have already been observed in experiments simulating climate change [10–12]. As biocrusts contribute to ecosystem multifunctionality [2,11,13,14], changes in their composition, cover and diversity could lead to a reduction of the capacity of drylands to provide essential ecosystem services such as atmospheric CO2 sequestration and the maintenance of soil fertility. For this reason, ﬁnding accurate and operational methods to estimate the cover and diversity of biocrust constituents is a key goal for any ecosystem monitoring program in drylands. Remote sensing has been highlighted several times as an important tool for biodiversity monitoring and conservation [15–18]. It is one of the most cost-eﬀective approaches to identify biodiversity hotspots and to predict changes in species composition, potentially providing repeated measurements and making it possible to study temporal changes in biodiversity [19]. In this context, the spectral variation hypothesis (SVH) proposes that the larger the spectral heterogeneity of an environment is, the higher the number of species found here will be [20]. This hypothesis has been validated several times using α-diversity metrics with vascular plants [21–25]. Even though measures of spectral diversity based on spectral dispersion (i.e., the coeﬃcient of variation (CV) or the standard deviation (SD)) have shown good correlations with the richness, diversity, evenness, and composition of vascular plants [25–27], the SVH has never been tested using lichens. 1. Introduction The use of optical remote sensing of biocrusts so far has mainly focused on mapping and monitoring their distribution [28–33] and only one study [34] investigated lichens’ α-diversity at the landscape level through correlation of high-resolution data with ﬁeld samples. Since biocrusts are spectrally characterized by narrow absorption features in speciﬁc spectral regions [35], hyperspectral data have shown potential when discriminating them from vegetation and other soil features (e.g., [35–37]). However, confounding factors such as water content and biocrust tridimensional structure might hinder their spectral characterization. For this reason, several authors [31,33,35,38–40] proposed the use of the continuum removal (CR) algorithm [41] to ensure the comparability of biocrusts’ absorption features. The CR quantiﬁes the absorption features at speciﬁc wavelengths, normalizing the reﬂectance spectra to a common baseline. This is achieved by approximating the continuum between local spectral maxima through straight-line segments: a value of 1 is assigned to the local maxima, and a value between 0 and 1 is obtained in correspondence of the absorption features. This approach might be particularly useful to enhance the discrimination of lichens, which are characterized by subtle spectral diﬀerences [42]. In this framework, the objective of this study was to apply the SVH to capture the α-diversity of lichens to support dryland monitoring by (i) evaluating the potential of hyperspectral high spatial resolution images to identify biocrust-forming lichens (at the genus level); (ii) exploring the relationships between spectral diversity metrics and the α-diversity of lichens within biocrust communities. 2.1. Study Area and Sampling Location of the study area in central Spain and some examples of the dominant lichen communities found in the area. 2.2. Hyperspectral Imagery Acquisition Hyperspectral images of the biocrust samples were acquired with a hyperspectral scanning imaging system [45] measuring spectral reflectance in 840 bands in the visible (VIS) and near-infrared (NIR) spectral region (i.e., 380–1000 nm), with a spectral resolution of 2–3 nm (calculated at full width at half maximum; Figure 2) The system consists of a custom high precision linear stage that embeds Following the sampling protocol described in [30], we collected 54 biocrust samples using Petri dishes of 8.5 cm of diameter from 18 plots (three samples per plot randomly chosen). These plots were distributed following two altitudinal transects in two separated slopes of the study area with diﬀerent exposures (north- vs. south-facing). Each transect was divided in three levels depending on the height of the transect respect to its base, placing three plots on each level. 2.1. Study Area and Sampling The study area is located in Aranjuez, central Spain (40◦01′53.9”N 3◦32′50.8”W; Figure 1). The climate is semi-arid Mediterranean, with mean annual temperature and rainfall of 15 ◦C and 349 mm, respectively. The plant cover is below 40%, and is dominated by Macrochloa tenacissima and other small shrubs, such as Helianthemum squamatum and Gypsophila struthium. A well-developed biocrust 3 of 16 3 of 16 Remote Sens. 2019, 11, 2942 Remote Sens. 2019, 11, x FOR community, a mixture of lichens and mosses, is present between vegetation, covering up to more than 50% of the plant interspaces. In lichen-dominated biocrusts, Acarospora spp., Buellia spp., Diploschistes spp., Fulgensia spp., Psora spp., and Squamarina spp. are the most abundant genera, while in moss-dominated biocrusts, Pleurochaete squarrosa, Tortula revolvens, and Didymodon acutus are the most abundant mosses. Since this study focused on lichens, we decided to cluster the mosses identiﬁed (under the term Moss). Finally, in patches of soil dominated by cyanobacteria, genera as Microcoleus spp., Tolypothrix spp., and Nostoc spp. are present [43]. See [44] for a species list of the biocrust-forming lichens and mosses in our study area. most abundant mosses. Since this study focused on lichens, we decided to cluster the mosses identified (under the term Moss). Finally, in patches of soil dominated by cyanobacteria, genera as Microcoleus spp., Tolypothrix spp., and Nostoc spp. are present [43]. See [44] for a species list of the biocrust-forming lichens and mosses in our study area. Following the sampling protocol described in [30], we collected 54 biocrust samples using Petri dishes of 8.5 cm of diameter from 18 plots (three samples per plot randomly chosen). These plots were distributed following two altitudinal transects in two separated slopes of the study area with different exposures (north- vs. south-facing). Each transect was divided in three levels depending on the height of the transect respect to its base, placing three plots on each level. Figure 1. Location of the study area in central Spain and some examples of the dominant lichen communities found in the area. Figure 1. Location of the study area in central Spain and some examples of the dominant lichen communities found in the area. Figure 1. Location of the study area in central Spain and some examples of the dominant lichen communities found in the area. Figure 1. g ) y a hyperspectral imaging spectromet d di t d h l t bl li ht 2.2. Hyperspectral Imagery Acquisition dedicated halogen stable light source (i.e. 600 or 1000 W, LOT Quantum Design). Powered by an electrical engine, both the spectrometer and the halogen light source are able to move back and forth at a defined speed. The system is a push-broom scanner and it measures lines of 1004 pixels while moving forward by means of the linear stage until an image of the whole studied object is collected. We used a water-filter tube between the lamp and the mirror reflecting the light to the samples to avoid sample overheating. Hyperspectral images of the biocrust samples were acquired with a hyperspectral scanning imaging system [45] measuring spectral reﬂectance in 840 bands in the visible (VIS) and near-infrared (NIR) spectral region (i.e., 380–1000 nm), with a spectral resolution of 2–3 nm (calculated at full width at half maximum; Figure 2). The system consists of a custom high-precision linear stage that embeds a hyperspectral imaging spectrometer (Hyperspec® VNIR, HeadWall Photonics, USA) and a dedicated halogen stable light source (i.e., 600 or 1000 W, LOT Quantum Design). Powered by an electrical engine, both the spectrometer and the halogen light source are able to move back and forth at a deﬁned speed. The system is a push-broom scanner and it measures lines of 1004 pixels while moving forward by means of the linear stage until an image of the whole studied object is collected. We used a water-ﬁlter tube between the lamp and the mirror reﬂecting the light to the samples to avoid sample overheating. 4 of 16 4 f 16 f 16 Remote Sens. 2019, 11, 2942 R t S 2019 11 FOR R t S 2019 11 FO Figure 2. Picture of the system used to acquire the hyperspectral imagery. The system consists of: (a) samples holder and motor driver; (b) HeadWall VNIR camera thermostated by heaters; (c) stable halogen lamp; (d) calibrated Lambertian Spectralon panel; (e) PC connected to the spectrometer by a dedicated interface. Figure 2. Picture of the system used to acquire the hyperspectral imagery. The system consists of: (a) samples holder and motor driver; (b) HeadWall VNIR camera thermostated by heaters; (c) stable halogen lamp; (d) calibrated Lambertian Spectralon panel; (e) PC connected to the spectrometer by a dedicated interface. Table 1. Diversity metrics used in this study. pi is the fractional cover of the ith class α-diversity metric. g ) y a hyperspectral imaging spectromet d di t d h l t bl li ht 2.2. Hyperspectral Imagery Acquisition Formula Species richness (S) S = Number of classes Shannon’s index (H’) H′ = −∑pi ∗ ln (pi) Reciprocal of Simpson’s index (D) D = 1 / ∑pi 2 Pielou’s index (J’) J′ = H′ / ln (S) 2.7. Statistical Analysis To evaluate the spectral diversity metrics that better capture the α-diversity of biocrust-forming ichens, we evaluated through linear regression models the relationships between spectral diversity calculated as the average CV and CR of the three samples from each plot) and α-diversity metrics species richness, Shannon’s index, Simpson’s index, and Pielou’s index), calculated using the ractional cover of each plot (the average of three samples). Due to the high heterogeneity of four plots, their samples were not averaged, and the values of the single samples were used in the analysis. Species richness was calculated as the total number of genera observed in the three samples from each plot. Due to the small sample size (n = 26) in this analysis, the cross-validated statistics obtained with the leave-one-out cross-validation procedure were also computed to compare performances of Figure 2. Picture of the system used to acquire the hyperspectral imagery. The system consists of: (a) samples holder and motor driver; (b) HeadWall VNIR camera thermostated by heaters; (c) stable halogen lamp; (d) calibrated Lambertian Spectralon panel; (e) PC connected to the spectrometer by a dedicated interface. Figure 2. Picture of the system used to acquire the hyperspectral imagery. The system consists of: (a) samples holder and motor driver; (b) HeadWall VNIR camera thermostated by heaters; (c) stable halogen lamp; (d) calibrated Lambertian Spectralon panel; (e) PC connected to the spectrometer by a dedicated interface. p ( g p ) g g y ots, their samples were not averaged, and the values of the single samples were used in the analysis pecies richness was calculated as the total number of genera observed in the three samples from ach plot. Due to the small sample size (n = 26) in this analysis, the cross-validated statistics obtained ith the leave-one-out cross-validation procedure were also computed to compare performances of Two sets of images were collected: one with the dry samples (dry set) and another 10 minutes after evenly watering the samples with 30 ml of distilled water (wet set). A total of 18 images (six samples on each set; Figure 3) with a spatial resolution of 0.091 mm were taken. g ) y a hyperspectral imaging spectromet d di t d h l t bl li ht 2.2. Hyperspectral Imagery Acquisition A calibrated white Lambertian Spectralon® panel (Labsphere, North Sutton, USA) was placed close to the samples to calculate the reflectance as the ratio between the radiance reflected by the biocrust samples and the panel. The instrument’s dark current signal was measured by manually closing the imaging spectrometer aperture prior to capturing each image and subtracted from the measured radiance. Two sets of images were collected: one with the dry samples (dry set) and another 10 min after evenly watering the samples with 30 mL of distilled water (wet set). A total of 18 images (six samples on each set; Figure 3) with a spatial resolution of 0.091 mm were taken. A calibrated white Lambertian Spectralon® panel (Labsphere, North Sutton, USA) was placed close to the samples to calculate the reﬂectance as the ratio between the radiance reﬂected by the biocrust samples and the panel. The instrument’s dark current signal was measured by manually closing the imaging spectrometer aperture prior to capturing each image and subtracted from the measured radiance. different spectral diversity metrics in predicting α diversity. 3. Results 3.1. Classifications and Accuracy Evaluation The classification (Figure 3) of both dry and wet images was highly accurate. The high values of the AUC derived from the ROC curves showed that the SVMs used were successfully trained to classify biocrust-forming lichens (Figure 4). .3. Images Processing and Classification Both sets of images were pre-processed and classified to characterize the composition of each ample. First, the parts in the images not belonging to the samples were masked and the reflectanc was computed between 420 and 900 nm. To reduce the noise in the measured signal, a Savitzky-Golay moothing filter [46] was applied using a 20 band-window width. A minimum noise fraction (MNF ransformation was applied to the smoothed reflectances to synthesize the main information in educed number of MNF components as well as to remove the residual noise in the data. The first 15 MNF components of each image transformation were used as input of the classification algorithm. The training set for the classification was created by selecting pure endmembers of each classe by visual-identification on the images of the samples. The following classes were considered in th lassification process: Acarospora (Acarospora spp.), Buellia (Buellia spp.), Diploschistes (Diploschiste Figure 3. True color composite samples (upper images) from one of the hyperspectral images used and the classification derived (lower images). Figure 3. p p ( spp.), Fulgensia (Fulgensia spp.), Psora ( Bi t l ifi ti f d The classification of the wet datas 2.3. Images Processing and Classiﬁcation Biocrust classification was performed with a supervised machine learning method, using a total of around 1000 pixels per class identified in each image to train the support vector machine (SVM) algorithm. The SVM is a supervised classification method based on statistical learning theory [47,48]. Using training samples from the classes of interest, it separates them by a decision surface, called hyperplane, that maximizes the margin between them. The closest training samples to the hyperplane are the ones used by the algorithm, called support vectors. SVM is a binary classifier in its simplest form but can also act as a multiclass classifier by creating a binary classifier for each possible pair of of 0.97 and an overall accuracy of 97.83% (Table 2). The lowest accuracies were achieved classifying Moss and Fulgensia, with AUCs of 0.91 and 0.92, respectively. The most accurate results were obtained for Buellia and Psora, with AUCs values of 0.99 and 0.98, respectively. Bare soil was the most confused class, mostly with moss, even though it was better classified than in the dry set of images, where it presented an AUC of 0.82. The dry set was less accurate, presenting an average AUC of 0.93, an average kappa coefficient of 0.95 and an overall accuracy of 95.69%. All the lichens were accurately classified, with values of AUC over 0.95, similar to those obtained from the wet set. Moss presented the lowest accuracy with an AUC of 0 75 and was the most misclassified being confused mostly Both sets of images were pre-processed and classiﬁed to characterize the composition of each sample. First, the parts in the images not belonging to the samples were masked and the reﬂectance was computed between 420 and 900 nm. To reduce the noise in the measured signal, a Savitzky-Golay smoothing ﬁlter [46] was applied using a 20 band-window width. A minimum noise fraction (MNF) transformation was applied to the smoothed reﬂectances to synthesize the main information in a reduced number of MNF components as well as to remove the residual noise in the data. The ﬁrst 15 MNF components of each image transformation were used as input of the classiﬁcation algorithm. form, but can also act as a multiclass classifier by creating a binary classifier for each possible pair of classes. SVM uses a penalty parameter that allows a certain degree of misclassification. g ) y a hyperspectral imaging spectromet d di t d h l t bl li ht 2.2. Hyperspectral Imagery Acquisition True color composite samples (upper images) from one of the hyperspectral images used and the classiﬁcation derived (lower images). The training set for the classification was created by selecting pure endmembers of each cla visual-identification on the images of the samples. The following classes were considered in sification process: Acarospora (Acarospora spp ), Buellia (Buellia spp ), Diploschistes (Diplosch Figure 3. True color composite samples (upper images) from one of the hyperspectral images used and the classification derived (lower images). Figure 3. True color composite samples (upper images) from one of the hyperspectral images used and the classiﬁcation derived (lower images). p p ( spp.), Fulgensia (Fulgensia spp.), Psora ( Bi t l ifi ti f d The classification of the wet datas 2.3. Images Processing and Classiﬁcation Fine tuning is important to avoid overfitting of the model because increasing its value increases the cost of misclassifying points and forces the creation of a more accurate model, which might be not the lowest accuracy, with an AUC of 0.75, and was the most misclassified, being confused mostly with bare soil. The training set for the classiﬁcation was created by selecting pure endmembers of each classes by visual-identiﬁcation on the images of the samples. The following classes were considered in the classiﬁcation process: Acarospora (Acarospora spp.), Buellia (Buellia spp.), Diploschistes (Diploschistes spp.), 5 of 16 Remote Sens. 2019, 11, 2942 Fulgensia (Fulgensia spp.), Psora (Psora spp.), Squamarina (Squamarina spp.), Moss and Bare Soil. Biocrust classiﬁcation was performed with a supervised machine learning method, using a total of around 1000 pixels per class identiﬁed in each image to train the support vector machine (SVM) algorithm. The SVM is a supervised classiﬁcation method based on statistical learning theory [47,48]. Using training samples from the classes of interest, it separates them by a decision surface, called hyperplane, that maximizes the margin between them. The closest training samples to the hyperplane are the ones used by the algorithm, called support vectors. SVM is a binary classiﬁer in its simplest form, but can also act as a multiclass classiﬁer by creating a binary classiﬁer for each possible pair of classes. SVM uses a penalty parameter that allows a certain degree of misclassiﬁcation. Fine tuning is important to avoid overﬁtting of the model because increasing its value increases the cost of misclassifying points and forces the creation of a more accurate model, which might be not generalizable. We used a radial basis kernel function, which usually performs well in remote sensing applications [49]. This kernel is controlled by two parameters that determine the ﬁnal classiﬁcation accuracy, the penalty parameter (C) and the width of the Gaussian kernel (c). A large C reduces the training dataset error, but may result in model overﬁtting to the training data, reducing their generalizability. We used the SVM in a pairwise classiﬁcation way to classify the images and evaluate the best parameters to use for each classiﬁcation using the LIBVSM library [31,50]. 2.4. Validation of Classiﬁcations The validation set for each classiﬁcation was created selecting 300 pixels per class of pure endmembers by visual-identiﬁcation. We assessed the accuracy of the classiﬁcations using the receiver operating characteristics (ROC) technique [51] and the area under the curve (AUC) [52,53] derived from it. A ROC graph is a two-dimensional depiction of a classiﬁer’s performance [54] and is constructed calculating the sensitivity and speciﬁcity of the resulting classiﬁcation for each possible classiﬁcation threshold, where sensitivity = a/a + c specificity = b/b + d, sensitivity = a/a + c specificity = b/b + d, specificity = b/b + d, with a and d as the true positives and the true negatives for a certain classiﬁcation, respectively, and b and c as the corresponding false positives and false negatives. The ‘sensitivity’ is the probability that a pixel of a particular class is correctly classiﬁed, while the ‘speciﬁcity’ is the probability that a pixel not belonging to that class is correctly classiﬁed. In this way, the best performing classiﬁcation would be that with the highest possible value of both sensitivity and speciﬁcity. We evaluated the probability of detection (calculated as the sensitivity) against the false positive rate (calculated as 1 – speciﬁcity). A ROC graph was calculated for each of the classiﬁcations produced (i.e., 18 classiﬁcations, six samples each), averaging the curves obtained for each class. From each ROC graph, we calculated its corresponding area under the curve (AUC), that is an overall quantitative performance score of the classiﬁcation that allows to reduce the ROC performance to a single scalar value independent of a single prediction threshold [52,54]. The AUC represents the probability that a randomly chosen positive sample is correctly classiﬁed with greater suspicion than a randomly chosen negative one [53]. This value might range from 0.5 (a random assignment to the class of interest) to 1 (a perfect classiﬁcation). The AUC values were calculated by joining the points of the ROC through a composite trapezoid rule using the AUC function from the R package DescTools [55]. To assess classiﬁcation accuracy, we also evaluated the average accuracy creating a confusion matrix with the average accuracies from the classiﬁcations of each set of images (i.e., dry and wet), extracting the overall accuracy and Cohen’s kappa coeﬃcient [56]. The points used to create these matrices were the same used to create the ROC curves. Remote Sens. 2019, 11, 2942 6 of 16 2.5. Computation of the Spectral Diversity Using the classiﬁcations previously obtained, we calculated, for each biocrust sample, the average spectral reﬂectance of each lichen genera from the reﬂectance images. We then calculated the pigment absorption features by the continuum removal method, normalizing the spectra to a common baseline. Several metrics were then calculated as indicators of spectral diversity for each sample: the coeﬃcient of variation (CV; i.e., ratio of the standard deviation to the mean), calculated between 420–900 nm (CV420-900), between 550–750 nm (CV550–750), and at 680 nm (CV680), and the standard deviation of the continuum removal (SD_CR), calculated between 550–750 nm (SD_CR550–750; i.e., absorption feature related to the presence of chlorophyll) and at 680 nm (SD_CR680; i.e., maximum peak of this absorption feature). 2.7. Statistical Analysis To evaluate the spectral diversity metrics that better capture the α-diversity of biocrust-forming lichens, we evaluated through linear regression models the relationships between spectral diversity (calculated as the average CV and CR of the three samples from each plot) and α-diversity metrics (species richness, Shannon’s index, Simpson’s index, and Pielou’s index), calculated using the fractional cover of each plot (the average of three samples). Due to the high heterogeneity of four plots, their samples were not averaged, and the values of the single samples were used in the analysis. Species richness was calculated as the total number of genera observed in the three samples from each plot. Due to the small sample size (n = 26) in this analysis, the cross-validated statistics obtained with the leave-one-out cross-validation procedure were also computed to compare performances of diﬀerent spectral diversity metrics in predicting α-diversity. 2.6. Biodiversity Metrics The fractional cover of each lichen class (classiﬁed at the genus level) was used to calculate the following α-diversity metrics for each sample: species richness (S), Shannon’s index (H’; [57]), Simpson’s index (D; [58]) and Pielou’s index (J’; [59]). The details of the calculation are reported in Table 1. These metrics were calculated using the ‘vegan’ R package, version 2.4–5 [60]. Table 1. Diversity metrics used in this study. pi is the fractional cover of the ith class Table 1. Diversity metrics used in this study. pi is the fractional cover of the ith class α-Diversity Metric. Formula Species richness (S) S = Number of classes Shannon’s index (H’) H′ = −Ppi ∗ln (pi) Reciprocal of Simpson’s index (D) D = 1 / Ppi 2 Pielou’s index (J’) J′ = H′ / ln (S) tical Analysis 3.1. Classiﬁcations and Accuracy Evaluation Fulgensia 0 0.04 0 0 99.31 0 0.05 0 Moss 0 0.76 0 0 0.05 99.65 0.3 0 Psora 0 0.02 0 0 0.05 0.03 97.29 0 quamarina 0 0 0.06 0.09 0 0 0 98.79 2 Spectral Characterization of Biocrusts Table 2. Average confusion matrix obtained crossing the ground truth (columns) with the results of the classiﬁcation (lines) performed on wet samples. 300 pixels per class in each image were randomly selected as validation set to create this matrix. A total of 20,400 random selected pixels were used. The ground truth (%) shows the class distribution in percent for each ground truth class. 3.2. Spectral Characterization of Biocrusts Dry biocrusts had increasing reflectances from the blue region until 700 nm. In the visible region the reflectances of various biocrust classes differ because of different pigment content and composition (Figure 5) differences in the NIR region are mainly related to the biocrust tridimensiona structure. Lichens presented higher reflectances than bare soil and moss across the whole spectrum Their higher reflectance in the NIR region is related to their more developed structure, which cause multi-scattering of light in this region. Acarospora, Squamarina, Buellia, and Diploschistes are light colored lichens that showed higher reflectances in the visible region; Psora presented the lowes reflectance among lichens. Mosses had the lowest reflectance due to their darker color and les developed structure, resulting in a reduction of light scattering. The application of the continuum Ground truth (%) Acarospora Bare Soil Buellia Diploschistes Fulgensia Moss Psora Squamarina Acarospora 99.96 0 0 0.04 0 0 0 0 Bare soil 0.04 97.55 0 0.72 0.4 0.32 1.06 0 Buellia 0 0 93.45 4.26 0 0 0 0.98 Diploschistes 0 1.63 6.49 94.89 0.24 0 1.3 0.23 Fulgensia 0 0.04 0 0 99.31 0 0.05 0 Moss 0 0.76 0 0 0.05 99.65 0.3 0 Psora 0 0.02 0 0 0.05 0.03 97.29 0 Squamarina 0 0 0.06 0.09 0 0 0 98.79 3.2. Spectral Characterization of Biocrusts g developed structure, resulting in a red 3.2. Spectral Characterization of Biocrusts removal algorithm in the spectral range 450–900 nm highlighted the absorption features caused by different pigments. The chlorophyll absorption feature at 680 nm was present in all the classes. Bare soil showed a weak absorption feature at 680 nm, evidencing the presence of cyanobacteria colonizing the soils interspace within lichens and moss patches. 3.1. Classiﬁcations and Accuracy Evaluation The classiﬁcation (Figure 3) of both dry and wet images was highly accurate. The high values of the AUC derived from the ROC curves showed that the SVMs used were successfully trained to classify biocrust-forming lichens (Figure 4). 7 of 16 7 f 16 Remote Sens. 2019, 11, 2942 O ote Sens. 2019, 11, x FOR PEER REVIEW 7 o Figure 4. ROC curves of the genera classifications and the derived AUC values of the wet samples. Figure 4. ROC curves of the genera classiﬁcations and the derived AUC values of the wet samples. Figure 4. ROC curves of the genera classifications and the derived AUC values of the wet samples. Figure 4. ROC curves of the genera classiﬁcations and the derived AUC values of the wet samples. Table 2. Average confusion matrix obtained crossing the ground truth (columns) with the results of the classification (lines) performed on wet samples. 300 pixels per class in each image were randomly selected as validation set to create this matrix. A total of 20400 random selected pixels were used. The ground truth (%) shows the class distribution in percent for each ground truth class. Ground truth (%) Acarospora Bare Soil Buellia Diploschistes Fulgensia Moss Psora Squamarina Acarospora 99.96 0 0 0.04 0 0 0 0 Bare soil 0.04 97.55 0 0.72 0.4 0.32 1.06 0 Buellia 0 0 93.45 4.26 0 0 0 0.98 Diploschistes 0 1.63 6.49 94.89 0.24 0 1.3 0.23 The classiﬁcation of the wet dataset had an average AUC of 0.95 (Figure 4), a kappa coeﬃcient of 0.97 and an overall accuracy of 97.83% (Table 2). The lowest accuracies were achieved classifying Moss and Fulgensia, with AUCs of 0.91 and 0.92, respectively. The most accurate results were obtained for Buellia and Psora, with AUCs values of 0.99 and 0.98, respectively. Bare soil was the most confused class, mostly with moss, even though it was better classiﬁed than in the dry set of images, where it presented an AUC of 0.82. The dry set was less accurate, presenting an average AUC of 0.93, an average kappa coeﬃcient of 0.95 and an overall accuracy of 95.69%. All the lichens were accurately classiﬁed, with values of AUC over 0.95, similar to those obtained from the wet set. Moss presented the lowest accuracy, with an AUC of 0.75, and was the most misclassiﬁed, being confused mostly with bare soil. 3.1. Classiﬁcations and Accuracy Evaluation Psora showed an absorption feature around 550 nm due to phycoerythrin, which is absent in the other lichens. An absorption feature at 500 nm related to the presence of carotenoids was observed in mosses. Fulgensia presented an absorption peak around 490 nm due to the presence of carotenoids or phycoerythrin. Dry biocrusts had increasing reﬂectances from the blue region until 700 nm. In the visible region the reﬂectances of various biocrust classes diﬀer because of diﬀerent pigment content and composition (Figure 5) diﬀerences in the NIR region are mainly related to the biocrust tridimensional structure. Lichens presented higher reﬂectances than bare soil and moss across the whole spectrum. Their higher reﬂectance in the NIR region is related to their more developed structure, which causes multi-scattering of light in this region. Acarospora, Squamarina, Buellia, and Diploschistes are light-colored lichens that showed higher reﬂectances in the visible region; Psora presented the lowest reﬂectance among lichens. Mosses had the lowest reﬂectance due to their darker color and less developed structure, resulting in a reduction of light scattering. The application of the continuum removal algorithm in the spectral range 450–900 nm highlighted the absorption features caused by diﬀerent pigments. The chlorophyll absorption feature at 680 nm was present in all the classes. Bare soil showed a weak absorption feature Remote Sens. 2019, 11, 2942 8 of 16 at 680 nm, evidencing the presence of cyanobacteria colonizing the soils interspace within lichens and moss patches. Psora showed an absorption feature around 550 nm due to phycoerythrin, which is absent in the other lichens. An absorption feature at 500 nm related to the presence of carotenoids was observed in mosses. Fulgensia presented an absorption peak around 490 nm due to the presence of carotenoids or phycoerythrin. Remote Sens. 2019, 11, x FOR PEER REVIEW 8 of 16 Remote Sens. 2019, 11, x FOR PEER REVIEW 8 of 16 Figure 5. Top: RGB composites of some of the samples used in the study, showing the change of color that takes place from dry (first row) to wet (second row) state of biocrusts. Down: Mean reflectance spectra and mean continuum removal absorption spectra between 450–900 nm of the biocrust classes and bare soil studied in this work. The red dashed line marks the absorption feature at 680 nm related to the presence of chlorophyll. (a) Dry samples; (b) Wet samples. 3.1. Classiﬁcations and Accuracy Evaluation Watering had a strong effect on biocrust optical properties (Figure 5b) causing a decrease of Figure 5. Top: RGB composites of some of the samples used in the study, showing the change of color that takes place from dry (ﬁrst row) to wet (second row) state of biocrusts. Down: Mean reﬂectance spectra and mean continuum removal absorption spectra between 450–900 nm of the biocrust classes and bare soil studied in this work. The red dashed line marks the absorption feature at 680 nm related to the presence of chlorophyll. (a) Dry samples; (b) Wet samples. Watering had a strong eﬀect on biocrust optical properties (Figure 5b), causing a decrease of Figure 5. Top: RGB composites of some of the samples used in the study, showing the change of color that takes place from dry (first row) to wet (second row) state of biocrusts. Down: Mean reflectance spectra and mean continuum removal absorption spectra between 450–900 nm of the biocrust classes and bare soil studied in this work. The red dashed line marks the absorption feature at 680 nm related to the presence of chlorophyll (a) Dry samples; (b) Wet samples Figure 5. Top: RGB composites of some of the samples used in the study, showing the change of color that takes place from dry (ﬁrst row) to wet (second row) state of biocrusts. Down: Mean reﬂectance spectra and mean continuum removal absorption spectra between 450–900 nm of the biocrust classes and bare soil studied in this work. The red dashed line marks the absorption feature at 680 nm related to the presence of chlorophyll. (a) Dry samples; (b) Wet samples. Watering had a strong effect on biocrust optical properties (Figure 5b), causing a decrease of reflectance across the whole spectrum. The change was particularly evident in the absorption feature caused by chlorophyll at ~680nm, which became deeper, evidencing the abrupt change in the reflectance from red to NIR, typical of vegetation (i.e., red edge region). The continuum removal algorithm allowed to enhance this absorption feature at ~680 nm related to the activation of chlorophyll after watering, increasing its variation (Figure 6). Watering had a strong eﬀect on biocrust optical properties (Figure 5b), causing a decrease of reﬂectance across the whole spectrum. 3.3. Fractional Cover of Biocrusts and Diversity Metrics 3.3. Fractional Cover of Biocrusts and Diversity Metrics The most abundant classes were moss and bare soil, covering more than 76% and 46% of th surface in some samples, respectively (Table 3). The most abundant lichen was Diploschistes, whil the least were Psora and Buellia, which appeared scarcely. Species richness ranged from 2 to 6 Shannon’s index ranged from 0.18 to 1.53, with a mean value of 1.03, and Simpson’s index from 0.0 to 0.77, with a mean of 0.56. Pielou’s index ranged from 0.14 to 0.95, with a mean value of 0.66. The most abundant classes were moss and bare soil, covering more than 76% and 46% of the surface in some samples, respectively (Table 3). The most abundant lichen was Diploschistes, while the least were Psora and Buellia, which appeared scarcely. Species richness ranged from 2 to 6. Shannon’s index ranged from 0.18 to 1.53, with a mean value of 1.03, and Simpson’s index from 0.08 to 0.77, with a mean of 0.56. Pielou’s index ranged from 0.14 to 0.95, with a mean value of 0.66. Table 3. Fractional cover values of the classes evaluated in this work. Samples: number of samples where the class was identified. Plots: number of plots where the class was identified. Mean Fc, Max Fc, Min Fc, SD Fc: mean, maximum, minimum and standard deviation of the fractional cover observed for each class. Classes: Acarospora: Acarospora spp.; Buellia: Buellia spp., Bare Soil: bare soil; Diploschistes: Diploschistes spp., Fulgensia: Fulgensia spp.; Moss: mosses; Psora: Psora spp.; Squamarina: Squamarina spp. Table 3. Fractional cover values of the classes evaluated in this work. Samples: number of samples where the class was identiﬁed. Plots: number of plots where the class was identiﬁed. Mean Fc, Max Fc, Min Fc, SD Fc: mean, maximum, minimum and standard deviation of the fractional cover observed for each class. Classes: Acarospora: Acarospora spp.; Buellia: Buellia spp., Bare Soil: bare soil; Diploschistes: Diploschistes spp., Fulgensia: Fulgensia spp.; Moss: mosses; Psora: Psora spp.; Squamarina: Squamarina spp. 3.1. Classiﬁcations and Accuracy Evaluation The change was particularly evident in the absorption feature caused by chlorophyll at ~680nm, which became deeper, evidencing the abrupt change in the reﬂectance from red to NIR, typical of vegetation (i.e., red edge region). The continuum removal algorithm allowed to enhance this absorption feature at ~680 nm related to the activation of chlorophyll after watering, increasing its variation (Figure 6). 9 of 16 9 of 16 Remote Sens. 2019, 11, 2942 Figure 6. Mean continuum removal at ~680 nm of the lichen classes identified. Bars indicate the first and third quartile of each class. (a) dry samples; (b) wet samples. Figure 6. Mean continuum removal at ~680 nm of the lichen classes identiﬁed. Bars indicate the ﬁrst and third quartile of each class. (a) dry samples; (b) wet samples. Figure 6. Mean continuum removal at ~680 nm of the lichen classes identified. Bars indicate the first and third quartile of each class. (a) dry samples; (b) wet samples. Figure 6. Mean continuum removal at ~680 nm of the lichen classes identiﬁed. Bars indicate the ﬁrst and third quartile of each class. (a) dry samples; (b) wet samples. 3.3. Fractional Cover of Biocrusts and Diversity Metrics 3.3. Fractional Cover of Biocrusts and Diversity Metrics 3.3. Fractional Cover of Biocrusts and Diversity Metrics 3.3. Fractional Cover of Biocrusts and Diversity Metrics Slopes, coefficients of determination (r2) and p-values (between brackets) of the linear regression models calculated between spectral diversity metrics (standard deviation of the continuum removal calculated between 550–750 nm (SD_CR550–750) and at 680 nm (SD_CR680); coefficient of variation calculated between 420–900 nm (CV420–900), between 550–750 nm (CV550–750), and at 680 nm (CV680)) and α-diversity metrics (Species richness (S), Shannon’s index (H’), Simpson’s index (D), and l ’ I d ( ’)) h h l α-Diversity Metric SD_CR550–750 SD_CR680 CV420–900 CV550–750 CV680 Species richness (S) 0.001 0.003 0.005 0.013 0.08 - - - - - Shannon’s Index (H’) 0.012 0.022 0.056 0.063 0.071 0.33(0.001) 0.41(0.0004) - - 0.16(0.03) Simpson’s Index (D) 0.02 0.049 0.156 0.164 0.184 0.39(0.0004) 0.47(0.0001) - - 0.26(0.007) Pielou’s Index (J’) 0.023 0.041 0.112 0.118 0.141 0.39(0.0004) 0.42(0.0002) - - 0.19(0.02) Pielou s Index (J )) when the samples were wet. α-diversity metric SD_CR550–750 SD_CR680 CV420–900 CV550–750 CV680 Species richness (S) 0.001 0.003 0.005 0.013 0.08 - - - - - Shannon’s Index (H’) 0.012 0.022 0.056 0.063 0.071 0.33(0.001) 0.41(0.0004) - - 0.16(0.03) Simpson’s Index (D) 0.02 0.049 0.156 0.164 0.184 0.39(0.0004) 0.47(0.0001) - - 0.26(0.007) Pielou’s Index (J’) 0.023 0.041 0.112 0.118 0.141 When the spectral diversity was calculated as SD_CR550-750 and SD_CR680, the relationships with α-diversity were positive and strongly signiﬁcant for all the diversity metrics (Table 4). The standard deviation of the CR at 680 nm (SD_CR680), which is related to the diﬀerence in chlorophyll content, was the spectral diversity metric better related to α-diversity metrics. Neither the SD_CR nor the CV captured the species richness. The Simpson’s index was positively correlated to the CV at all the spectral ranges analyzed (420–900, 550–750, and 680 nm), especially at 680 nm. While Simpson’s and Pielou’s indices were the metrics that correlated best with SD_CR680 (r2 = 0.47 and r2 = 0.42, respectively; Table 4, Figure 7), they did not show any signiﬁcant correlation with CV420–900 or CV550–750. The statistics in cross-validation (Table 5) of the linear regressions presented similar values to the original analysis, showing the stability of the predictions made by the linear regression models between spectral diversity and α-diversity of lichens. 0.39(0.0004) 0.42(0.0002) - - 0.19(0.02) When the spectral diversity was calculated as SD_CR550-750 and SD_CR680, the relationships with α-diversity were positive and strongly significant for all the diversity metrics (Table 4). 3.3. Fractional Cover of Biocrusts and Diversity Metrics 3.3. Fractional Cover of Biocrusts and Diversity Metrics Class Samples Plots Mean Fc (%) Max Fc (%) Min Fc (%) SD Fc (%) Acarospora 37 17 3.8 32.8 0.3 5.8 Bare Soil 54 18 21.6 46.1 5.1 8.1 Buellia 33 13 2.4 13.8 0.5 3 Diploschistes 54 18 14.6 53.1 0.1 11 Fulgensia 53 18 12 25.4 0.7 7.3 Moss 54 8 43.5 76.9 4.9 17 Psora 41 16 1.9 12.6 0.1 2.6 Squamarina 27 13 4.3 4.3 0.3 4.5 Class Samples Plots Mean Fc (%) Max Fc (%) Min Fc (%) SD Fc (%) Acarospora 37 17 3.8 32.8 0.3 5.8 Bare Soil 54 18 21.6 46.1 5.1 8.1 Buellia 33 13 2.4 13.8 0.5 3 Diploschistes 54 18 14.6 53.1 0.1 11 Fulgensia 53 18 12 25.4 0.7 7.3 Moss 54 8 43.5 76.9 4.9 17 Psora 41 16 1.9 12.6 0.1 2.6 Squamarina 27 13 4.3 4.3 0.3 4.5 Squamarina 27 13 4.3 3.4. Relationships between Biodiversity and Spectral Diversity 3.4. Relationships between Biodiversity and Spectral Diversity The correlation analysis between spectral diversity (CV and SD_CR) and α-diversity metrics (i.e. species richness, Shannon’s Index, Simpson’s Index, and Pielou’s Index) showed that there were no significant correlations with the dry samples (results not shown). Conversely, positive and ll f l h f d h h l bl The correlation analysis between spectral diversity (CV and SD_CR) and α-diversity metrics (i.e., species richness, Shannon’s Index, Simpson’s Index, and Pielou’s Index) showed that there were no signiﬁcant correlations with the dry samples (results not shown). Conversely, positive and statistically signiﬁcant relationships were found when the samples were wet (Table 4). 10 of 16 Remote Sens. 2019, 11, 2942 Table 4. Slopes, coeﬃcients of determination (r2) and p-values (between brackets) of the linear regression models calculated between spectral diversity metrics (standard deviation of the continuum removal calculated between 550–750 nm (SD_CR550–750) and at 680 nm (SD_CR680); coeﬃcient of variation calculated between 420–900 nm (CV420–900), between 550–750 nm (CV550–750), and at 680 nm (CV680)) and α-diversity metrics (Species richness (S), Shannon’s index (H’), Simpson’s index (D), and Pielou’s Index (J’)) when the samples were wet. Table 4. 3.3. Fractional Cover of Biocrusts and Diversity Metrics 3.3. Fractional Cover of Biocrusts and Diversity Metrics The standard deviation of the CR at 680 nm (SD_CR680), which is related to the difference in chlorophyll content, was the spectral diversity metric better related to α-diversity metrics. Neither the SD_CR nor the CV captured the species richness. The Simpson’s index was positively correlated to the CV at all the spectral ranges analyzed (420–900, 550–750, and 680 nm), especially at 680 nm. While Simpson’s and Pielou’s indices were the metrics that correlated best with SD_CR680 (r2 = 0.47 and r2 = 0.42, respectively; Table 4, Figure 7), they did not show any significant correlation with CV420–900 or CV550– 750. The statistics in cross-validation (Table 5) of the linear regressions presented similar values to the original analysis, showing the stability of the predictions made by the linear regression models between spectral diversity and α-diversity of lichens Figure 7. Linear regression between the spectral diversity measured as the Standard Deviation of the Continuum removal at 680 nm and three α-diversity metrics: (a) Shannon’s index, (b) Simpson’s index, and (c) Pielou’s index. Shaded areas represent ±95% symmetrical confidence interval. Figure 7. Linear regression between the spectral diversity measured as the Standard Deviation of the Continuum removal at 680 nm and three α-diversity metrics: (a) Shannon’s index, (b) Simpson’s index, and (c) Pielou’s index. Shaded areas represent ±95% symmetrical conﬁdence interval. Figure 7. Linear regression between the spectral diversity measured as the Standard Deviation of the Continuum removal at 680 nm and three α-diversity metrics: (a) Shannon’s index, (b) Simpson’s index, and (c) Pielou’s index. Shaded areas represent ±95% symmetrical confidence interval. Figure 7. Linear regression between the spectral diversity measured as the Standard Deviation of the Continuum removal at 680 nm and three α-diversity metrics: (a) Shannon’s index, (b) Simpson’s index, and (c) Pielou’s index. Shaded areas represent ±95% symmetrical conﬁdence interval. Table 5. Summary of statistics in fitting (r2 and RMSE) and cross-validation (r2cv and RMSEcv) of the linear regression models between the spectral diversity measured as the standard deviation of the continuum removal at 680 nm and the α-diversity metrics (Shannon’s, Simpson’s, and Pielou’s i di ) RMSE 2 ffi i f d i i Table 5. Summary of statistics in ﬁtting (r2 and RMSE) and cross-validation (r2cv and RMSEcv) of the linear regression models between the spectral diversity measured as the standard deviation of the continuum removal at 680 nm and the α-diversity metrics (Shannon’s, Simpson’s, and Pielou’s indices). 4. Discussion The high accuracies obtained using SVM to classify hyperspectral imagery reinforce their use to diﬀerentiate biocrusts [31]. Diﬀerences in the reﬂectance of biocrust constituents are subtle (Figure 5), but SVMs are capable of diﬀerentiating spectrally similar classes when the inputs are spectral signatures [61–63]. Even though the accuracy metrics from both sets of images were similar, the classiﬁcation improved when biocrusts were wet. This result is explained by the enhanced diﬀerences that appear in the reﬂectance spectra when biocrusts are metabolically activated after irrigation [35]. Several studies have characterized the optical properties of biocrust communities in the same optical range used in this study [42]. However, the spatial scale of these studies only allowed characterization of biocrust communities. Conversely, the ﬁne spatial resolution of the imaging system used in this work allowed to characterize for the ﬁrst time the pure spectral signature of six lichen genera, to compare their characteristics and to capture the spectral diversity among them. In fact, we used a pixel size lower than the size of each object of interest (i.e., thallus of lichens), as suggested by [64,65]. j ( ) gg y [ ] Lichens present diﬀerent structural and biochemical traits, which create wavelength-dependent variations that can be integrated by spectral diversity, as shown in vascular plants by [66]. Diﬀerent genera of lichens are characterized by particular biochemical traits that promote spectral variations, so their spectral diversity can be exploited to infer their α-diversity. However, it is important to understand which are the best metrics to represent the relationship between lichen spectral diversity and α-diversity. Since the absorption peak around 680 nm has been widely investigated as a spectral feature of biocrusts and is present in all lichens (e.g., [35,40,67–69]), we tested if a spectral diversity metric focused on this feature may be suitable for monitoring changes in their composition. We found that using a spectral diversity metric based on this absorption feature (i.e., the SD_CR680) increases the spectral variability determined by the chlorophyll content of diﬀerent lichens, while removing the confounding inﬂuence of other factors such as the structure of lichens. In fact, some lichen genera are characterized by a strong tridimensional component (e.g., Diploschistes spp., Squamarina spp.) relative to others, which can determine a higher intra-genera variability compared to the inter-genera variability. 3.3. Fractional Cover of Biocrusts and Diversity Metrics 3.3. Fractional Cover of Biocrusts and Diversity Metrics RMSE: root mean square error; r2: coeﬃcient of determination. indices). RMSE: root mean square error; r : coefficient of determination. α-Diversity Metric r2 RMSE rCV2 RMSECV hannon’s index (H’) 0.41 0.01 0.32 0.01 mpson’s index (D) 0.47 0.009 0.39 0.01 Pielou’s index (J’) 0.42 0.009 0.35 0.01 α-Diversity Metric r2 RMSE rCV2 RMSECV Shannon’s index (H’) 0.41 0.01 0.32 0.01 Simpson’s index (D) 0.47 0.009 0.39 0.01 Pielou’s index (J’) 0.42 0.009 0.35 0.01 Remote Sens. 2019, 11, 2942 11 of 16 11 of 16 4. Discussion The normalization of the reﬂectance spectra to a common baseline through the CR algorithm minimized these structural eﬀects, allowing to capture the spectral variability of lichens determined by the chlorophyll content. Conversely, the CV is inﬂuenced by both variations in the content of pigments and the structure of biocrusts, which may hide inter-genera variability. This might be the reason why the CV did not perform as well as expected in previous studies conducted with vascular plants (e.g., [23–25]), where the structural component constitutes were the main source of spectral variability. Conversely, the results obtained in this study highlight the dominant role of the chlorophyll content to determine the diﬀerences between lichen genera and the importance of using the absorption feature centered at ~680 nm to capture their spectral diversity. Nevertheless, using absorption features that are found in the shortwave infrared region of the spectra of lichens [35] might help to improve the results obtained in this study, that investigated only the visible and near infrared spectral domain. Among the α-diversity metrics tested, the Simpson’s index correlated the most with the spectral diversity of lichens. The better performance of the Simpson’s index compared to the Shannon’s index is in agreement with recent studies conducted in tropical forests [21] and in a prairie grassland [24]. The latter found similar and weaker relationships between spectral diversity and evenness (calculated as Pielou’s index) as we did here, although they correlated these metrics with the CV instead of the SD_CR. This might be due to the fact that Shannon’s index assumes that all the species are present and randomly sampled [70], and the Simpson’s index is more sensitive to dominant or common species, as noted by [24], making it more suitable when this is the case. The similar relationships obtained in cross-validation show that these results are reliable despite their sample size. The imagery used in this study captured with high detail the lichen genera present in our samples, allowing to identify positive and signiﬁcant relationships between the spectral diversity and the α-diversity of lichens. The methodology proposed in this study should be in principle applied to monitor α-diversity of lichens at wider scales, a key task to understand the shifts in the composition Remote Sens. 2019, 11, 2942 12 of 16 that these communities are undergoing in the actual context of global change [9–12]. 4. Discussion However, the upscaling of this methodology may be not trivial due to the decreased spatial and/or spectral resolution when working on wider scales. Many ecological processes maintain scale-dependent relations [24,71–73] and a sampling scale bigger than the object studied might cause a loss of information that is provided at ﬁner spatial resolutions [26]. In order to understand if the relations found in this work hold with decreasing spatial resolution, the spatial sensitivity of the spectral diversity–biodiversity relationship should be investigated. A previous work in a prairie grassland [24] identiﬁed a strong scale dependence of the spectral diversity–biodiversity relationships and suggested that the optimal pixel size for distinguishing α-diversity in prairie plots was similar to the size of an individual herbaceous plant (1 mm to 10 cm). This might hamper the monitoring of the α-diversity of lichen-dominated biocrusts at landscape scale with sensors installed on satellite or airborne platforms, which most likely would have a bigger pixel size than the one used in this work. Conversely, the use of unmanned aerial vehicles (UAVs), which can carry light-weight imaging sensors of diﬀerent spatial (reaching 2–4 cm/pixel in many cases) and spectral resolutions [74] may allow replicating studies like ours at landscape level [75]. Many of these sensors do not have very high spectral resolution, but have at least one band at ~680 nm [74], which would allow to calculate the SD_CR680 (the spectral diversity metric that presented the highest predictability of lichens’ α-diversity in our study) to monitor α-diversity of lichens at larger spatial scales. Achieving the results shown in this work at landscape scale might also be hampered by the spectral properties that other components (e.g., vascular plants, mosses, bare soil) have on the spectral reﬂectance measured from remote sensors, which might hinder the separation of the pure spectral component of lichens and to estimate their α-diversity. This issue could be however solved using spectral mixture analysis [76], a technique that models a mixed spectrum as a combination of its spectral components weighted by the correspondent subpixel fractional covers [77] and has already proved to be successful for mapping biocrusts [31]. 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Conclusions The very high accuracies obtained classifying the hyperspectral images using SVMs showed the reliability of this methodology to identify diﬀerent biocrust-forming lichens. Therefore, we were able to extract pure spectral signatures of diﬀerent biocrust constituents and to evaluate the relationships between α-diversity and spectral diversity of lichens. We found that the SD_CR680 nm was the spectral diversity metric that predicted the best the α-diversity metrics that include richness and evenness in their calculations (i.e., Pielou’s and Simpson’s indices). As such, we suggest that this index could help to track spatio-temporal changes in lichen-dominated biocrust communities. In this context, the results of this study will help to improve future works upscaling the methodology here shown to coarser scales in drylands, a key task in any monitoring program aiming to assess the impacts of ongoing climate change and desertiﬁcation processes in these environments. Author Contributions: J.B.-S., C.P., G.T., R.G., M.L.d.G., F.T.M., and M.R. designed the ﬁeld sampling; J.B.-S., C.P., G.T., R.G., M.L.d.G., conducted the ﬁeld sampling; J.B.-S. and C.P. conducted imaging measurements; J.B.-S. analyzed the data; J.B.-S., C.P., G.T., and M.R. interpreted the results. All authors contributed to the discussion of the results and the ﬁnal version of the manuscript. Funding: The research has received funding from the European Union’s Horizon 2020 research and innovation 514 program under the Marie Sklodowska-Curie grant agreement no. 721995. F.T.M. acknowledges support from the European Research Council grant agreement no. 647038 (BIODESERT). Acknowledgments: We thank Roberto Garzonio and Khelvi Biriukova for their help during the hyperspectral images acquisition, and Biagio Di Mauro for his comments during the early stages of this manuscript. We are also grateful to the three reviewers for their comments and suggestions, which improved this manuscript. 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https://openalex.org/W3185494368	https://www.frontiersin.org/articles/10.3389/fvets.2021.652847/pdf	English	null	Case Report: Trans-Articular External Skeletal Fixation of the Hip for a Highly Comminuted Juxta-Articular Fracture of the Proximal Femur Caused by Gunshot Injury in a Cat	Frontiers in veterinary science	2,021	cc-by	6,405	Case Report: Trans-Articular External Skeletal Fixation of the Hip for a Highly Comminuted Juxta-Articular Fracture of the Proximal Femur Caused by Gunshot Injury in a Cat Hyun-Jung Han 1, Kyeongpung Lee 2 and Hun-Young Yoon 3* 1 Department of Veterinary Emergency and Critical Care, Konkuk Veterinary Medical Teaching Hospital, Konkuk University, Seoul, South Korea, 2 Department of Materials Science and Engineering, Seoul National University, Seoul, South Korea, 3 Department of Veterinary Surgery, College of Veterinary Medicine, Konkuk University, Seoul, South Korea Keywords: cat, comminuted fracture, external skeletal ﬁxation, gunshot, juxta-articular, proximal femur, trans- articular, coxofemoral joint Received: 13 January 2021 Accepted: 24 June 2021 Published: 19 July 2021 Received: 13 January 2021 Accepted: 24 June 2021 Published: 19 July 2021 CASE REPORT published: 19 July 2021 doi: 10.3389/fvets.2021.652847 CASE REPORT published: 19 July 2021 doi: 10.3389/fvets.2021.652847 This report describes a novel surgical technique for trans-articular external skeletal ﬁxation (TA-ESF) of the hip to stabilize a rare, highly comminuted juxta-articular fracture of the proximal femoral segment involving the metaphysis and diaphysis in a cat. A 2-year-old, castrated male, Korean shorthair cat was admitted for a left femoral fracture caused by gunshot injury. Radiographs and computed tomography (CT) scans revealed a highly comminuted proximal femoral diaphyseal fracture that involved the femoral neck and the greater trochanter. The left femoral head was intact on CT. Under ﬂuoroscopic guidance, an intramedullary Steinmann pin was placed to align the femur, and ﬁve positive-proﬁle, end-threaded pins were placed in the left hemipelvis and distal femur. A triangulated frame of connecting bars was constructed using Steinmann pins and epoxy resin for TA-ESF. This secured the pins placed in the pelvis and femur as well as the intramedullary pin, providing proximal femoral stabilization by trans-articular ﬁxation of the hip. The cat began placing weight on the left pelvic limb 4 days postoperatively, and progressively obtained near-normal limb function by day 112. The TA-ESF was partially dismantled to a linear tie-in construct on day 64 and was completely removed on day 161. Final radiographs on day 161 revealed lack of bone healing in the proximal segment, especially femoral head and neck, despite functional recovery of the injured leg. At one year postoperatively, the cat had normal limb function without any noticeable complications. On follow-up 29 months after surgery, the owner reported that the cat had normal limb function without any noticeable complications. Despite insufﬁcient bone healing in the cat, TA-ESF of the hip allowed for satisfactory functional recovery of this challenging juxta-articular fracture of the proximal femur, which was not amenable to stabilization with a traditional non-load-sharing ﬁxation system. Edited by: Young-Sam Kwon, Kyungpook National University, South Korea Reviewed by: Jason Bleedorn, University of Wisconsin-Madison, United States Haebeom Lee, Chungnam National University, South Korea *Correspondence: Hun-Young Yoon yoonh@konkuk.ac.kr Specialty section: This article was submitted to Veterinary Surgery and Anesthesiology, a section of the journal Frontiers in Veterinary Science CASE DESCRIPTION A 2-year-old castrated, male, Korean shorthair cat weighing 4.4 kg was admitted with a highly comminuted proximal femoral fracture caused by a gunshot injury sustained 3 days earlier. On presentation, the cat could not bear weight on the left pelvic limb. Additionally, there was a small skin puncture wound, suspected to be the bullet trajectory, over the left hemipelvis. The cat showed signs of pain during palpation of the left pelvis and pelvic limb, but did not show any neurological deﬁcits. Abnormalities on a complete blood count and biochemistry panel included a decreased packed cell volume of 24.7% (reference range, 30.3– 52.3%) and an elevated creatine kinase level of 675 U/L (reference range, 0–314 U/L). To construct the TA-ESF of the hip, two 2.5 mm and one 1.8 mm positive-proﬁle end-threaded pins were placed in the ilium and ischium, respectively. Then, one 2.0 mm and one 1.8 mm positive-proﬁle end-threaded pins were also placed perpendicular to the distal femur (Figures 2C,D). The three pins placed in the ilium and ischium were connected to the ﬁrst connecting rod, and the two pins in the distal femur were connected to the second connecting rod. The ﬁrst and second connecting rods were linked to each other, and the third connecting rod was placed to link the ﬁrst and second bars cranially. The preplaced intramedullary pin was tied into the ﬁrst connecting rod. The connecting rods were fashioned using 3.0 mm Steinmann pins and epoxy resin (Figures 3A,B). For a stable triangular conﬁguration, the magnitudes of forces and moments at intersection points were considered to distribute the external loads equally. A proximal segmental fracture of the left femur and multiple radiopaque objects of various sizes were identiﬁed on both plain radiographs and computed tomography scans (Figure 1). A comminuted spiral diaphyseal fracture and comminution of the neck and greater trochanter with an intact femoral head were identiﬁed on computed tomography. Injuries to the major organs were not identiﬁed. However, there was parenchymal damage to the left gluteal and thigh muscles. The comminuted proximal femoral fracture was stabilized with a closed approach. Written informed consent for the surgical procedure and complications was obtained from the owner. The major bullet fragments embedded in the abdominal wall were removed. Abbreviations: ESF, External Skeletal Fixation; TA-ESF, Trans-articular External Skeletal Fixation. INTRODUCTION FIGURE 1 \| Preoperative radiographs and computed tomography scan of the left hind limb. (A,B) Craniocaudal (A) and mediolateral (B) radiographic views showing highly comminuted fractures at the proximal femur, including the neck, trochanteric, subtrochanteric, and the proximal half of the diaphysis. (C,D) Volume-rendered computed tomography scan of the cranial (C) and caudoventral (D) views showing the comminuted bones of the neck and trochanter and the comminuted spiral fracture of the proximal half of the femoral diaphysis. The hip joint is conﬁrmed to be intact (white arrowheads). Surgical stabilization of highly comminuted fractures of the proximal or distal juxta-articular bones has rarely been reported in veterinary literature. These comminuted juxta-articular fractures are diﬃcult to stabilize since the presence of a short proximal or distal segment limits the types of ﬁxation that can be used (1). A circular external skeletal ﬁxation (ESF) is preferred for fractures with very short proximal or distal segments in dogs and cats, particularly if the fracture cannot be reconstructed (1– 3). The ring components of these ESFs, which use ﬁxation wires, are particularly advantageous in such comminuted fractures with short segments since they provide the ability to use multiple points of ﬁxation in extremely small fracture segments (1). However, the application of such circular ESFs is limited by the anatomical location. In the cat described in the present case, application of ring components of the circular ESF to the proximal femur was limited due to the proximity to the body wall (1). FIGURE 1 \| Preoperative radiographs and computed tomography scan of the left hind limb. (A,B) Craniocaudal (A) and mediolateral (B) radiographic views showing highly comminuted fractures at the proximal femur, including the neck, trochanteric, subtrochanteric, and the proximal half of the diaphysis. (C,D) Volume-rendered computed tomography scan of the cranial (C) and caudoventral (D) views showing the comminuted bones of the neck and trochanter and the comminuted spiral fracture of the proximal half of the femoral diaphysis. The hip joint is conﬁrmed to be intact (white arrowheads). This report describes a novel surgical technique for the successful management and functional recovery of a highly comminuted juxta-articular fracture of the proximal femur caused by a gunshot injury using trans-articular ESF (TA-ESF) of the hip in a cat. These results were obtained despite a lack of bone healing. Citation: Han H-J, Lee K and Yoon H-Y (2021) Case Report: Trans-Articular External Skeletal Fixation of the Hip for a Highly Comminuted Juxta-Articular Fracture of the Proximal Femur Caused by Gunshot Injury in a Cat. Front. Vet. Sci. 8:652847. doi: 10.3389/fvets.2021.652847 July 2021 \| Volume 8 \| Article 652847 Frontiers in Veterinary Science \| www.frontiersin.org 1 Han et al. TA-ESF for Juxta-Articular Femur Fracture INTRODUCTION bullet fragments were not removed due to the costs involved and since they did not pose any immediate clinical problems. Under general anesthesia, the cat was placed in the right lateral recumbent position, and the left pelvic limb was prepared using a routine hanging limb preparation. Under ﬂuoroscopic guidance, the femoral condyles were secured with a pair of AO forceps and indirect reduction was achieved by applying traction to the distal femur. A small skin incision was made medial to the greater trochanter, and a 2.4-mm Steinmann pin was inserted into the medullary canal of the femur in a normograde fashion (Figure 2A). The intramedullary pin occupied 47% of the medullary cavity at the isthmus (5.1 mm) and stabilized the major diaphyseal segments but not the comminuted femoral neck or the greater trochanter (Figure 2B). CASE DESCRIPTION However, the other small intraabdominal During this process, the limb was temporarily supported by an assistant to maintain appropriate rotation and angulation of the femur based on palpation of the condyles and the comminuted greater trochanter. This also allowed for ﬂuoroscopic identiﬁcation of the femoral head, femoral diaphysis, and linea aspera. Additionally, the coxofemoral joint July 2021 \| Volume 8 \| Article 652847 Frontiers in Veterinary Science \| www.frontiersin.org 2 TA-ESF for Juxta-Articular Femur Fracture Han et al. bandaged, and the cat was then re-positioned in left lateral recumbency to remove the bullet from the right abdominal wall. FIGURE 2 \| Intraoperative ﬂuoroscopic series showing closed reduction and implant insertion in a 2-year-old cat with a comminuted proximal femur fracture. (A,B) The intramedullary pin is inserted into the trochanteric region, normograde from proximal to distal (A), and it stabilizes the proximal and the distal femoral diaphyseal fragments (B). (C,D) For trans-articular external skeletal ﬁxation of the hip, three of the ﬁve positive-proﬁle end-threaded pins are inserted to the left pelvis, including two pins in the ileum (white arrows) and the third pin in the ischium (black arrow), while the other two pins are placed in the distal fragments of the fractured femur (white arrowheads). The cat recovered from anesthesia and surgery without complications and was administered a constant-rate infusion of fentanyl for 24 h (fentanyl: loading, 4 µg/kg; constant-rate infusion, 4 µg/kg/h), followed by meloxicam for 4 days (0.1 mg/kg PO for 24 h) for analgesia. Postoperative radiographs were taken to verify appropriate placement of the pins and alignment of the femur (Figure 4A). Craniocaudal imaging was not possible due to the immovable left hip joint. The day after surgery, the cat would not place weight on the left pelvic limb, which was very painful, and could not tolerate palpation of the left femur (Supplementary Video 1). On postoperative day 4, the cat was more comfortable, ambulatory, and could place nominal weight on the left pelvic limb. The cat was discharged 4 days after surgery with instructions to the owners to restrict the cat’s activity, including conﬁning the cat to a small room and taking it for short walks on a leash. The owners were also instructed to clean the ﬁxation pin–skin interfaces daily with saline and chlorhexidine solution. The cat was re-evaluated on postoperative days 18, 32, 46, 64, 80, 112, and 161. CASE DESCRIPTION (A,B) The intramedullary pin is inserted into the trochanteric region, normograde from proximal to distal (A), and it stabilizes the proximal and the distal femoral diaphyseal fragments (B). (C,D) For trans-articular external skeletal ﬁxation of the hip, three of the ﬁve positive-proﬁle end-threaded pins are inserted to the left pelvis, including two pins in the ileum (white arrows) and the third pin in the ischium (black arrow), while the other two pins are placed in the distal fragments of the fractured femur (white arrowheads). FIGURE 3 \| (A,B) Triangular frame composed of connecting bars with epoxy-putty molded pins for trans-articular external skeletal ﬁxation of the hip: the 3.0-mm Steinmann pin (#1, blue dashed line) links the ﬁxation pins placed in the left pelvis (black arrows); the other 3.0-mm Steinmann pin (#2, red dashed line) links to the ﬁxation pins placed in the distal segment of the fractured femur (white arrows); #1 and #2 connecting bars are linked to each other at the intersection; the 2.4-mm Steinmann pin (#3, yellow dashed line) links to the #1 and #2 connecting bars; the other 2.4-mm Steinmann pin (#4, black dashed line) is tied into the intramedullary pin (yellow arrow) and links to the #1 connecting bar. Epoxy-putty covers all the pins composed of connecting bars including column of the pins and those connections. FIGURE 3 \| (A,B) Triangular frame composed of connecting bars with epoxy-putty molded pins for trans-articular external skeletal ﬁxation of the hip: the 3.0-mm Steinmann pin (#1, blue dashed line) links the ﬁxation pins placed in the left pelvis (black arrows); the other 3.0-mm Steinmann pin (#2, red dashed line) links to the ﬁxation pins placed in the distal segment of the fractured femur (white arrows); #1 and #2 connecting bars are linked to each other at the intersection; the 2.4-mm Steinmann pin (#3, yellow dashed line) links to the #1 and #2 connecting bars; the other 2.4-mm Steinmann pin (#4, black dashed line) is tied into the intramedullary pin (yellow arrow) and links to the #1 connecting bar. Epoxy-putty covers all the pins composed of connecting bars including column of the pins and those connections. The cat’s condition progressively improved over the next couple of weeks, and it did not appear to be in pain when examined on postoperative day 80. The examination on day 112 showed nearly normal gait (Supplementary Video 4). CASE DESCRIPTION During these evaluations, the ﬁxator was monitored for complications. Pain and limb function were subjectively assessed using an ordinal scoring system (Supplementary Table 1). Serial radiographs were also obtained to assess progression of the fracture toward union. By postoperative day 32, the cat had progressed to weight- bearing lameness. However, it still had a distinct head nod and mild pain on palpation of the left femur. On postoperative day 64, perceived pain and lameness were similar to those in the previous evaluation, and radiographs revealed an interfragmentary callus and cortical remodeling, bridging the diaphyseal femoral fracture gap. The femoral neck and greater trochanter were remodeling, exhibiting corticalization with heterogeneous opacity (Figure 4B). The ﬁxation pin placed in the ischium had broken, and the triangular construct was partially disassembled to a linear tie-in construct by removing the ﬁxation pins placed in the pelvis using Jacob’s hand chuck. Additionally, the ﬁrst and third connecting rods were cut near the area of linkage using a saw and pin cutter, while avoiding impairment of the remaining linear tie-in frame (Figure 4B). This disassembly re-established motion in the left coxofemoral joint. The cat showed more pain immediately after disassembly than before disassembly of the frame. However, it still placed weight on the left pelvic limb with notable lameness and a distinct head nod (Supplementary Video 2). Although, regained the range of motion of the left hip joint and passive manipulation, including ﬂexion and extension of the hindlimb, were reduced, showing mild discomfort (Supplementary Video 3). FIGURE 2 \| Intraoperative ﬂuoroscopic series showing closed reduction and implant insertion in a 2-year-old cat with a comminuted proximal femur fracture. (A,B) The intramedullary pin is inserted into the trochanteric region, normograde from proximal to distal (A), and it stabilizes the proximal and the distal femoral diaphyseal fragments (B). (C,D) For trans-articular external skeletal ﬁxation of the hip, three of the ﬁve positive-proﬁle end-threaded pins are inserted to the left pelvis, including two pins in the ileum (white arrows) and the third pin in the ischium (black arrow), while the other two pins are placed in the distal fragments of the fractured femur (white arrowheads). FIGURE 2 \| Intraoperative ﬂuoroscopic series showing closed reduction and implant insertion in a 2-year-old cat with a comminuted proximal femur fracture. CASE DESCRIPTION The range of motion of the left coxofemoral joint steadily improved and was nearly normal (40◦of ﬂexion and 140◦of extension) by day 112. When evaluated on day 161, the cat did not have an appreciable lameness, no longer experienced pain was ﬂexed and maintained at a normal weight-bearing angle while the epoxy resin polymerized. After polymerization, the left coxofemoral joint was ﬁxed at ∼90◦. The ﬁxator was lightly July 2021 \| Volume 8 \| Article 652847 Frontiers in Veterinary Science \| www.frontiersin.org 3 Han et al. TA-ESF for Juxta-Articular Femur Fracture FIGURE 4 \| Postoperative serial radiographic views of the left femur. (A) Lateromedial radiograph acquired immediately after surgery shows good alignment and normal length of the left femur with trans-articular external skeletal ﬁxation of the hip and a tied-in intramedullary pin. (B) On postoperative day 64, periosteal callus bridges the femoral fracture gap with cortical remodeling. Heterogenous opacity of the femoral neck and greater trochanter are identiﬁed on the craniocaudal radiographic view (white arrows). Trans-articular external skeletal ﬁxation of the hip is disassembled to the linear tie-in frame by removal of ﬁxation pins in the left pelvis and the associated connecting bars. The connecting bars are cut at the linkage area (green arrows) to preserve the linear tie-in frame. The ﬁxation pin in the ischium is broken and partially embedded in the ischium (black arrow). (C) On day 161, the whole frame of the external skeletal ﬁxation is removed, leaving the intramedullary pin and embedded part of the broken pins (black arrows). Fracture union showing good progression in bone healing is identiﬁed as a heterogeneous opacity with a mottled appearance at the femoral head and neck (white arrows). FIGURE 4 \| Postoperative serial radiographic views of the left femur. (A) Lateromedial radiograph acquired immediately after surgery shows good alignment and normal length of the left femur with trans-articular external skeletal ﬁxation of the hip and a tied-in intramedullary pin. (B) On postoperative day 64, periosteal callus bridges the femoral fracture gap with cortical remodeling. Heterogenous opacity of the femoral neck and greater trochanter are identiﬁed on the craniocaudal radiographic view (white arrows). Trans-articular external skeletal ﬁxation of the hip is disassembled to the linear tie-in frame by removal of ﬁxation pins in the left pelvis and the associated connecting bars. The connecting bars are cut at the linkage area (green arrows) to preserve the linear tie-in frame. CASE DESCRIPTION The ﬁxation pin in the ischium is broken and partially embedded in the ischium (black arrow). (C) On day 161, the whole frame of the external skeletal ﬁxation is removed, leaving the intramedullary pin and embedded part of the broken pins (black arrows). Fracture union showing good progression in bone healing is identiﬁed as a heterogeneous opacity with a mottled appearance at the femoral head and neck (white arrows). on manipulation of the left pelvic limb, and could jump up onto an examination table. Radiographs demonstrated advanced remodeling of the comminuted greater trochanter and a callus bridging the diaphyseal fracture. Bone resorptions were identiﬁed in the femoral head and neck, represented by a mottled appearance of the head and thinning, sclerotic changes of the neck, and the rotational malalignment of the femoral head was conﬁrmed as the head was superimposed over the femoral shaft (Figure 4C). The proximal ﬁxation pin in the distal femur had broken. The remainder of the ﬁxator was removed, leaving the intramedullary pin and broken segments of the ﬁxation pins embedded in the bone (Figure 4C). The length of the left femur and the left pelvic limb muscle volume were comparable to those of the contralateral limb. through a closed approach. This is the biggest beneﬁt of ESF since it preserves the fracture hematoma and periosteal vascularity while providing longitudinal distraction forces to hold the fragment closer together using surrounding soft tissues (16, 17). The TA-ESF frame in the present study was creatively developed based on the anatomical features of the pelvis–distal femoral interconnection and physical distribution of the load. For stabilizing the comminuted proximal juxta-articular femur, the pelvis was utilized as the proximal segment instead of the comminuted proximal segment of the femur. The ﬁxation pins in the pelvis were placed in the sacropelvic area of the ilium and the ischiatic tuberosity based on a previous study (18), considering stress concentration, amount of bony stock, and the risk of iatrogenic injury to the sciatic nerve. This TA-ESF frame of the hip was reinforced by making a triangular conﬁguration, which placed a third connecting rod to induce load distribution. It is considered that this triangular conﬁguration could reduce the load of the conventional TA-ESF frame, ultimately increasing the stability of the TA-ESF frame. Telephone follow-up was conducted for 29 months postoperatively. CASE DESCRIPTION The owner reported that the cat represented normal gait without any noticeable pain and lameness (Supplementary Video 5). Frontiers in Veterinary Science \| www.frontiersin.org DISCUSSION y A previous case report revealed a similar TA-ESF frame of the hip in a cat (19). However, there are signiﬁcant diﬀerences in the frame between the previous and present studies. Whereas, the previous frame secured the proximal femoral segment, the frame of this study was a more complex fracture that could not secure the proximal femoral segment due to its high comminution. Thus, the previous TA-ESF frame of the hip only served to support the primary ESF placed in the femur, while the TA- ESF frame of the hip in the present study played a primary role in stabilizing the comminuted proximal juxta-articular femur in the pelvis utilized as the proximal segment instead of the comminuted proximal segment of the femur. Further, to restore the femoral length and to enhance the bending resistance, the tied-in conﬁguration, which was not used in the previous frame, was additionally constructed in the present study. ESF, as chosen in this cat, is the preferred mode of trans- articular ﬁxation in small animals for supporting tendon (4, 5) and ligament injuries (4, 6, 7). Further, it supports the primary implants for fractures (6, 8) of various joints, such as the stiﬂe (4, 9–14), tarsal (5, 7, 10), and elbow (15). In particular, since the trans-articular ﬁxation using ESF preserves the joint during the procedure (4–8), it is valuable for temporary immobilization of the associated joint without the articular invasion. Therefore, ESF was considered the most feasible option for temporary ﬁxation of the coxofemoral joint in this cat, providing suﬃcient stabilization of the proximal femur, while preserving the coxofemoral joint. Additionally, with ESF, biological osteosynthesis, and ligamentotaxis can be enhanced July 2021 \| Volume 8 \| Article 652847 Frontiers in Veterinary Science \| www.frontiersin.org 4 TA-ESF for Juxta-Articular Femur Fracture Han et al. Femoral malalignment and deformities were the major concerns for this patient. To align the fractured femur and avoid postoperative deformities, the anatomical landmarks, including the greater trochanter, femoral condyle, and linea aspera, were consistently assessed by palpation and ﬂuoroscopic guidance. The greater trochanter, though highly comminuted, could be easily palpated since its fragments were not scattered and remained closely enough to maintain a near-normal shape. Thus, it could be used as a landmark to evaluate the rotational alignment of the femur with the condyles and the linea aspera. DISCUSSION Using these landmarks, intramedullary pinning was performed to restore the length and axial alignment of the femur, thus, helping the cat gain functional recovery of the injured gait. However, due to the limitations of the closed approach in this kind of comminuted fracture for bridging and biological osteosynthesis, each bone fragment could not be opposed accurately, and this might lead to delayed bone healing and the residual rotational malalignment of femoral head. Additionally, since the femoral neck had a highly comminuted fracture, the anteversion and inclination angle of the femoral neck could not be accurately assessed perioperatively. Therefore, the appropriate angles were estimated intraoperatively under ﬂuoroscopic guidance by comparing the locations of the center of the femoral head, greater trochanter, and ideal axis of the shaft to avoid increasing these angles markedly. If an abnormal anteversion and inclination angle occurred despite these preventive eﬀorts, a salvage procedure was planned, involving femoral head and neck osteotomy. head of this cat. Arterial branches that supply blood to the femoral head propagate from extra osseous vessels, such as the lateral and medial circumﬂex femoral arteries and the caudal and cranial gluteal and iliolumbar arteries, which are supplied from the surrounding soft tissue to the femoral head through the femoral neck and trochanteric fossa (22, 23). Compromise of this vascular network may induce changes in the femoral head and neck, such as resorption and degenerative joint disease (24–26). Although, avascular necrosis of the femoral head was not a common sequela in a dog and cat with femoral head and neck fractures (25), the comminuted neck, trochanter, and the surrounding soft tissue injury from the gunshot in the present case may have caused more extensive compromise of this vascular network than in cases with only neck fracture, markedly disrupting the blood supply of the femoral head and neck, and potentially led to the development of avascular necrosis in this cat. Additionally, the femoral neck was not stabilized in this cat because the comminuted neck fragments could not hold the implants engaging the neck. Thus, this consistent instability may also contribute to sclerotic changes and non-union of the neck and degenerative changes of the femoral head (25). However, it did not become a serious clinical problem causing pain and lameness since the cat is currently very active with a normal gait, 29 months after surgery. DISCUSSION At the last follow-up, lack of bone healing was identiﬁed in the proximal diaphyseal segments of the radiograph, despite functional recovery of the injured leg. This is thought to be due to the characteristics of a comminuted fracture and impaired blood supply caused by high energy-induced trauma, such as gunshot injury. Further, the sustained instability of the proximal segment caused by the inability to place implants in the comminuted proximal segment was also considered to have contributed to delayed bone healing. To increase the stability of the proximal segment, the preferred surgeries for biologic osteosynthesis of comminuted fractures, such as minimally invasive plate osteosynthesis or an interlocking nail, were considered. However, these conventional methods were unsuitable for this cat since the proximal segment was comminuted and not durable enough to place the implants. And, the femoral head and neck osteotomy with repair of diaphyseal fracture were also considered as a salvage procedure, however, since the femoral head was intact, it was decided to withhold and considered as a later option. Due to the limitations of conventional surgery, a TA-ESF of the hip was used for bridging osteosynthesis and a triangular conﬁguration was developed to reduce the load and increase the stability of the TA-ESF. Nevertheless, since a secure connection between TA-ESF and the proximal segment was not possible due to the comminuted and longitudinally fractured proximal diaphyseal fragment, it might have led to constant instability of the proximal segment inducing deﬁcient bone healing. Another major concern was postoperative degenerative changes of the femoral head, possibly due to immobilization of the hip joint and ischemic osteonecrosis secondary to reduced blood supply. Joint immobilization is the most signiﬁcant cause of degenerative joint disease associated with trans-articular ESF (11, 15, 20), but it was not considered the primary cause of femoral head degeneration in this cat. Previous experimental and clinical studies have reported that prolonged joint immobilization alters the biochemical characteristics of synovial joints (3, 20), inducing softening of the articular cartilage and decreasing cartilage thickness, leading to the development of degenerative joint disease (10, 14). These changes were apparent after 11 weeks of immobilization. However, they were reversible even after 11 weeks of immobilization. Thus, no signiﬁcant diﬀerence was seen between the immobilized joint and the age- matched controls after a remobilization period of 50 weeks (21). Additionally, weight-bearing during immobilization of the limb might promote recovery of articular cartilage (13). Frontiers in Veterinary Science \| www.frontiersin.org DISCUSSION In this cat, the left hip joint was immobilized for 64 days, which was not long enough to induce degenerative changes in the articular cartilage, relative to the immobilization period of previous studies (21). Moreover, consistent weight bearing-use of the limb during the immobilization period may have helped minimize these changes and facilitated recovery of the articular cartilage. Therefore, it is less likely that joint immobilization would have contributed signiﬁcantly to degenerative changes of the aﬀected femoral head in this cat. Performed surgery and contributed to the conception and design There are some limitations to the present case report. Although, this cat had several problems requiring long-term follow-up, this was not possible. In the radiograph at the last follow-up visit, lack of fracture healing, degenerative change of the aﬀected femoral head and thinning and sclerotic changes of the femoral neck were identiﬁed. These Damaged blood supply to the proximal femur was considered to be the main cause of degenerative changes in the femoral July 2021 \| Volume 8 \| Article 652847 5 Han et al. TA-ESF for Juxta-Articular Femur Fracture abnormalities should have been consistently monitored because they might progress to malunion of the fractured femur, especially non-union of the femoral neck and degenerative joint disease of the hip joint. Moreover, the intramedullary pin, which was left due to insuﬃcient remodeling of the femoral diaphysis, was to be removed as soon as complete union was conﬁrmed. For this reason, constant long-term follow- up was strongly recommended to the owner. However, the owner did not presented after ESF removal, since the cat could walk normally, without any signs and the owner had ﬁnancial and long-distance restrictions. Additionally, partial failure of this TA-ESF of the hip due to several weak points, including fracture of some ﬁxation pins and instability of the proximal segment, were seen in this case. Although, these problems did not aﬀect functional recovery in this cat, further, investigation on the number and location of taxation pins and frame of connecting bars with more cases is necessary to improve the stability and conﬁguration of this TA-EFS of the hip. proximal femur, in which the implant cannot be secured to the proximal segment. AUTHOR CONTRIBUTIONS H-JH performed surgery and contributed to the conception and design of the work, analyses of the data, and writing the manuscript. KL contributed to providing the concept of the most stable frame for TA-ESF based on physics theory and preparing the relevant part of manuscript, while H-YY supervised aftercare, and critically revised the manuscript. All authors contributed substantially to the study design, preparation, and ﬁnal approval of the manuscript. ETHICS STATEMENT Ethical review and approval were not required for the animal study because this is a case report which has been written using clinical data. Written informed consent was obtained from the owners for the participation of their animal in this study. CONCLUDING REMARKS In summary, despite the lack of fracture healing, TA- ESF provided a satisfactory clinical outcome, including successful biological osteosynthesis and functional recovery of a comminuted fracture of the proximal femur caused by gunshot injury in this cat. The ESF conﬁguration was determined based on the principle of physical force distribution, and the tied-in conﬁguration enhanced its stability. Further, the use of epoxy made it possible to construct this complicated conﬁguration easily without the limitations of angles, planes, or the position of ﬁxation pins. This conﬁguration is a feasible surgical option for such highly comminuted juxta-articular fractures of the SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fvets. 2021.652847/full#supplementary-material DATA AVAILABILITY STATEMENT The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s. REFERENCES 8. Boone EG, Johnson AL, Hohn RB. Distal tibial fractures in dogs and cats. J Am Vet Med Assoc. (1986) 188:36–40. 1. Farese JP, Lewis DD, Cross AR, Collins KE, Anderson GM, Halling KB. 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This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Copyright © 2021 Han, Lee and Yoon. 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. 21. Vanwanseele B, Lucchinetti E, Stüssi E. The eﬀects of immobilization on the characteristics of articular cartilage: current concepts and future directions. Osteoarthr Cartil. (2002) 10:408–19. doi: 10.1053/joca.2002.0529 July 2021 \| Volume 8 \| Article 652847 Frontiers in Veterinary Science \| www.frontiersin.org 7
https://openalex.org/W2972149456	https://www.sciendo.com/pdf/10.2478/bsrj-2019-016	English	null	Determinants of Tax Morale in Croatia: an Ordered Logit Model	Business systems research	2,019	cc-by	8,023	Abstract Background: A lower tax morality leads to an increased readiness to become active in the unofficial economy and causes the lack of public revenues. Objectives: The aim of this paper is to investigate determinants that shape tax morale of Croatian citizens. Methods/Approach: An ordered logit model is employed to evaluate which determinants shape tax morale of Croatian citizens. Data for the research were collected from 2,000 face-to-face interviews conducted in Croatia in late 2015. Results: The descriptive analysis illustrates that 52 percent of respondents reported a high level of tax morale, 26 percent of respondents have a low tax morale, while 8 and 14 percent have a mid-low and a mid-high tax morale, respectively. The ordered logit analysis revealed that gender, age, financial situation, region, and participation in the unofficial economy have an impact on the tax morale. Conclusions: Besides socio-demographic, socio-economic, and spatial determinants, a great number of sanctions for participation in informal activities also shapes tax morale of the Croatian citizens. More precisely, marginal effects show that those perceiving the expected sanctions as "normal tax or social security contributions due, plus a fine or a prison sentence" have by 6.3 percentage points higher probability of reporting the highest tax morale than others. Keywords: Croatia, ordered logit model, tax evasion, tax morale JEL classification: C25, H26, K42, O17 Paper type: Research article Received: Jun 04, 2018 Accepted: Jan 03, 2019 Citation: Bejaković, P., Bezeredi, S. (2019), “Determinants of Tax Morale in Croatia: an Ordered Logit Model”, Business Systems Research, Vol. 10, No. 2, pp. 37-48. DOI: 10.2478/bsrj-2019-016 Determinants of Tax Morale in Croatia: an Ordered Logit Model Predrag Bejaković Institute of Public Finance, Zagreb, Croatia Slavko Bezeredi Institute of Public Finance, Zagreb, Croatia Business Systems Research \| Vol. 10 No. 2 \|2019 Business Systems Research \| Vol. 10 No. 2 \|2019 Introduction Luttmer et al. (2014) in their seminal work define tax morale “as an umbrella term capturing nonpecuniary motivations for tax compliance as well as factors that fall outside the standard, expected utility framework”. They argue that Allingham et al. (1972) have constructed the benchmark economic model of tax evasion, in which the most important parameters having an effect on tax evasion are the tax rate, the detection risk, and the punishment for the detected individuals who evade paying taxes. In the mentioned model, a rational taxpayer evades to pay taxes as long as the 37 Business Systems Research \| Vol. 10 No. 2 \|2019 Business Systems Research \| Vol. 10 No. 2 \|2019 cost of being caught and punished is smaller than the benefit from tax evasion. Since this contribution, there has been an increased interest in analysing and enhancing the knowledge on “tax morale”. Modern fiscal literature by it means improving voluntary compliance with tax regulation and achieving a social norm of compliance (Wenzel, 2005a; 2005b). ) Luttmer et al. (2014) state that taxpayers may have some intrinsic motivation, which is a key instrument that helps them to respect the law and pay taxes or feel some moral guilt for failure to comply. They may respect the law due to the groups of various reasons. The first group is reciprocal motivations, i.e. the readiness to pay taxes in exchange for goods and services that the state provides to all citizens even though their monetary benefits would be bigger if they did not pay taxes (Luttmer et al., 2014). The second group may be various forms of the pressure from peer behaviour and the prospect of social recognition or sanctions from friends, neighbours, family members or co-workers. The third group consists of cultural or social norms like traditions, prevailing values and believes that can affect compliance and morale, but are also under the influence of various forms of motivation and the peers’ sensitivity. The fourth group includes the level of tax knowledge or tax literacy that influences compliance attitudes and decisions of current and potential taxpayers (Nichita, 2015). The fifth group contains deviations from utility maximization and information imperfections (for example, taxpayers may over- or underestimate the probability of being caught in tax evasion or may show loss aversion). Introduction Finally, the last group contains a policy and administration aspect: the behaviour and practice of the tax administration and the perceived tax burden, the tax structure in general as well as the tax enforcement strategy can influence taxpayers’ decision to pay taxes (Torgler et al., 2005). There are two main types of tax behaviour: compliance, which is either voluntary or enforced; and noncompliance that can be in the form of tax evasion or tax avoidance (OECD/FIIAPP, 2015). Many authors (Lago-Peñas et al., 2010; Kogler et al., 2013; Luttmer et al., 2014) believe that tax morale plays an important role in the tax compliance, although very often they see enforcement as the main reason of compliance. The main determinants of the tax morale expressing dissent against formal institutions are often connected with the existing or perceived infringement of the principle of equality by the same public authority (Chong et al., 2007). In particular, the tax policy and social security system are defined by regulations, which a part of the population judges as unfair according to the principle of equality and to its corollary, the ability-to-pay principle. As an outcome, people who withdraw from their fiscal obligations and accept a negative tax morale are able to justify their noncompliance with the rule. Such a negative tax morale is not only a causal element of the informal economy; it is very often also a cause of exclusion from a legal system expecting duties, but also from various rights. These considerations suggest that designing tax measures in accordance with the principle of equality is fundamental for increasing their effectiveness and for promoting a shift from the negative to the positive tax morale. The shift from the negative to the positive tax morale is the transformation of noncompliance into compliance, which is an essential condition for promoting the stable inclusion into the formal world of work and compliance regarding tax and social security obligations. The aim of this paper is to evaluate the determinants that shape tax morale of Croatian citizens. To do this, an ordered logit model is conducted based on data collected in Croatia in 2015. Croatia is a post-transition society, which emerged from a socialist system, and experienced damages of the Homeland war in the 1990s. Literature review Nerré (2008) deems that a tax culture is a specific characteristic of a particular country. It was coined by the tradition of taxation (having in mind various forms of taxes) on the one side, and by the interaction of the stakeholders and the cultural values like honesty, justice or also a sense of duty on the other side. Lago-Peñas et al. (2010) divide the literature on the tax culture into two categories. First group consists of those studies oriented only on one country, but after then authors usually compare international or regional variations and changes over time. Examples of such approach are researches Torgler (2005a), Martínez-Vázquez et al. (2009), Nerré et al. (2014), and Williams et al. (2014). Torgler (2005a) examine the impact of direct democracy on tax morale in Switzerland, and concluded that “direct democratic rights have a significantly positive effect on tax morale”. Martínez-Vázquez et al. (2009) argue that an increased level of the tax morale in Spain was an endogenous variable influenced primarily by improved tax policy and tax administration reforms. Nerré et al. (2014), as several more important reasons for low tax morale in Serbia, mention high tax rates, unjust distribution of tax burden and lack of detection, while other less important reasons were corruption among civil servants and a lack of civic conscience. Williams et al. (2014) conducted a resesarch for Bulgaria in 2013 and have propounded an explanation for the undeclared economy, which claims the following “the greater is the asymmetry between formal and informal institutions (i.e., citizens’ civic morality and state morality), the greater is the likelihood of participation in the undeclared economy, and vice versa”. y Similar to one measurement approach on the tax morale, there is a possibility to evaluate the magnitude of tax morale via surveys. Torgler et al. (2005) state that “The World Values Survey provides a valuable data source for getting internationally comparable information on tax morale”. They explain that the mentioned Survey uses the following question: “Please tell us for each of the following statements whether you think it can always be justified, never be justified, or something in between: … Cheating on tax if you have the chance”. The question leads to a ten-scale index of tax morale with the two extreme points “never justified” and “always justified”. Introduction It is important to underline that the practices and principles of tax systems under socialism have significantly influenced the path of tax reform during the transition period (Nerré 38 Business Systems Research \| Vol. 10 No. 2 \|2019 Business Systems Research \| Vol. 10 No. 2 \|2019 et al., 2014). Furthermore, Schneider (2017) estimates that Croatia is a country with one of the highest level of the undeclared economy among all EU-28 member states in 2015 (27.7% of GDP), only Bulgaria (30.6 % of GDP) and Romania (28.0 % of GDP) have higher levels of the undeclared economy, while the EU-28 average is 18.3% of GDP. Accordingly, the mentioned facts are the main motivation and reason for choosing Croatia as a country for research. This paper is the first that examines the impact of expected size of sanction for participation in informal activities and various socio-demographic, socio-economic, and spatial factors on tax morale in Croatia. The literature about informal economy reveals that higher tax morale leads to a smaller shadow economy (Torgler et al., 2007; Torgler et al., 2009) and the lower levels of participation in the informal economy (Williams et al., 2015; Williams et al., 2016a; Windebank et al., 2017; Franić et al., 2017). Thus, this article contributes to a better understanding of the determinants of tax morale as well as informal economy in Croatia. The paper provides an overview of the literature in Section 2 and a summary of the empirical evidence of the socio- economic and institutional determinants of tax morale in the following sections. Literature review The ten-point scale has been integrated into a four-point scale (from 0 to 3), with the value 3 for “never justifiable”. According to results from various years, the highest share of the people that state how tax evasion is never justified has been recorded in Japan (more than 80%), followed by USA (around 70%) and Switzerland (around 60%). This 39 Business Systems Research \| Vol. 10 No. 2 \|2019 value decreases to around 40% in Portugal, the Netherlands and Finland and is even lower in Belgium (below 35%). Another approach to measuring tax morale is by using the International Social Survey Programme (ISSP). This cross-national collaboration began in 1983 and has grown to more than 30, mostly European, countries (Croatia has been a member since 2005) and enables various types of social science projects. From time to time, the Programme asks the following question: “Do you feel it is wrong or not wrong if a taxpayer does not report all of his or her income in order to pay less personal income taxes? (1= not wrong, 2= a bit wrong, 3= wrong, 4=seriously wrong)”. ISSP Mean Tax Morale Values are low for Austria, Germany, Ireland and France and high for Australia, Sweden, Spain and Japan. Unfortunately, there is little overlapping of the results from various surveys and it is almost impossible to compare the variables from different sources (Schneider, 2011). ( ) Unlike the researchers oriented only on one country, the second group includes researches that make use of cross-national data. Using data from six countries, Torgler (2004) concludes that tax morale increases with age and is lower for self-employed, unemployed, and upper class taxpayers. Financial satisfaction, strong GDP growth, institutional quality, national pride, trust in government and legal system increase tax morale and decrease tax evasion. Similar results were obtained by other researches that added to the existing knowledge or provide possible explanations. Thus, Tittle (1980) deems that significantly higher tax morale recorded by women is caused by the facts that females are more compliant than males, and thus, are more risk averse. Furthermore, the modern female generations are much more independent than the former generations, and it is assumed that they will comply less (Torgler et al., 2005). As for elderly people, Tittle (1980) argues that they are more sensitive to the threats of sanctions than others. Furthermore, Torgler et al. Literature review (2005) argue that older people attained their social stakes over the years, as property or social status, and that they are more sensitive to the reactions from others. Consequently, they also state that their potential costs of sanction due to tax evasion increase. Chong et al. (2007) argue that high or increased income inequality, in combination with unsatisfactory quality of institutions, is one of the most important determinants of low tax morale. After the analysis of the tax culture of the 50 countries surveyed, Tsakumis et al. (2007) state that cultural framework based on male-oriented individualism, causes lower readiness to pay taxes. Finally, Lago-Peñas et al. (2010) claim that well educated taxpayers are supposed to know more about the fiscal legislations and tax because they are more aware of the importance of public goods provided by the state. Source: Authors’ work Methodology To evaluate which socio-economic, socio-demographic, and spatial determinants shape tax morale of the Croatian citizens, data were collected from 2,000 face-to- face interviews conducted in Croatia between August 20th and October 9th 2015. The survey was conducted by well-known Croatian market research agency “Ipsos Puls”. To get a nationally representative sample of the population, the sample was drawn according to official data gather in Census 2011 by the Croatian Bureau of Statistics and a two-way stratified random sample was used according to regions and settlement size. In each surveyed household, one respondent aged 18 years and older was selected using the “first birthday” method. To collect the data, trained professional interviewers used face-to-face methodology (TAPI – Tablet Assisted Personal Interview). Furthermore, to balance under- and over-representation in the sample, weighting procedure based on age, gender, education, region and settlement size was applied. The main advantage of the cross-sectional surveys is that 40 Business Systems Research \| Vol. 10 No. 2 \|2019 these surveys target different members of the population and together with the appropriate sampling method, these are the main factors that make the survey as representative as possible. On the other hand, a weakness of cross-sectional sample surveys is their inability to track change over time. surveys is their inability to track change over time. Table 1 The list of variables used in the analysis Variables Definition Values # of missing values Dependent variable: Tax morale A categorical variable which estimates the level of tax morale using the following survey question: “Now I would like to know how you assess various behaviours. For each of them, please tell me to what extent you find it acceptable or not. Please use the following 10-point Likert scale: ‘1’ means that you find it ‘absolutely unacceptable’ and ‘10’ means that you find it ‘absolutely acceptable’:... Someone evades taxes by not or only partially declaring income.” The 10-point scale has been recoded into a four-point scale: 1 = low tax morale (responses from 4 through 10); value 2 = mid low tax morale (response 3); value 3 = mid high tax morale (response 2); value 4 = high tax morale (response 1 “completely disagree”). 38 Explanatory variables: Gender A dummy variable indicating the respondent’s gender. 0 = men; 1 = women. 0 Age An interval variable indicating the exact age of the respondent. Methodology Exact age of the respondent (18 or more) 0 Employment status A categorical variable for the employment status of the respondents. 1 = employed; 2 = self-employed; 3 = unemployed; 4 = retired; 5 = student and inactive. 0 Financial situation A categorical variable for the respondent perception regarding its financial situation. 1 = struggling; 2 = maintaining; 3 = just comfortable or no money problems. 45 Household size A categorical variable which measures the size of the respondent’s household. 1 = one person; 2 = two persons; 3 = three persons; 4 = four or more people. 0 Region A categorical variable for the region to which the respondent belongs. 1 = Zagreb and surroundings; 2 = North Croatia; 3 = Slavonia; 4 = Lika and Banovina; 5 = Gorski Kotar, Hrvatsko Primorje and Istria; 6 = Dalmatia. 0 Participation in undeclared work A dummy variable which indicates participation in undeclared work using the following survey question: “Did you yourself carry out any undeclared paid activities in the last 12 months?” 0 = no; 1 = yes. 41 Purchasing goods and services on the informal market A dummy variable which indicates purchasing goods and services on the informal market using the following survey question: “Have you in the last 12 months paid for any goods or services of which you had a good reason to assume that they included undeclared work (e.g. because there was no invoice or VAT receipt)?” 0 = no; 1 = yes. 72 Expected sanctions A categorical variable describing the expected size of sanctions when someone is engaged in unregistered activities. 1 = normal tax or social security contributions due, but no fine; 2 = normal tax or social security contributions due, plus a fine or a prison. 344 Source: Authors’ work Table 1 The list of variables used in the analysis Variables Definition Values # of missing values Dependent variable: Tax morale A categorical variable which estimates the level of tax morale using the following survey question: “Now I would like to know how you assess various behaviours. For each of them, please tell me to what extent you find it acceptable or not. Please use the following 10-point Likert scale: ‘1’ means that you find it ‘absolutely unacceptable’ and ‘10’ means that you find it ‘absolutely acceptable’:... Methodology Someone evades taxes by not or only partially declaring income.” The 10-point scale has been recoded into a four-point scale: 1 = low tax morale (responses from 4 through 10); value 2 = mid low tax morale (response 3); value 3 = mid high tax morale (response 2); value 4 = high tax morale (response 1 “completely disagree”). 38 Explanatory variables: Gender A dummy variable indicating the respondent’s gender. 0 = men; 1 = women. 0 Age An interval variable indicating the exact age of the respondent. Exact age of the respondent (18 or more) 0 Employment status A categorical variable for the employment status of the respondents. 1 = employed; 2 = self-employed; 3 = unemployed; 4 = retired; 5 = student and inactive. 0 Financial situation A categorical variable for the respondent perception regarding its financial situation. 1 = struggling; 2 = maintaining; 3 = just comfortable or no money problems. 45 Household size A categorical variable which measures the size of the respondent’s household. 1 = one person; 2 = two persons; 3 = three persons; 4 = four or more people. 0 Region A categorical variable for the region to which the respondent belongs. 1 = Zagreb and surroundings; 2 = North Croatia; 3 = Slavonia; 4 = Lika and Banovina; 5 = Gorski Kotar, Hrvatsko Primorje and Istria; 6 = Dalmatia. 0 Participation in undeclared work A dummy variable which indicates participation in undeclared work using the following survey question: “Did you yourself carry out any undeclared paid activities in the last 12 months?” 0 = no; 1 = yes. 41 Purchasing goods and services on the informal market A dummy variable which indicates purchasing goods and services on the informal market using the following survey question: “Have you in the last 12 months paid for any goods or services of which you had a good reason to assume that they included undeclared work (e.g. because there was no invoice or VAT receipt)?” 0 = no; 1 = yes. 72 Expected sanctions A categorical variable describing the expected size of sanctions when someone is engaged in unregistered activities. 1 = normal tax or social security contributions due, but no fine; 2 = normal tax or social security contributions due, plus a fine or a prison. Methodology 344 Source: Authors’ work Table 1 The list of variables used in the analysis Variables Definition Table 1 The list of variables used in the analysis Variables Definition Values # of missing values Dependent variable: Tax morale A categorical variable which estimates the level of tax morale using the following survey question: “Now I would like to know how you assess various behaviours. For each of them, please tell me to what extent you find it acceptable or not. Please use the following 10-point Likert scale: ‘1’ means that you find it ‘absolutely unacceptable’ and ‘10’ means that you find it ‘absolutely acceptable’:... Someone evades taxes by not or only partially declaring income.” The 10-point scale has been recoded into a four-point scale: 1 = low tax morale (responses from 4 through 10); value 2 = mid low tax morale (response 3); value 3 = mid high tax morale (response 2); value 4 = high tax morale (response 1 “completely disagree”). 38 Explanatory variables: Gender A dummy variable indicating the respondent’s gender. 0 = men; 1 = women. 0 Age An interval variable indicating the exact age of the respondent. Exact age of the respondent (18 or more) 0 Employment status A categorical variable for the employment status of the respondents. 1 = employed; 2 = self-employed; 3 = unemployed; 4 = retired; 5 = student and inactive. 0 Financial situation A categorical variable for the respondent perception regarding its financial situation. 1 = struggling; 2 = maintaining; 3 = just comfortable or no money problems. 45 Household size A categorical variable which measures the size of the respondent’s household. 1 = one person; 2 = two persons; 3 = three persons; 4 = four or more people. 0 Region A categorical variable for the region to which the respondent belongs. 1 = Zagreb and surroundings; 2 = North Croatia; 3 = Slavonia; 4 = Lika and Banovina; 5 = Gorski Kotar, Hrvatsko Primorje and Istria; 6 = Dalmatia. 0 Participation in undeclared work A dummy variable which indicates participation in undeclared work using the following survey question: “Did you yourself carry out any undeclared paid activities in the last 12 months?” 0 = no; 1 = yes. Business Systems Research \| Vol. 10 No. 2 \|2019 In this paper the dependent variable is tax morale that measures individuals’ attitude about the moral obligation to pay taxes. To estimate the level of tax morale from the survey, the following question is used: “Now I would like to know how you assess various behaviours. For each of them, please tell me to what extent you find it acceptable or not. Please use the following 10-point Likert scale: ‘1’ means that you find it ‘absolutely unacceptable’ and ‘10’ means that you find it ‘absolutely acceptable’:... Someone evades taxes by not or only partially declaring income.” As in Torgler (2004), and Torgler et al. (2009), the 10-point scale has been redefined into a four point scale (1, 2, 3, 4), with the value 4 for “absolutely unacceptable”. Responses 4 through 10 were integrated into a value 1 due to lack of variance among them. The explanatory variables used to analyse the determinants of tax morale were selected based on previous studies of the informal economy and tax morale (Torgler 2004; Williams et al., 2017; Williams et al., 2018). The list of used variables is shown in Table 1. Given that the data contain missing values (i.e. ‘don’t know’ and refusal), multiple imputation procedure was applied to predict the values (Rubin, 1987, Royston, 2004; Schafer et al., 2002). More precisely, using a system of chained equations, twenty five imputations were simulated for each missing value. g Given that the dependent variable is an ordered categorical variable, an ordered logit model is here applied and then the average marginal effects are calculated. The ordered logit model is defined as (Cameron et al., 2005): * * 1 if i i i i j i j y x y j y           , (1) (1) where 1, , j M  and 1, , i N  . M denotes the total number of alternatives of the dependent variable and N denotes the sample size. Furthermore, ix is a column vector of explanatory variables,  is a row vector of parameters to be estimated and i is the logistic distributed random error. The unknown parameters j’s represent threshold parameters to be estimated along with , where 0  and M   (Cameron et al., 2005). Business Systems Research \| Vol. 10 No. 2 \|2019 Finally, the probability that observation i will choose alternative j is:       * 1 1 i j i j j i j i P y j P y F x F x                     , (2) (2) where F is the standard logistic cumulative distribution function   . 1 e F e     (3) (3) Finally, the marginal effect of an increase in an independent variable for the j -th response is given by (Mallick, 2009) Finally, the marginal effect of an increase in an independent variable for the j -th response is given by (Mallick, 2009)       ' ' 1 \| . k j i j i k P y j x F x F x x                  (4) (4) Methodology 41 Purchasing goods and services on the informal market A dummy variable which indicates purchasing goods and services on the informal market using the following survey question: “Have you in the last 12 months paid for any goods or services of which you had a good reason to assume that they included undeclared work (e.g. because there was no invoice or VAT receipt)?” 0 = no; 1 = yes. 72 Expected sanctions A categorical variable describing the expected size of sanctions when someone is engaged in unregistered activities. 1 = normal tax or social security contributions due, but no fine; 2 = normal tax or social security contributions due, plus a fine or a prison. 344 Source: Authors’ work “Have you in the last 12 months paid for any goods or services of which you had a good reason to assume that they included undeclared work (e.g. because there was no invoice or VAT receipt)?” p ) A categorical variable describing the expected size of sanctions when someone is engaged in unregistered activities. Source: Authors’ work 41 Business Systems Research \| Vol. 10 No. 2 \|2019 Findings g This study of a nationally representative sample in Croatia reveals that 52.1 percent of respondents reported that tax evasion is never justifiable (high level of tax morale), 26.0 percent of respondents have low tax morale, while 8.4 and 13.5 percent have mid low and mid high tax morale, in turn. To examine which population groups more frequently state the high level of tax morale, Table 2 illustrates the descriptive results. 42 Business Systems Research \| Vol. 10 No. 2 \|2019 The descriptive statistics reveals that women more frequently report high level of tax morale than man (54.6 percent compared with 49.4 percent) and that the proportion of declaring high level of tax morale increases as age increases. Having a high level of tax morale is less common among those struggling financially, those participating in the undeclared work and among those purchasing on informal markets. Reporting high level of tax morale also appears to be more prevalent in some regions (i.e. Zagreb and surroundings, Lika and Banovina) than the rest of the country. Those who perceive the size of sanctions when someone is engaged in informal activities to be higher more frequently reported high level of tax morale. Findings Table 2 Distribution of tax morale in Croatia, % of survey respondents Tax morale Low Mid low Mid high High Total 26.0 8.4 13.5 52.1 Gender Male 28.7 9.0 12.9 49.4 Female 23.6 7.8 14.0 54.6 Age 15-24 31.3 11.4 17.6 39.7 25-39 29.6 9.3 15.7 45.4 40-54 26.9 9.6 13.0 50.5 55-64 23.0 5.6 12.0 59.4 65+ 19.4 6.0 9.4 65.2 Employment status Employed 26.6 9.4 13.7 50.3 Self-employed 32.6 15.5 14.1 37.8 Unemployed 31.6 8.0 14.3 46.1 Retired 20.4 5.2 11.0 63.4 Student and inactive 29.6 11.5 17.8 41.1 Financial situation Struggling 29.6 6.4 14.9 49.1 Maintaining 22.6 8.9 12.6 55.9 Just comfortable/ No money problems 26.1 10.6 12.7 50.6 Household size One person 22.8 4.6 15.9 56.7 Two persons 27.1 8.8 12.2 51.9 Three persons 26.7 9.7 16.6 47.0 Four or more people 26.8 9.4 11.1 52.7 Region Zagreb and surroundings 22.5 5.2 13.9 58.4 North Croatia 24.4 9.3 12.3 54.0 Slavonia 27.5 9.7 14.8 48.0 Lika and Banovina 16.4 11.5 14.8 57.3 Gorski Kotar, Hrvatsko Primorje and Istria 36.3 8.7 21.8 33.2 Dalmatia 28.8 9.1 6.7 55.4 Participation in undeclared work No 23.3 8.1 14.0 54.6 Yes 46.9 10.3 9.9 32.9 Purchasing goods and services on the informal market No 21.5 7.4 13.8 57.3 Yes 38.4 10.9 12.3 38.4 Expected sanctions Normal tax or social security contributions due, but no fine 27.6 8.4 12.7 51.3 Normal tax or social security contributions due, plus a fine/ prison 20.8 8.4 14.6 56.2 Source: Authors’ work based on the representative survey of 2,000 individuals in Croatia Table 2 Distribution of tax morale in Croatia, % of survey respondents 43 Business Systems Research \| Vol. 10 No. 2 \|2019 To determine which types of Croatian citizens display a greater statistically significant association to have a high level of tax morale, Table 3 illustrates the results of an ordered logit regression analysis. As a robustness check of the results, an ordered probit model and linear regression model were estimated to see if the results are robust considering the change of the empirical methodology. The significance and the signs of the coefficients are the same in all three models. Furthermore, ordered logit and ordered probit models provide almost the same size of the marginal effect. Therefore, it can be concluded that the results are robust. Findings The results show that the propensity to have a high level of tax morale is greater among women, older age groups, and those who are not struggling financially. Many researchers show that tax morale rises with age. On the other hand, some of them illustrate that tax morale is stronger in the case of women (Torgler, 2006; Lago-Peñas et al., 2010; Hug et al., 2011; Torgler, 2012; Williams et al., 2016b), while Torgler (2005b) and Daude et al. (2010) show the opposite for the countries of the Latin America. Many researchers also state that financial situation increases tax morale (Torgler, 2005b; 2006; Lago-Peñas et al., 2010; Ristovska et al., 2013), while Daude et al. (2010) get negative correlation between satisfaction with financial situation and the high level of tax morale. Findings 10 No. 2 \|2019 The findings also illustrate that there are statistically significant correlations between the regions and the level of tax morale. More precisely, according to our research those living in Slavonia or in Gorski Kotar, Hrvatsko Primorje and Istria have a lower level of tax morale than others. As Williams et al. (2016b) show for the European Union, the results indicate that participants in the undeclared work and purchasers of goods and services on the informal market have a significantly lower level of tax morale than others. Indeed, participation in undeclared work decreases the probability of reporting the highest tax morale by 13.2 percentage points, while purchasing goods and services on the informal market decreases it by 14.2 percentage points (please see Table 3 and Figure 1). The additional finding is that the expected size of sanctions when someone is engaged in informal activities have also an impact on the tax morale. Furthermore, marginal effects show that those perceiving the expected sanctions for participation in informal activities as "normal tax or social security contributions due, plus a fine or prison" have by 6.3 percentage point higher probability of reporting the highest tax morale than others. Figure 1 Marginal effects on probability of high level of tax morale, % Source: Authors’ work based on the representative survey of 2,000 individuals in Croatia Notes: For categorical variable: Financial situation the marginal effect reflects the difference between the financial situation “Maintaining” and “Struggling”. 7.7 6.3 3.7 0.4 -13.2 -14.2 -20 -15 -10 -5 0 5 10 Financial situation: Maintaining Expected sanction: Normal tax or social security contributions due, plus a fine/ Prison Female Age Participation in undeclared work Purchasing goods and services on the informal market Figure 1 Marginal effects on probability of high level of tax morale, % g Marginal effects on probability of high level of tax morale, % Source: Authors’ work based on the representative survey of 2,000 individuals in Croatia Notes: For categorical variable: Financial situation the marginal effect reflects the difference between the financial situation “Maintaining” and “Struggling”. Findings Table 3 Ordered logit regression analysis of the tax morale determinants in Croatia Coefficient (Standard error) Marginal effect Female 0.161 (0.095)* 0.037 Age 0.017 (0.004)* 0.004 Employment status (RC: Unemployed) Employed 0.097 (0.144) 0.022 Self-employed -0.259 (0.245) -0.059 Retired 0.160 (0.184) 0.037 Student and inactive -0.081 (0.185) -0.019 Financial situation (RC: Struggling) Maintaining 0.336 (0.111)* 0.077 Just comfortable/ No money problems 0.206 (0.128)* 0.047 Household size (RC: One person) Two persons -0.138 (0.136) -0.031 Three persons 0.056 (0.147) 0.013 Four or more people 0.218 (0.145) 0.049 Region (RC: Zagreb and surroundings) North Croatia -0.189 (0.148) -0.043 Slavonia -0.329 (0.143) -0.076 Lika and Banovina 0.003 (0.176) 0.001 Gorski Kotar, Hrvatsko Primorje and Istria -0.856 (0.150)* -0.196 Dalmatia -0.185 (0.144) -0.042 Participation in undeclared work -0.578 (0.174)* -0.132 Purchasing goods and services on the informal market -0.622 (0.110)* -0.142 Expected sanctions (RC: Normal tax or social security contributions due, but no fine) Normal tax or social security contributions due, plus a fine / prison 0.278 (0.105)*** 0.063 Number of observations 2,000 Number of imputations 25 Prob > F 0.000 Source: Authors’ work based on the representative survey of 2,000 individuals in Croatia Notes: (1) We report the marginal effects for the highest score of the tax morale index (4) (2) Significance: p<0.05, p<0.01, *p<0.001 Table 3 Ordered logit regression analysis of the tax morale determinants in Croatia Coefficient (Standard error) Marginal effect Female 0.161 (0.095) 0.037 Age 0.017 (0.004)* 0.004 Employment status (RC: Unemployed) Employed 0.097 (0.144) 0.022 Self-employed -0.259 (0.245) -0.059 Retired 0.160 (0.184) 0.037 Student and inactive -0.081 (0.185) -0.019 Financial situation (RC: Struggling) Maintaining 0.336 (0.111)* 0.077 Just comfortable/ No money problems 0.206 (0.128)* 0.047 Household size (RC: One person) Two persons -0.138 (0.136) -0.031 Three persons 0.056 (0.147) 0.013 Four or more people 0.218 (0.145) 0.049 Region (RC: Zagreb and surroundings) North Croatia -0.189 (0.148) -0.043 Slavonia -0.329 (0.143) -0.076 Lika and Banovina 0.003 (0.176) 0.001 Gorski Kotar, Hrvatsko Primorje and Istria -0.856 (0.150)* -0.196 Dalmatia -0.185 (0.144) -0.042 Participation in undeclared work -0.578 (0.174)* -0.132 Purchasing goods and services on the informal market -0.622 (0.110)* -0.142 Expected sanctions (RC: Normal tax or social security contributions due, but no fine) Normal tax or social security contributions due, plus a fine / prison 0.278 (0.105)*** 0.063 Number of observations 2,000 Number of imputations 25 Prob > F 0.000 Source: Authors’ work based on the representative survey of 2,000 individuals in Croatia Notes: (1) We report the marginal effects for the highest score of the tax morale index (4) (2) Significance: p<0.05, p<0.01, **p<0.001 44 Business Systems Research \| Vol. Discussion and conclusion Torgler et al. (2009) define tax morale "as the intrinsic motivation to pay taxes. It measures an individual’s willingness to pay taxes, in other words, the moral obligation to pay taxes or the belief that paying taxes contributes to society". Using ordered logit model, this research analyses the determinants of the tax morale in Croatia. The results of the research are in line with those obtained by various previously realised surveys in other countries - that tax morale is greater among women and older age groups (Torgler, 2005b; Torgler, 2006; Lago-Peñas et al., 2010). Being in a better financial situation and perceiving the high level of sanction for participation in informal activities also increases the probability of reporting the highest level of tax morale. Tax morale itself has many aspects, which are under the influence of a variety of factors. The measures for enhancement of the tax moral are in the field of the tax legislation and tax policy, but also out of it. The tax system should be simple, stable, and predictable, have low tax rates and a broader tax base, almost without exemptions and reductions; while tax laws should be clear and fully respecting its citizens’ rights. 45 Business Systems Research \| Vol. 10 No. 2 \|2019 Unnecessary complicated labour law and constant changes in the tax structure and regulations are often used as a justification by those who considered playing against the rules of the game or are operating in the unofficial economy as morally acceptable. Such behaviour is also stimulated by the fact that the law-makers and responsible authorities (sometimes) fail to provide or implement penalties for undesirable forms of the tax conduct. However, the most important factor for achieving an improved tax morale is a strong and sustained economic growth; because in a prosperous and developed economy, most citizens are ready to bear their tax obligation if they feel that tax system is just and/or that the tax burden is distributed fairly among the population. A well-designed and administered tax system enables adequate incentives for investment and growth, and helps - together with targeted social spending policies - to improve income distribution and the fairness of the tax system. Discussion and conclusion A lower tax morality and a feeling of unfairness of the state regarding the use of collected taxes leads to greater willingness of individuals and business units to participate in the hidden economy and increase the tax evasion. The way to raise tax morale is by rational behaviour in the spending of the state budget. Taxpayers who notice that taxes are wisely and efficiently spent on quality public services have greater incentives to comply with tax laws. However, if the state spends public revenues in a reckless mode, does not respect the rule of law, provides inadequate public services, citizens will likely be evading the payment of taxes. Stubborn taxpayers that still attempt to evade payment of taxes would have to be convinced that compliance with tax laws and the payment of taxes brings a greater benefit to society than avoiding the paying taxes. Business Systems Research \| Vol. 10 No. 2 \|2019 12. Nerré, B. (2008), “Tax Culture: A Basic Concept for Tax Politics”, Economic Analysis and Policy, Vol. 38, No. 1, pp. 153-167. 13. Nerré, B., Dragojlović, A., Randjelović, S., Djenić, M. (2014), “Tax Reform in Serbia: experiences and perspectives”, in proceedings of the Tax Reforms: Experiences and Perspectives Conference, Institute of Public Finance, Zagreb, pp. 79-96. 14. Nichita, R. 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(2007), “The relation between national cultural dimensions and tax evasion”, Journal of International Accounting, Auditing and Taxation, Vol. 16, No. 2, pp. 131-147. pp 32. Wenzel, M. (2005a), “Misperceptions of social norms about tax compliance: From theory to intervention”, Journal of Economic Psychology, Vol. 26, No. 6, pp. 862-883. 33. Wenzel, M. (2005b), “Motivation or rationalisation? Causal relations between ethics, norms and tax compliance”, Journal of Economic Psychology, Vol. 26, No. 4, pp. 491-508. 34. Williams, C. C., Franić, J., Dzekova, R. (2014), “Explaining the undeclared economy in Bulgaria: an institutional asymmetry perspective”, The South East European Journal of Economics and Business, Vol. 9, No. 2, pp. 33-45. 47 About the authors Predrag Bejaković, PhD is employed at the Institute of Public Finance, Zagreb. He has taken part in a number of projects and has published in scientific and professional journals. He is the author and co-author of a number of books from the areas of the economy, education (particularly VET and adult education), public finance, underground economy and labour economics. His main fields of interests are labour economics, education, social policy, poverty reduction and economic development. Recently he was a team leader for Croatia for the Project Grey related to the underground economy. The author can be contacted at predrag.bejakovic@ijf.hr. Slavko Bezeredi, univ.spec.oec. is a Researcher at the Institute of Public Finance, Croatia, employed since 2011. Currently, he is a PhD student at Faculty of Economics and Business, University of Zagreb. He has been involved as a member of the Croatian national team in the project EUROMOD (a Europe-wide tax-benefit microsimulation model) since 2013. Recently he was employed as a Marie Curie Research Fellow at the University of Sheffield for a year. His research interests include microsimulations of taxes and social benefits, make work pay policies, labour market and informal economy. The author can be contacted at slavko.bezeredi@ijf.hr. 48
https://openalex.org/W4384561836	https://westminsterresearch.westminster.ac.uk/download/961599f46a1956ae558f824c1ecc0934c7f3e1a5f15482d006ceb5410510a8b7/1706063/s10020-023-00682-x%20%281%29.pdf	English	null	The role of CD180 in hematological malignancies and inflammatory disorders	Molecular medicine	2,023	cc-by	19,929	© The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Abstract Toll-like receptors play a significant role in the innate immune system and are also involved in the pathophysiology of many different diseases. Over the past 35 years, there have been a growing number of publications exploring the role of the orphan toll-like receptor, CD180. We therefore set out to provide a narrative review of the current evidence surrounding CD180 in both health and disease. We first explore the evidence surrounding the role of CD180 in physiology including its expression, function and signaling in antigen presenting cells (APCs) (dendritic cells, monocytes, and B cells). We particularly focus on the role of CD180 as a modulator of other TLRs including TLR2, TLR4, and TLR9. We then discuss the role of CD180 in inflammatory and autoimmune diseases, as well as in hematological malignancies of B cell origin, including chronic lymphocytic leukemia (CLL). Based on this evidence we produce a cur- rent model for CD180 in disease and explore the potential role for CD180 as both a prognostic biomarker and thera- peutic target. Throughout, we highlight specific areas of research which should be addressed to further the under- standing of CD180 biology and the translational potential of research into CD180 in various diseases. K d CD180 T ll lik t Ch i l h ti l k i I t i it I fl ti S t i l Keywords CD180, Toll-like receptors, Chronic lymphocytic leukemia, Innate immunity, Inflammation, Systemic lupus erythematosus, Hematological malignancies notably, toll-like receptors (TLRs) are gaining traction as important immunological players which regulate various physiological functions and play a role in many different diseases, including cancer and inflammatory disorders. Over the past 35 years, there have been a growing num- ber of publications on the expression and function of the orphan TLR, CD180, with significant literature exploring the role of the receptor in disease. Molecular Medicine Molecular Medicine Edwards et al. Molecular Medicine (2023) 29:97 https://doi.org/10.1186/s10020-023-00682-x Open Access Kurtis Edwards1, Peter M. Lydyard1,2,3* , Nino Kulikova4, Tamar Tsertsvadze5, Emanuela V. Volpi1, Nicholas Chiorazzi6 and Nino Porakishvili1 Kurtis Edwards1, Peter M. Lydyard1,2,3 , Nino Kulikova4, Tamar Tsertsvadze5, Emanuela V. Volpi1, Nicholas Chiorazzi6 and Nino Porakishvili1 Kurtis Edwards1, Peter M. Lydyard1,2,3 , Nino Kulikova4, Tamar Tsertsvadze5, Emanuela V. Volpi1, Nicholas Chiorazzi6 and Nino Porakishvili1 Introduction Research into the innate immune system has undergone a renaissance in recent times with a growing appre- ciation of the importance of this immunological system outside of its classical ‘innate’ functions. This has been hallmarked by an ever-increasing interest towards innate immune receptor expression and function. Perhaps most CD180 was initially discovered on human B cells through reactivity with a mouse monoclonal anti- body (clone G28.8) prepared for one of the annual International Human Leukocyte Antigen workshops in the 1980s (Valentine et al. 1988). The same group also showed that the binding of the G28.8 antibody to its target induced activation of human B cells (Valen- tine et al. 1988). The target antigen, identified later on murine B cells, was characterized as a radioprotective molecule of approximately 105 kDa (RP105) (Miyake © The Author(s) 2023. Open Access permits use, sharing, adaptation, dist original author(s) and the source, pro other third party material in this artic to the material. If material is not inclu regulation or exceeds the permitted licence, visit http://creativecommon Correspondence: Peter M. Lydyard p.lydyard@ucl.ac.uk Emanuela V. Volpi e.volpi@westminster.ac.uk 1 School of Life Sciences, University of Westminster, London, UK 2 The University of Georgia, Tbilisi, Georgia 3 Division of Infection of Immunity, University College London, Gower Street, London WC1E 6BT, UK 4 Agricultural University of Georgia, Tbilisi, Georgia 5 Javakhishvili Tbilisi State University, Tbilisi, Georgia 6 The Feinstein Institute, New York, NY, USA The molecular structure of CD180 In humans and mice, some TLRs including CD180 are found on the cell surface, whereas others are intracel- lular and located within endosomes (Medzhitov 2001). CD180 is a type 1 single-pass transmembrane protein with a molecular mass of 105 kDa and is made up of 661 amino acids in humans (Fugier-Vivier et al. 1997) and 641 in mice (Miyake et al. 1995); the amino acid sequence shares a high degree of sequence and struc- tural homology with other TLRs (Miyake et al. 1995). CD180’s extracellular LRR motif (Takeda et al. 2003) forms a horseshoe-like topology (Miura et al. 1996; Ohto et al. 2011) which is typical of TLRs (Fig. 1). CD180 exhibits strong similarity with TLR4 (Divanovic et al. 2005) and it forms a 2:2 heterodimer with myeloid differentiation 1 (MD-1) at its LRR motif (Ohto et al. 2011; Yoon et al. 2011). The association of CD180 with MD-1 is essential for its surface membrane expression in mice and humans (Nagai et al. 2002; Miyake et al. 1998; Miura et al. 1998). Similarly, TLR4 is also a het- erodimer and requires myeloid differentiation 2 (MD-2) for stable membrane expression (Visintin et al. 2006).f More recently, CD180 was shown to be present on malignant hematological cells. Functional studies using primary cells and immortalized cell lines have indicated that CD180 plays a significant role in the immunopathology of these cancers. Various publica- tions on autoimmune and inflammatory disorders have also implicated CD180 in the pathophysiology of these diseases. In this review, we discuss the characteristics and function(s) of CD180 in both normal physiology and pathological conditions and compare features of CD180 to other TLRs. We discuss the role of CD180 in hema- tological malignancies, autoimmune diseases, and other inflammatory diseases. In addition, we highlight TLR4 and CD180 also differ in their intracellular domains. All classical TLRs, including TLR4, con- tain a toll-like/interleukin-1 receptor (TIR) domain which enables signal propagation preferentially via myeloid differentiation primary-response protein 88 Fig. 1 The structure of CD180 compared to TLR4. The extracellular domain of CD180/MD-1 contains leucine rich repeats (LRR) and dimerizes at the C-terminus, thus forming a head-to-head topology (A), which contrasts TLR4/MD-2 that dimerizes at the N-terminus (B). CD180/MD-1 complex forms a 2:2 heterodimer, perhaps in the presence of a lipid molecule (L), which acts as a ligand and binds to the hydrophobic cavity of MD-1. Further studies showed that CD180 is present on a number of cell types, other than B lymphocytes, includ- ing dendritic cells (DCs) and macrophages (Divanovic et al. 2005) and has several distinct physiological roles. © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Edwards et al. Molecular Medicine (2023) 29:97 Page 2 of 22 how this knowledge may translate into further research and/or practice in the future. et al. 1995) and was eventually dubbed CD180 in the new cluster of differentiation (CD) nomenclature. Ear- lier studies using transfection to isolate the RP105/ CD180 antigen detected the presence of a leucine rich repeat (LRR) motif, which was the first indication that RP105 could be homologous with the TLR family (Miyake et al. 1995). Later, sequencing data, coupled with phylogenetic analysis, confirmed that CD180 had a high homology with TLRs, especially TLR4, and it was therefore included into the TLR family (Miura et al. 1996; Divanovic et al. 2005; Fugier-Vivier et al. 1997). Further studies showed that CD180 is present on a number of cell types, other than B lymphocytes, includ- ing dendritic cells (DCs) and macrophages (Divanovic et al. 2005) and has several distinct physiological roles. et al. 1995) and was eventually dubbed CD180 in the new cluster of differentiation (CD) nomenclature. Ear- lier studies using transfection to isolate the RP105/ CD180 antigen detected the presence of a leucine rich repeat (LRR) motif, which was the first indication that RP105 could be homologous with the TLR family (Miyake et al. 1995). Later, sequencing data, coupled with phylogenetic analysis, confirmed that CD180 had a high homology with TLRs, especially TLR4, and it was therefore included into the TLR family (Miura et al. 1996; Divanovic et al. 2005; Fugier-Vivier et al. 1997). The molecular structure of CD180 This is analogous to TLR4/MD-2 which binds to lipopolysaccharide (LPS) via a lipid-binding cavity found within MD-2. Whilst CD180 has a small intracellular portion, the intracellular portion of TLR4/MD-2 contains a toll-like/interleukin-1 receptor (TIR) box domain which enables the initiation of intracellular signaling via two alternative pathways: myeloid differentiation primary-response protein 88 (MyD88)-dependent and MyD88-independent Fig. 1 The structure of CD180 compared to TLR4. The extracellular domain of CD180/MD-1 contains leucine rich repeats (LRR) and dimerizes at the C-terminus, thus forming a head-to-head topology (A), which contrasts TLR4/MD-2 that dimerizes at the N-terminus (B). CD180/MD-1 complex forms a 2:2 heterodimer, perhaps in the presence of a lipid molecule (L), which acts as a ligand and binds to the hydrophobic cavity of MD-1. This is analogous to TLR4/MD-2 which binds to lipopolysaccharide (LPS) via a lipid-binding cavity found within MD-2. Whilst CD180 has a small intracellular portion, the intracellular portion of TLR4/MD-2 contains a toll-like/interleukin-1 receptor (TIR) box domain which enables the initiation of intracellular signaling via two alternative pathways: myeloid differentiation primary-response protein 88 (MyD88)-dependent and MyD88-independent Page 3 of 22 Edwards et al. Molecular Medicine (2023) 29:97 Edwards et al. Molecular Medicine (MyD88)- or toll/interleukin-1 receptor domain-con- taining adaptor-inducing interferon-β (TRIF)-depend- ent pathways (Kawasaki and Kawai 2014; Miyake 2004). However, CD180 does not have a TIR domain but instead possesses a very short intracytoplasmic tail (Miyake et al. 1995) (Fig. 1). Thus, CD180 cannot recruit the typical TLR-associated intracellular signal- ing components (Yazawa et al. 2003). are released upon tissue damage, cell death and oxida- tive stress. These molecules include the phospholipid OxPAPC, high mobility group Box 1 (HMGB1), S100 proteins, heat shock proteins, and many more that are also sensed by TLRs (Yu et al. 2010). It is therefore possi- ble that CD180 has both natural microbe-associated and endogenous ligands that are yet to be discovered. Whilst both the endogenous and exogenous ligands for CD180 are yet to be established, it is possible that CD180 detects its ligands through homodimerization (Ohto et al. 2011; Yoon et al. 2011) (see section on CD180 structure). CD180 might also heterodimerize with other TLRs, thereby allowing interactions with ligands through well characterized mechanisms. For example, TLR1/2 and TLR2/6 form heterodimers to detect lipoproteins/ lipopeptides, and TLR1/10, TLR2/TLR10, and TLR4/ TLR6 can heterodimerize to detect endogenous DAMPs. CD180 is known to physically associate with TLR4 and TLR2 (Divanovic et al. Potential ligands for CD180 CD180 is referred to as an ‘orphan’ TLR since its natu- ral ligands have not yet been identified. The other TLRs have a wide variety of ligands ranging from flagellin to DNA and RNA of microbes (Kawasaki and Kawai 2014). These microbial-associated ligands are categorized as microbe-associated molecular patterns (MAMPs)— formerly called pathogen-associated molecular pat- terns (PAMPs)—and their recognition is a pivotal part of innate immune responses. The TLR4/MD-2 complex detects lipopolysaccharide (LPS) from Gram-negative bacteria through the lipid A binding pocket found within MD-2 (Fig. 1); however, early research indicated that LPS did not bind to MD-1 (Tsuneyoshi et al. 2005). Though, this was subsequently challenged by crystallographic studies which demonstrated direct LPS interactions with MD-1 via a lipid-like moiety (Ohto et al. 2011; Yoon et al. 2010) (Fig. 1), suggesting the CD180/MD-1 complex also can directly bind and respond to LPS (see Function of CD180). However, LPS is unlikely to be a CD180 ligand, but rather is sensed in tandem with TLR4, since TLR4/ MD-2 CD180/MD1-negative human embryonic kidney cells (HEK) 293 cells are unresponsive to LPS (Liu et al. 2013). The molecular structure of CD180 2005; Nagai et al. 2005) and, therefore, these TLRs are candidates which could form heterodimers with CD180 for ligand detection. Moreo- ver, a model by which CD180 heterodimerizes with other TLRs could explain how CD180 initiates signaling events, despite its short intracytoplasmic tail. The search for CD180 ligands is ongoing and will be crucial for develop- ing our understanding of CD180 in health and disease. CD180‑mediated B cell signaling and activation Signaling through most TLRs results in activation through TIRAP (toll/interleukin 1-receptor adaptor pro- tein) and MyD88 leading to induction of pro-inflamma- tory cytokines. TLR4 also has another signaling pathway mediated by the adaptor proteins TRIF and TRAM (TRIF related adaptor molecule), leading to production of type 1 interferons. Although CD180 lacks a TIR domain, it possesses a short intracellular portion of around 11 amino acids. It is possible that CD180 interacts with the B cell receptor (BCR) through this tail. PIM-1L-kinase is also phosphorylated upon ligation of CD180 on human B cells and increases the phosphoryla- tion of the anti-apoptotic protein BAD. PIM-L1 kinase also co-localizes, and is physically associated with CD180 (Egli et al. 2015), implying that it is an early adaptor mol- ecule in CD180 signal transduction (Fig. 3). The exact CD180 signaling pathway(s) is/are still unclear in normal B cells (Fig. 3). Signaling events in knockout mice (KO) and human B cells showed that CD180 ligation leads to the phosphorylation of vari- ous intracellular protein kinases including Syk, Lyn, p38MAPK, ERK and AKT (Porakishvili et al. 2011; Chan et al. 1998). Whilst these intracellular signaling patterns are reminiscent of BCR signaling, CD180 activation fails to effectively mobilize intracellular calcium as IgM engagement does (Porakishvili et al. 2011; Chan et al. 1998). Following ligation of CD180 (with murine monoclo- nal antibody to CD180—mAb) in healthy B cells, both the AKT and mitogen activated protein (MAP) kinase pathways become activated, ultimately converging into the upregulation of the Nuclear factor kappa-light-chain- enhancer of activated B cells (NF-kB) pathway (Yazawa et al. 2003; You et al. 2017) (Fig. 3). In turn, healthy B cells upregulate expression of the activation and prolif- eration markers CD86 and Ki-67 (Porakishvili et al. 2005, 2011) and increase their production of immunoglobu- lins (Chaplin et al. 2011). More detailed signaling stud- ies, especially with identified ligands, will be important to link the early signaling events to downstream players and eventually to transcription factors other than NF-kB. CD19 is important in CD180 signaling, based on evi- dence in CD19 mouse KO experiments and on the fact that ligation of CD180 leads to CD19 phosphorylation; CD19 deficiency therefore leads to hyporesponsiveness to CD180 ligation (Yazawa et al. 2003). It is also notewor- thy that we have observed strong correlations between the density of CD180 and CD19 in normal B cells (Fig. 2). CD180 in different cell populations: expression, signaling and function CD180 is readily expressed by cells of the haematopoi- etic system, in particular antigen presenting cells: DCs, macrophages and B cells (Zarember and Godowski 2002). Thus, the human spleen and lymph nodes are the anatomical sites with the greatest expression of CD180 (along with other TLRs) (Zarember and Godowski 2002; Roshak et al. 1999). However, since CD180, unlike sev- eral of the TLRs, is not expressed by epithelial cells lining the digestive (Testro and Visvanathan 2009) and urinary tracts (Samuelsson et al. 2004), and the uterus (Schaefer et al. 2004), it is assumed that CD180 does not provide the classical ‘sentinel’ function of TLRs at entry points into the body. Crystallographic studies have been utilized to investi- gate how MD-1 may bind to lipid molecules, such as lipid IVa in chickens (Yoon et al. 2010) and phosphatidylcho- line in mice (Harada et al. 2010). Using mass spectrom- etry, Ohto, Miyake and Shimizu later also determined electron densities which were consistent with the pres- ence of lipid molecules bound to the MD-1 portion of the murine CD180/MD-1 complex (Ohto et al. 2011). In accordance with the conservation in structure between CD180 and TLR4, the evidence to date suggests that the ligand for CD180 is likely to bind to the MD-1 portion of the complex, as is the case with TLR4/MD-2 and LPS (Fig. 1). Schultz et al. also showed that synthetic lipopep- tide molecules, that mimicked the lipoproteins expressed by Mycobacterium tuberculosis and Mycobacterium bovis, resulted in CD180-dependent activation of macrophages (Schultz et al. 2018). Therefore, Mycobacterium-derived MAMPs may be natural CD180 ligands. CD180 is expressed at the highest levels on both human (Valentine et al. 1988) and murine mature B cells (Miyake et al. 1995), although certain heterogeneity exists in the expression of CD180 amongst some defined B cell subsets. Murine follicular (FO) B cells exhibit a reduced expression of CD180 compared to marginal zone (MZ) B cells (Nagai et al. 2005). Furthermore, based on immunohistochemistry studies of normal human tonsils, our group demonstrated that CD180 is highly expressed within the marginal zone, but not in the germinal cen- tres (Edwards et al. 2021), a pattern which is mirrored In addition to MAMPs, many endogenous danger mol- ecules (damage-associated molecular patterns, DAMPs) Edwards et al. Molecular Medicine (2023) 29:97 Page 4 of 22 Fig. 2 The relationship between CD180 and CD19 in normal B cells. CD180 in different cell populations: expression, signaling and function Using Pearson’s correlation, the density of CD180 correlated strongly with CD19 on normal B cells. PBMC samples from healthy donors were stained with anti-CD180 (PE) and anti-CD19 (FITC) monoclonal antibodies (mAb). Antigen densities were measured by flow cytometry and expressed as a ratio to the fluorescence of the isotype control—relative fluorescent intensity (RFI) in murine lymph node tissues (Miura et al. 1998). The expression of CD180 was also found to be around two- fold higher by memory B cells compared to naïve B cells (Good et al. 2009). In humans, there appears to be no difference in the expression of CD180 in the CD5+ B cell subset compared with the CD5− subset in the peripheral blood (Porakish- vili et al. 2005). Furthermore, fully differentiated human B cells (plasma cells) do not normally express CD180 (Kikuchi et al. 2018). Early data indicated that some molecules found on the surface of B cells might regulate expression of CD180. Activation of human B cells with anti-IgM stimulating antibodies decreases the expression of CD180 (Porakish- vili et al. 2005), suggesting that activated B cells express less CD180 than resting B cells. Studies of murine B cells also showed that ligation of TLR7 or TLR9 can downreg- ulate the expression of CD180 (You et al. 2017). Further, the expression of CD180 on B cells can differ, and tends to be downregulated, in malignant, autoimmune, and inflammatory pathologies. Fig. 2 The relationship between CD180 and CD19 in normal B cells. Using Pearson’s correlation, the density of CD180 correlated strongly with CD19 on normal B cells. PBMC samples from healthy donors were stained with anti-CD180 (PE) and anti-CD19 (FITC) monoclonal antibodies (mAb). Antigen densities were measured by flow cytometry and expressed as a ratio to the fluorescence of the isotype control—relative fluorescent intensity (RFI) Fig. 2 The relationship between CD180 and CD19 in normal B cells. Using Pearson’s correlation, the density of CD180 correlated strongly with CD19 on normal B cells. PBMC samples from healthy donors were stained with anti-CD180 (PE) and anti-CD19 (FITC) monoclonal antibodies (mAb). Antigen densities were measured by flow cytometry and expressed as a ratio to the fluorescence of the isotype control—relative fluorescent intensity (RFI) which facilitates the recruitment of Lyn and Vav upon CD180 activation (Yazawa et al. 2003). Lyn and Vav phosphorylation are important early signaling events in CD19 activation (Sato et al. CD180 in different cell populations: expression, signaling and function 1997); thus, CD180 may activate a similar signaling program. Dependency of murine B cells on Lyn signaling has been evidenced in KO mice studies. Lyn−/− mice showed an attenuated activation of MAPK isoforms compared to wild-type mice, indicating a significant role of Lyn in early CD180-mediated signal transduction (Chan et al. 1998). CD180‑mediated B cell signaling and activation Importantly, CD19 may serve as an adaptor molecule Edwards et al. Molecular Medicine (2023) 29:97 Page 5 of 22 Fig. 3 CD180 signaling in human B cells. Upon CD180 engagement with anti-CD180, CD19 becomes phosphorylated and associates with CD180 in the membrane. Downstream signaling members Vav appear to be crucial for transducing CD180 signals. CD180 concomitantly activates mitogen activated protein kinases and Akt signaling in healthy B cells, both of which appear to contribute to the upregulation of costimulatory molecules and cell survival. CD180 also associates with the PIM-1L kinase at the cell surface, which, upon activation of CD180 with anti-CD180, results in increasing levels of BAD. B cells can also receive signals from CD40 and interleukin 4 (IL-4) receptors to promote proliferation and survival whilst CD40 activation may promote upregulation of PIM-1L Fig. 3 CD180 signaling in human B cells. Upon CD180 engagement with anti-CD180, CD19 becomes phosphorylated and associates with CD180 in the membrane. Downstream signaling members Vav appear to be crucial for transducing CD180 signals. CD180 concomitantly activates mitogen activated protein kinases and Akt signaling in healthy B cells, both of which appear to contribute to the upregulation of costimulatory molecules and cell survival. CD180 also associates with the PIM-1L kinase at the cell surface, which, upon activation of CD180 with anti-CD180, results in increasing levels of BAD. B cells can also receive signals from CD40 and interleukin 4 (IL-4) receptors to promote proliferation and survival whilst CD40 activation may promote upregulation of PIM-1L Murine B cells the targeting of CD40, which resulted in the prolifera- tion of antigen non-specific B cells (Chaplin et al. 2013). Data from these experiments, therefore indicated a syn- ergy between CD180 and the BCR for effective antigen responses. Some of the earliest functional studies on CD180 dem- onstrated that this receptor is related to B cell activation. Ligation of CD180 on murine B cells (Roshak et al. 1999) led to cell activation, proliferation, and protection from radiation and dexamethasone-induced apoptosis (Miyake et al. 1995; Miura et al. 1998). In 2005, Nagai et al. first demonstrated additional CD180 functions. Iinjection of mice with anti-CD180 upregulated transcription factors associated with plasma cell differentiation and increased the number of transitional and mature B cells but did not affect memory B cells (Nagai et al. 2005). It also increased production of IgG1 and IgG3, but not IgG2b or IgA (Chaplin et al. 2011). Further, coupling an antigen (Ag) to CD180 antibodies increased IgG production by B cells, even in CD40−/− and T cell-deficient mice (Chaplin et al. 2013). Antigen targeting to CD180 also increased the number of antigen-specific B cells, compared to The physiological function of CD180 is still unclear, and it is likely that resolution will ultimately require iden- tification of its in vivo ligand(s). As described above, in mice, LPS seems to bind to the CD180/MD-1 complex, but the role of LPS in generating CD180-dependent cell signaling is still debatable. There are, however, data sug- gesting that CD180 on B cells regulates the function/ expression of some of the classical TLRs. CD180 regulates TLR2 and TLR4 function Unlike TLR4 and other TLRs, CD180 ligation alone does not lead to cytokine production (Chaplin et al. 2011); instead, CD180 regulates cytokine production through Edwards et al. Molecular Medicine (2023) 29:97 Page 6 of 22 Edwards et al. Molecular Medicine modulation of TLR function in murine B cells. In 2000, Ogata et al. first showed that CD180-deficient B cells exhibit hyporesponsiveness to LPS (Ogata et al. 2000). It was later shown that wild type murine B cells responded to LPS or lipid A with anti-CD180 mAb by upregula- tion of CD86, whereas B cells from MD-1−/− mice had an attenuated response (Nagai et al. 2005). Interest- ingly, CD180−/− mice also had reduced Ig production in response to LPS compared to wild type (WT) mice. Later, Yazawa et al. also showed that, by using antibod- ies to block MD-1, LPS-mediated phosphorylation of CD19 was completely inhibited. This further indicated the importance of CD180/MD-1 in the regulation of LPS detection by murine B cells (Yazawa et al. 2003). proliferation and modulation of TLR responses. B cells from CD180−/− mice also show an attenuated upregu- lation of CD69 and CD86 in response to synthetic lipo- peptides compared to wild type (WT) controls, which further implicates CD180 in the modulation of TLR2 function (Blumenthal et al. 2009). Unlike TLR-mediated cytokine production in murine B cells, ligation of CD180 alone fails to induce cytokine production, yet, together with TLR2 and TLR4 ligands, CD180 engagement increases production of IL-6, IL-10 and tumor necrosis factor alpha (TNFα) (Chaplin et al. 2011). CD180 in murine B cells, therefore, appears to be involved in enhancement/modulation of specific cytokine function through TLR2 and TLR4, as well as inducing proliferation, activation, and antibody production. The exact role which CD180 plays in the regulation of TLR-mediated LPS signaling in mice has, however, been challenged. Allen et al. showed that, whilst B cells from CD180−/− mice have a reduced proliferative response to LPS, the mice remain responsive to other proliferative stimuli (Allen et al. 2012). Moreover, MZ and FO B cells from CD180−/− mice show comparable upregulation of CD69 and MHC II as well as no differences in the pro- duction of IL-10 or IL-6 in response to LPS challenge. Consistently, the same study also found that basal B cell proliferation was increased, especially within the MZ B cell subset. Human B cells Antibody-mediated stimulation of CD180 on human peripheral blood B cells activates a programme of intra- cellular signaling and leads to an increase in the expres- sion of activation markers and proliferation (Porakishvili et al. 2005, 2011). Direct stimulation through CD180 also leads to increased cell survival, perhaps in part via anti-apoptotic proteins such as BAD (Egli et al. 2015) or activation of NF-kB in an AKT-dependent manner (Yamazaki et al. 2010; Porakishvili et al. 2015). Therefore, the function of CD180 on human B cells is directed pri- marily towards cell activation, survival, proliferation, and antibody production. B cell activating factor (BAFF) is a key survival fac- tor for B cells and is also important in TLR signaling. CD180−/− mice show an elevated expression of BAFF by qPCR, and neutralisation of BAFF reversed both the LPS hyporesponsive phenotype and elevated basal pro- liferation (Allen et al. 2012), implying a role of BAFF in CD180-mediated LPS responses. Whilst the data from KO mice studies indicate a role of CD180 in LPS respon- siveness, the findings by Allen et al. (2012) cannot be ignored. They suggest that absence of CD180 might not be the only reason for attenuation of murine B cell LPS responsiveness but rather that disrupted expression/ function of other factors related to CD180 activation may be at the root of defective LPS detection within this gen- otype. Exploration of other signaling players which func- tion downstream of CD180 will enable more accurate evaluation of how CD180 disruption contributes to LPS hyporesponsiveness in murine B cells. CD180 regulates the function of TLR9 in human B cells Yamazaki et al. (2010) showed that TLR9 expression within the endosomes increased following treatment of both naïve and memory B cells with anti-CD180 antibod- ies. The same authors also demonstrated that treatment with anti-CD180 and the TLR9 ligand CpG drastically increased the proportion of viable B naïve cells in culture, compared to naïve B cells treated with CpG alone. More- over, the effect of the engagement of CD180 plus TLR9 was synergistic, increasing the phosphorylation of AKT. However, stimulation of B cells with anti-CD180 alone failed to significantly induce AKT phosphorylation. These results are discordant with our own studies in nor- mal human B cells, since our group (Porakishvili et al. 2015) and another (Gordiienko et al. 2017a) have found that CD180 activation induced significant phosphoryla- tion of AKT (and p38MAPK). CD180 regulates TLR2 and TLR4 function Together, these findings suggest that defec- tive responses to LPS by murine B cells may not be com- pletely explained by the CD180−/− genotype, as suggested by Allen et al. Human B cells Like TLR4, CD180 appears to exert regulatory influ- ence on TLR2 in murine B cells. CD180−/− mice have poor proliferative responses, diminished plasma cell dif- ferentiation, and defective polyclonal Ig production in response to TLR2 (but not TLR9) ligands (Nagai et al. 2005), suggesting a dichotomous role of CD180 in B cell CD180 and TLR9 regulate antibody production CD180 and TLR9 regulate antibody production y CpG activation of TLR9 serves as a stimulus to promote polyclonal Ig production following infection (Bernasconi et al. 2199). Yamazaki et al. found that CpG treatment Edwards et al. Molecular Medicine (2023) 29:97 Page 7 of 22 Edwards et al. Molecular Medicine regulation of murine B cell differentiation and Ig pro- duction (Arnold et al. 2016). The addition of ATRA to B cells stimulated with anti-CD180 and CpG was found to significantly increase the formation of autophagosomes in both naïve and memory B cells. Moreover, B cells treated with ATRA, CpG, and anti-CD180 mAb induced autophagosome and lysosome colocalization, as well as the number of autophagosomes, indicating a role for these three stimuli in the remodelling and self-digestion of B cells. The production of IgG was also dependent on the transcription of the Unc-51 Like Autophagy Acti- vating Kinase 1 (ULK1), which is increased significantly by stimulating cells with ATRA, CpG, and anti-CD180 together (Eriksen et al. 2015). Therefore, in combination, CD180 and TLR9 function to regulate ATRA-mediated autophagy of B cells, which may be important for effec- tive Ig responses. increased the production of IgA, IgD, and IgM (Yamazaki et al. 2010). The same authors also showed that anti-CD180 antibodies did not enhance Ig production in the presence of anti-CD40, CpG, or SAC (Staphylococcus aureus Cowan strain 1) or IL-21 treatment. These findings suggest that production of immunoglobulins by human and murine B cells diverges in its response to the CD180 ligation. g g Interestingly, the vitamin A metabolite, all trans reti- noic acid (ATRA), a nutrient significantly correlated with immunological health as well as B cell function (Blomhoff et al. 1992; Ertesvag et al. 2007), may help to regulate the Ig production via CD180 and TLR9. Erik- sen et al. reported that ATRA treatment of both naïve and memory B cells increased the proliferative effects of anti-CD180 treatment alone and together with the TLR9 ligand CpG (Eriksen et al. 2012). Further, ATRA treatment attenuated the CD180-dependent production of IgG. However, it also drastically enhanced IgG pro- duction in memory B cells synergistically activated with anti-CD180 and CpG. Consistently, B cells stimulated with both, anti-CD180 and CpG, significantly increased production of IL-10 following ATRA treatment, whereas ATRA almost completely abrogated the production of IL-10 in B cells treated with anti-CD180 alone. Together, these findings suggest that TLR9, CD180, and ATRA work in concert for optimum antibody production. CD180 and TLR9 regulate antibody production Moreover, the fact that CD180 activation alone leads to a dampening of memory B cell activation could indicate that CD180 serves as a negative regulator to suppress excessive immune responses. This concept aligns with the studies of CD180 in autoimmune disease where loss of CD180 tends to be associated with a more severe dis- ease phenotype (see section on CD180 in disease). Together, the data accrued so far implicate a signifi- cant role for CD180 in the biology of B cells. CD180 has stand-alone functions, whereby it can promote activa- tion and proliferation of B cells as well as production of immunoglobulins. Further, CD180 works in concert with classical members of the TLR family, apparently exerting regulatory function over immune activation in response to microenvironmental stimuli. Differences in CD180 function also exist between mice and man, particularly with respect to TLR function, given that murine B cells and human B cells differ in their TLR expression profiles. Furthermore, CD180 may play a role in modulating the function of other TLRs expressed on B cells and indeed other classes of receptors. Myeloid cells B cell proliferation and Ig production by the proposed “CD180/TLR9 axis” appears to be regulated, at least par- tially, by reactive oxygen species (ROS). By-products of mitochondrial respiration act as second messengers in intracellular signaling systems, including BCR signaling (Tsubata 2020). It was shown recently that CD180/TLR9- mediated production of Ig was accompanied by the acti- vation of NADPH oxidase (NOX) (Gilljam et al. 2020). N-acetyl cystine (NAC), a ROS scavenger, also selectively reduced the production of IgG, rather than IgM, initiated by CD180/TLR9 activation (Gilljam et al. 2020). There- fore, the respiratory function, mitochondrial health, and production of ROS in B cells may also regulate isotype- specific Ig production and proliferation via combined CD180 and TL9 signaling. CD180 is highly expressed on both murine and human monocytes and macrophages (Zarember and Godowski 2002; Roshak et al. 1999), but not granulocytes (Tserts- vadze et al. 2015). Myeloid DCs express CD180 and TLR4 (Divanovic et al. 2005), whereas plasmacytoid DCs only appear to express CD180 intracellularly (Schuster et al. 2010) and do not produce TLR4 (Krug et al. 2001). The CD180/MD-1 complex has also been shown to be physically associated with both TLR4/MD-2 and with TLR2 in immunofluorescence and immunoprecipitation studies with murine macrophages and TLR expression models using human embryonic kidney (HEK) 293 cells (Divanovic et al. 2005; Blumenthal et al. 2009). The expression of CD180 in B lymphocyte malignanciesh The expression of CD180 on B cells has led to the study of the molecule in B lymphocyte malignancies where CD180 expression and function appears to diverge from its normal B cell counterparts. CD180 expression and function have also been studied in other non-malignant pathologies, such as autoimmune and inflammatory pathologies, and these will be addressed in the next sec- tion. There is now a growing body of evidence implicat- ing CD180 in disease, and this research has begun to highlight how CD180 could be exploited as a diagnostic, a prognostic, or even a therapeutic target. The apparently conflicting data on the CD180-medi- ated regulation of TLR2 or TLR4 may be explained by concentrations/availability of the respective ligands. Liu et al. found that cytokine responses in murine mac- rophages, pre-treated with blocking anti-MD-1 antibod- ies and then concomitantly treated with the TLR2 ligand Pam3CSK4, and TLR4 ligand LPS, were similar to con- trols (Liu et al. 2010). However, when the dosage of Pam3CSK4 was increased, production of pro-inflamma- tory cytokines was significantly enhanced. This is consist- ent with previous findings where TLR2 activation was found to overcome CD180-dependent negative regula- tion of TLR4-mediated LPS detection (Divanovic et al. 2005). Regulation of antigen presentationh The role of CD180 in myeloid cells has also been explored within the context of antigen processing. In 2013, Chap- lin, Chappell and Clark showed that mice with CD180- sufficent B cells, but CD180-deficient non-B cells, produced significantly less IgG compared to mice where CD180 was expressed in both B cells and non-B-cells (Chaplin et al. 2013). This suggested that CD180 expres- sion by APCs from both the myeloid and lymphoid lin- eages is required for optimum antibody response. The fact that DCs, treated with antigen-coupled anti-CD180, were able to more robustly stimulate the proliferation of antigen-specific T cells, compared to B cells also lends credence to this concept (Chaplin et al. 2013; Roe et al. 2019). Further, antigen targeting of CD180 also improved the capacity of DCs to stimulate antigen-specific T helper cells compared to targeting of CD40 (Roe et al. 2019). Therefore, CD180 functions to promote effective anti- body responses through both DCs and B cells. Experimental model systems utilizing the human HEK293 cell line to study TLR signaling also sup- port the negative role of CD180 on TLR4 in myeloid cells. HEK293 cells, transfected with cDNA in order to express the CD180/MD-1 and TLR4/MD-2 complexes, show reduced IL-8 production in response to LPS chal- lenge, compared to cells expressing TLR4/MD-2 alone (Divanovic et al. 2005). CD180 is also expressed by murine myeloid derived suppressor cells (MDSCs) and can negatively regulate responses to endotoxin via TLR4 (Dong et al. 2019). In contrast, Nagai et al. showed that knockout of CD180 did not impair the production of TNFα in response to TLR4 or TLR2 ligands in either murine DCs or macrophages (Nagai et al. 2005). This report was consistent with a later study which also found that mac- rophages from CD180−/− mice did not have attenuated cytokine responses following treatment with TLR2 or TLR4 ligands (Blumenthal et al. 2009).hl Regulation of TLR function g CD180 is an activation molecule on B cells and is involved in antibody production, cell activation, and modulation of TLR signaling. CD180-deficent murine DCs and macrophages show greater responsiveness to LPS (in terms of cytokine production) compared to their Building on the work investigating the role of ATRA in B cell function with respect to CD180 and TLR9, Eriksen et al. investigated the role of ATRA on CD180 and TLR9-mediated autophagy (Eriksen et al. 2015), since autophagy is a process apparently involved in the Page 8 of 22 Page 8 of 22 Edwards et al. Molecular Medicine (2023) 29:97 Edwards et al. Molecular Medicine (2023) 29:97 WT counterparts (Divanovic et al. 2005), indicating a role of CD180 in the negative regulation of TLR4. Con- sistently, murine macrophages, pre-treated with block- ing anti-MD-1 and then stimulated with LPS, produced more TNFα and IL-12-p40 but less IL-10 and chemokine ligand 5 (CCL-5) compared to unstimulated or isotype control-stimulated cells (Liu et al. 2013). Regulation of antigen presentationh CD180 signaling in CLL A multicentre study suggested that the high density of CD180 in marginal zone lymphoma (MZL) can be used in a test panel as a marker to distinguish MZL from other lymphomas and leukemias with similar characteristics (Mayeur-Rousse et al. 2016). Other combinations of markers used in conjunction with CD180, such as CD148 and CD200 (Miguet et al. 2015) or CD148 alone (Gautam et al. 2021), have been suggested to be useful for distin- guishing MZL from other malignancies such as mantle cell lymphoma (MCL). These marker combinations could be especially helpful for determining a differential diag- nosis in malignant B cell neoplasms when there are atypical presentations of immunophenotypic markers (Gautam et al. 2021). Notably, CD43 and CD180 have recently been explored as markers which can be used to diagnose atypical CLL (Li et al. 2021). This combination may be particularly useful in Asian populations where atypical immunophenotypic presentations are more common (Tomomatsu et al. 2010). As for normal B cells, studies on ligation of CD180 through anti-CD180 showed upregulation of CD86 and MHC class II and Ki67 (Porakishvili et al. 2011, 2004). Interestingly, only 50% of CD180-positive CLL samples showed this effect (responder samples), and this group of cases included both CLL cells with mutated IGVH genes (M-CLL) and unmutated IGVH genes (U-CLL). Impor- tantly, it was established that those CD180-positive but unresponsive M-CLL and U-CLL samples also responded poorly to the ligation of CD40 or the addition of recom- binant IL-4 (rIL-4) (Porakishvili et al. 2011). Thus, unre- sponsiveness to CD180 ligation might delineate CLL cell anergy. Ligation of CD180 on CD180-positive responder CLL samples resulted in cell activation, cell cycling, and phos- phorylation of intracellular protein kinases (Porakish- vili et al. 2011). The level of cell activation achieved by CD180 ligation was also found to be equivalent, or in some cases superior to the activation achieved with anti- CD40 or rIL-4 (Porakishvili et al. 2011), which are known to robustly induce CLL cell activation and protect from apoptosis (Falco et al. 2018). This occurs despite CD180- positive samples expressing much less CD180 compared to normal B cells, underscoring the potent signaling capacity of CD180 in CLL cells. Moreover, the combined ligation of CD180 and CD40, along with rIL-4 treat- ment, results in significantly increased activation and cell cycling (Porakishvili et al. 2005, 2011). CD180 signaling in CLL These data sug- gest that CD180 can be independently but synergistically Table 1 shows the relative expression of CD180 in B cell leukemias and lymphomas in the peripheral blood. Heterogeneity of CD180 expression in leukemias and lymphomasi However, even in CLL cases where CD180 is considered to be highly expressed, its density is lower than that on normal B cells (Porakishvili et al. 2005; Arvaniti et al. 2011). In the majority of CLL cases where CD180 was not expressed on the cell surface, the protein was found within the cytoplasm (Gordiienko et al. 2017a) (and unpublished data). Heterogeneity of CD180 expression in leukemias and lymphomasi CD180 appears to be mainly confined to malignancies of the B cell lineage (Valentine et al. 1988; Miyake et al. 1995; Divanovic et al. 2005; Zarember and Godowski 2002; Porakishvili et al. 2005) with only one report on acute myeloid leukemia which demonstrated that CD180 gene expression is high in leukemic stem cells (Saito et al. 2010). It is notable that HEK293 cells expressing only the CD180/MD-1 complex do not produce cytokines in response to LPS or Pam3CSK4, indicating that these ligands cannot directly induce signaling via CD180. Fur- thermore, murine macrophages and DCs, but not B cells, respond poorly to anti-CD180 mAbs (Divanovic et al. 2005; Nagai et al. 2005). Therefore, the data accrued so far suggest that CD180/MD-1 regulates the function of TLR4 and TLR2 in macrophages and DCs rather than acting as a lone activation molecule, and this differs from B cells, which robustly respond to anti-CD180 (Porakish- vili et al. 2005, 2011). CD180 and other TLRs are variably expressed in malignancies of B cell origin. In both leukemia and lym- phomas, the patterns of CD180 expression are heterog- enous. Using flow cytometry, it was shown that CD180 was weakly expressed by malignant cells in chronic lym- phocytic leukemia (CLL), mantle cell lymphoma and lymphoplasmacytic lymphoma, compared with control B cells (Miguet et al. 2013; Mayeur-Rousse et al. 2016). The level of expression of CD180 in hairy cell leukemia, marginal zone lymphoma (MZL), follicular lymphoma Edwards et al. Molecular Medicine (2023) 29:97 Page 9 of 22 Edwards et al. Molecular Medicine (2023) 29:97 (FL) and splenic diffuse red pulp small B cell lymphoma was found to be comparable to that of control B cells (Miguet et al. 2013; Mayeur-Rousse et al. 2016; Mestral- let et al. 2016). Interestingly, FL cells isolated from lymph nodes showed higher expression of CD180 compared to FL cells isolated from the peripheral blood, whereas CD180 expression was comparable in the different tissue compartments in MZL, MCL and CLL (Mestrallet et al. 2016). However, even in CLL cases where CD180 is considered to be highly expressed, its density is lower than that on normal B cells (Porakishvili et al. 2005; Arvaniti et al. 2011). In the majority of CLL cases where CD180 was not expressed on the cell surface, the protein was found within the cytoplasm (Gordiienko et al. 2017a) (and unpublished data). Chronic lymphocytic leukemia Using Pearson’s correlation, the density of CD180 and CD19 expression was found to be highly correlated on CLL cells. PBMC samples from CLL donors were stained with anti-human CD180 (PE) and anti-human CD19 (FITC). Antigen densities were measured by flow cytometry and expressed as a ratio to the isotype control. Relative fluorescent intensity (RFI) Importantly, AKT-S samples signal for survival, whilst p38MAPK-S samples tend to undergo apoptosis upon CD180 ligation (Porakishvili et al. 2015). This is consist- ent with the fact that activation of the AKT axis is pro- survival in CLL (Chapman et al. 2017; Zhuang et al. 2010) and thus, AKT-S could represent a compartment of CLL cases with more aggressive disease. However, the patho- physiological role of p38MAPK in CLL is unclear. Sup- port for a role of p38MAPK in a pro-apoptotic pathway comes from studies showing that apoptosis caused by novel cell cycle inhibitors (Paiva et al. 2015) and immu- nosuppressive agents (Bai et al. 2011) is mediated by p38MAPK. In addition, CD180 overexpression in car- diomyocytes can induce apoptosis via p38MAPK phos- phorylation in ischemia/reperfusion injury rat models (Li et al. 2016; Yang et al. 2015). Fig. 5 Categorization of CLL cells into signaler type. CLL cells can be categorized into four distinct groups depending on the phosphorylation (p) patterns of intracellular protein kinases p-AKT and p-p38MAPK following treatment with anti-CD180 mAb. double-signaler (DS), non-signaler (NS) g The dichotomous signaling pattern in CLL seems to be an acquired feature, and it is unknown if this pat- tern is observed in other B cell malignancies. The role of this dichotomy in CLL is presently unclear but does not appear to relate to surface density of CD180 or the co- expression of IgM, IgD or CD38 (Porakishvili et al. 2015). One of the putative kinases downstream from p38MAPK could include JNK1/2 and mTORc1, since CD180 ligation activates mTORc1 and JNK1/2 (Gordiienko et al. 2017a). Moreover, cyclin-dependent kinase inhibitor P1446A induces apoptosis of CLL cells via JNK/p38 (Paiva et al. 2015). It is possible that CD180 ligation could induce a similar chain of signaling events resulting in apoptosis. Fig. 5 Categorization of CLL cells into signaler type. CLL cells can be categorized into four distinct groups depending on the phosphorylation (p) patterns of intracellular protein kinases p-AKT and p-p38MAPK following treatment with anti-CD180 mAb. double-signaler (DS), non-signaler (NS) activated alongside CD40 and IL-4 receptor (Chaplin et al. 2011). Chronic lymphocytic leukemia Should these pathways cooperate together in vivo, this may result in increased CLL cell activation and tumor burden expansion. As previously mentioned, CD19 is also a major regulator of CD180 signaling, becoming phosphorylated and forming a complex with Vav and Lyn following CD180 activation (Yazawa et al. 2003). We have also found that the expression of CD180 and CD19 are highly correlated on CLL cells (Fig. 4). Chronic lymphocytic leukemia Chronic lymphocytic leukemia (CLL) is probably the most studied of the hematological malignancies with regard to the evaluation of the expression and function of CD180. Around 60% of CLL cases express CD180, with the range of expression density varying as a continuum. Table 1 The expression of CD180 in leukemias and lymphomas Malignancy Expression References Normal B cells + + + + (Valentine et al. 1988; Porakishvili et al. 2005) Chronic lymphocytic leukemia + (Porakishvili et al. 2005; Miguet et al. 2013; Mayeur-Rousse et al. 2016; Mestrallet et al. 2016) Follicular lymphoma (lymph node) + + + (Mayeur-Rousse et al. 2016; Mestrallet et al. 2016) Follicular lymphoma (peripheral blood) + + (Mayeur-Rousse et al. 2016; Mestrallet et al. 2016) Hairy cell leukemia + + + (Mayeur-Rousse et al. 2016) Mantle zell lymphoma + (Miguet et al. 2013; Mayeur-Rousse et al. 2016; Mestrallet et al. 2016) Marginal zone lymphoma (splenic) + + + + (Miguet et al. 2013) Marginal zone lymphoma (nodal) + + (Miguet et al. 2013) Splenic diffuse red pulp small B cell lymphoma + + + + (Miguet et al. 2013) Table 1 The expression of CD180 in leukemias and lymphomas Edwards et al. Molecular Medicine (2023) 29:97 Fig. 4 The relationship between CD180 and CD19 expression on CLL cells. Using Pearson’s correlation, the density of CD180 and CD19 expression was found to be highly correlated on CLL cells. PBMC samples from CLL donors were stained with anti-human CD180 (PE) and anti-human CD19 (FITC). Antigen densities were measured by flow cytometry and expressed as a ratio to the isotype control. Relative fluorescent intensity (RFI) Edwards et al. Molecular Medicine (2023) 29:97 Page 10 of 22 2015). Patterns of expression are shown in Fig. 5 based on their capacity to increase phosphorylated (p) p-AKT (AKT-signalers, AKT-S) and p-p38MAPK (p38MAPK- signalers or p38MAPK-S) above basal levels. Impor- tantly, in our hands, upon CD180 ligation AKT-S and p38MAPK-S CLL samples reduce phosphorylation of the alternative protein kinases to below basal levels, indicating that this reduction might condition the sign- aling dichotomy observed in CLL. Studies on larger cohorts of CLL patients are required to validate the proportions of patients categorized within these signaling groups at diagnosis and provide informa- tion as to whether treatment can affect CD180 signaling dichotomy. Fig. 4 The relationship between CD180 and CD19 expression on CLL cells. CD180 and the B cell receptor (IgM) Signaling through the BCR of B cells and CLL cells occurs mainly via the AKT pathway leading to proliferation and survival. Studies have shown that cross-talk between TLRs and the BCR can promote pro-survival signal- ing in lymph node-resident CLL cells (Dadashian et al. 2019; Herishanu et al. 2011). CD180 and the BCR can also interact in mouse B cells and in CLL cells to modify intracellular signaling. As described above, CD180 ligation on normal B cells leads to simultaneous signaling through AKT and p38MAPK pathways. However, most individual CLL samples use one of these signaling pathways, with a minority engaging both or neither (Porakishvili et al. Edwards et al. Molecular Medicine (2023) 29:97 Page 11 of 22 Edwards et al. Molecular Medicine Fig. 6 CD180 signaling in CLL. CD180 can signal dichotomously via a pro-survival BTK/PI3K/AKT pathway or via a pro-apoptotic pathway mediated by p38MAPK, which is accompanied by a downregulation of AKT. Activation of the AKT axis, which is thought to also involve activation of PI3K, mediates an increase in anti-apoptotic Mcl-1 and BCL-XL activity; and is also coupled with a downregulation in p38MAPK. Pre-engagement of CD180 can inhibit AKT-mediated signaling via IgM (dashed line), which tends to be accompanied by an increase in the phosphorylation of p38MAPK and the number of apoptotic CLL cells. Co-ligation of both CD150 and CD180 together attenuates both of AKT and p38MAPK pathway signaling Fig. 6 CD180 signaling in CLL. CD180 can signal dichotomously via a pro-survival BTK/PI3K/AKT pathway or via a pro-apoptotic pathway mediated by p38MAPK, which is accompanied by a downregulation of AKT. Activation of the AKT axis, which is thought to also involve activation of PI3K, mediates an increase in anti-apoptotic Mcl-1 and BCL-XL activity; and is also coupled with a downregulation in p38MAPK. Pre-engagement of CD180 can inhibit AKT-mediated signaling via IgM (dashed line), which tends to be accompanied by an increase in the phosphorylation of p38MAPK and the number of apoptotic CLL cells. Co-ligation of both CD150 and CD180 together attenuates both of AKT and p38MAPK pathway signaling unpublished observations. Moreover, CD150 and CD180 colocalize together in the surface membrane with CD19 (Gordiienko et al. 2017a). Pre-engagement of CD180 followed by sIgM ligation caused AKT-S samples to rewire signals from a pro-sur- vival (AKT-S) to a pro-apoptotic pathway (p38MAPk- S) (Porakishvili et al. 2015) (Fig. 6). CD180 and the B cell receptor (IgM) Similarly, in murine B cells, pre-treatment with anti-CD180 antibodies resulted in apoptosis following subsequent BCR activa- tion (Yamashita et al. 1996). This cross-talk could also be amplified by IL-4, which increases the expression of IgM and enhances its function in CLL cells (Aguilar-Hernan- dez et al. 2016). IL-4 can be produced by CLL cells in an autocrine fashion or by T cells (Kay et al. 2001). Co-ligation of CD150 with CD180 results in attenu- ation of the phosphorylation of both the AKT and p38MAPK pathways in CLL cells (Fig. 6). Notably, co- activation of CD40 and CD150 does not attenuate AKT or p38MAPK phosphorylation, indicating that usage of these pathways is receptor specific. It is not clear, how- ever, if the reduction in the activation of these pathways is important for modulating CLL cell survival, as seen previously with CD180-mediated dichotomous intracel- lular signaling (Porakishvili et al. 2015). Multiple myeloma Whereas bone marrow plasma cells do not normally express CD180, multiple myeloma (MM) cells do, and this increased expression is promoted by the hypoxic bone marrow microenvironment (Kikuchi et al. 2018; Egli et al. 2015). Both LPS challenge and anti-CD180 mAb treatment promoted the growth of MM cells, in vitro (Kikuchi et al. 2018). Moreover, LPS appeared to induce clonal expansion of CD180-positive MM cells and notably, knockdown of CD180 reduced the LPS responsiveness of MM cells further indicating the role of CD810 in the detection of LPS by TLR4. It is likely that these two receptors operate together to promote MM cell growth (Kikuchi et al. 2018). Since CD19 can enhance LPS-mediated growth by the CD180/MD1 complex, it might also play a major role in regulating signaling via CD180 (Yazawa et al. 2003). However, it should be noted that CD19 is only expressed by a very small proportion of MM cells as measured by flow cytometry (Tembhare et al. 2014). Although, super-resolution microscopy has revealed that MM cells do express CD19, albeit at very low densities (Nerreter et al. 2019). TLRs contribute to the pathophysiology of SLE. As dis- cussed above, CD180 can negatively regulate TLR activ- ity. Therefore, loss of CD180 could enable TLR-mediated cellular activation and inflammation, thus promoting SLE progression. In support of this, Yang et al. showed that CD180 activation inhibits expression of TLR7 and TLR9 in murine macrophages and DCs; notably, patients with SLE also have increased frequencies of CD180-negative macrophages and DCs (Yang et al. 2018a). Given that TLR7 and TLR9 are expressed by B cells, and CD180 can interact with the downstream pathways of these recep- tors in B cells, it will be important to study the functional role of TLR7 and TLR9 in CD180-positive and CD180- negative SLE B cells.f CD180 and CD150 IRF4 is a transcription factor important in regu- lating plasma cell differentiation (Shapiro-Shelef and Calame 2005) and IRF8 and PU.1 are involved in the activation of follicular and germinal centre B cells (Wang et al. 2019) and the development of pre-B cells (Pang et al. 2016). This could be important to CLL cells present in proliferation centres in lymph nodes (Edwards et al. 2021), indicating that activation and proliferation of CLL cells might result from a concordant ligation of CD180 and CD150. CD180 in systemic lupus erythematosus (SLE) CD180 in systemic lupus erythematosus (SLE) Systemic lupus erythematosus (SLE) is a multisystem dis- ease characterized by immune dysregulation and produc- tion of autoantibodies, typically against double stranded DNA (dsDNA) (Wang and Xia 2019). A high propor- tion of CD180-negative B cells are found in SLE patients (15 ± 11.7%) compared with healthy controls (1.7 ± 1.1%) (Koarada et al. 1999). Moreover, clinical observations show that the higher proportions of CD180-negative B cells associate with active disease (Koarada et al. 1999). Furthermore, increased proportions of CD180-negative B cells correlate with increased renal legions in murine lupus-like nephritis models (Fujita et al. 2012) and CD180-negative SLE B cells are primarily responsible for the production of anti-double stranded DNA autoanti- bodies (Koarada et al. 1999; Kikuchi et al. 2002). Interest- ingly, CD180-negative B cells from patients with SLE also exhibit increased production of antibodies to dsDNA in response to IL-6 or challenge with SAC, whereas CD180- positive B cells are unresponsive to such stimuli (Kikuchi et al. 2002). Mechanistically, the elevated autoimmune responses could relate to B cell activation since CD180-nega- tive SLE B cells overexpress B cell maturation antigen (BCMA) (Koarada et al. 2010). Additionally, sCD40L trimers, which are elevated in SLE, reduce the levels of viable CD180-negative (and CD180-positive) SLE B cells, and this effect is reversed by treatment of B cells with the BCMA ligand APRIL (a proliferation-inducing ligand) (Koarada et al. 2010). Therefore, the preferential expres- sion of BCMA by CD180-negative B cells could promote plasma cell differentiation, thereby perpetuating a more severe disease phenotype. CD180 and CD150 Interestingly, CD150-mediated AKT activation in CLL cells is accompanied with an increase in the phospho- rylation of the transcription factors FoxO1 and FoxO3a. Simultaneous ligation of CD150 with CD180 with mAbs results in a decrease in FOXO transcription (Gordiienko et al. 2017a), suggesting that together they promote a CD150 is part of the signaling lymphocyte activation molecule family (SLAMF1). It positively correlates with the expression of CD180 in CLL, and around 59% of cases express both CD150 and CD180 (Gordiienko et al. 2017a), consistent with independent studies (Porakish- vili et al. 2005, 2011; Bologna et al. 2016) and our own Page 12 of 22 Edwards et al. Molecular Medicine (2023) 29:97 Edwards et al. Molecular Medicine pro-survival phenotype. Independent ligation of CD150 or CD180 leads to upregulation of BCL6 mRNA (Gor- diienko et al. 2017b). Since, in general, BCL6 is an anti- apoptotic protein and a transcriptional regulator of TP53 (Phan and Dalla-Favera 2004), this may represent one modality that contributes to a pro-survival pheno- type following anti-CD180 or anti-CD150 treatment of CLL cells. Ligation of CD180 or CD150, individually or together, leads to a strong down regulation of IRF4 and upregulation of IRF8 and PU.1 (Gordiienko et al. 2017b), further supporting the idea that CD180 and CD150 can share intracellular signaling and transcriptomic path- ways. IRF4 is a transcription factor important in regu- lating plasma cell differentiation (Shapiro-Shelef and Calame 2005) and IRF8 and PU.1 are involved in the activation of follicular and germinal centre B cells (Wang et al. 2019) and the development of pre-B cells (Pang et al. 2016). This could be important to CLL cells present in proliferation centres in lymph nodes (Edwards et al. 2021), indicating that activation and proliferation of CLL cells might result from a concordant ligation of CD180 and CD150. pro-survival phenotype. Independent ligation of CD150 or CD180 leads to upregulation of BCL6 mRNA (Gor- diienko et al. 2017b). Since, in general, BCL6 is an anti- apoptotic protein and a transcriptional regulator of TP53 (Phan and Dalla-Favera 2004), this may represent one modality that contributes to a pro-survival pheno- type following anti-CD180 or anti-CD150 treatment of CLL cells. Ligation of CD180 or CD150, individually or together, leads to a strong down regulation of IRF4 and upregulation of IRF8 and PU.1 (Gordiienko et al. 2017b), further supporting the idea that CD180 and CD150 can share intracellular signaling and transcriptomic path- ways. CD180 in autoimmune disorders of the central nervous system (CNS) Data from anti-CD20 B cell studies have suggested that B cells play a role in the pathogenesis of neuromyelitis optica spectrum disorder (NMOSD) and multiple scle- rosis (MS). Hayden et al. showed that CD180 expres- sion was exclusively decreased in the CD19+CD27+IgD+ non-switched (NS) memory B cells in both NMOSD and MS compared to healthy controls (Hayden et al. 2021). Furthermore, the same group showed that there were increased titres of anti-citrate synthase (anti-CS) autoan- tibodies in patients with autoimmune disease (Czömpöly et al. 2006). Anti-CS IgM autoantibody serum level was also lower in both NMOSD and MS suggesting that reduced CD180 expression of NS B cells could contrib- ute to the deficient IgM autoantibody production in these disorders. There is an indication that children with KD are char- acterized with expansion of peripheral blood B cells with CD180-positive phenotype, compared to control sam- ples (Imayoshi et al. 2006). Notably, patients with viral infections similarly exhibit upregulated proportions of CD180-positive B cells, although to a lesser degree, fur- ther indicating a link between viral infection and KD. From the limited number of observations, the impor- tance of CD180 expression on expanding population of B cell was unclear. However, CD180 expression was upreg- ulated on KD B cells compared to normal controls and those with viral infections (Imayoshi et al. 2006). It is known that B cells are activated in KD, produc- ing antibodies, perhaps in response to viral or bacterial infection. Given the preferential expression of CD180 by memory B cells and the involvement of CD180 in B cell activation and immunoglobulin production, it is possible that CD180 plays a role in the development and patho- physiology of KD. CD180-overexpressing B cells in KD could therefore represent a subset of activated B cells. Kawasaki disease Kawasaki disease (KD) affects infants and children, lead- ing to inflammatory changes of blood vessels which is often accompanied by fever and lymph node swelling. Patients also develop inflammation of cardiac tissues and valves, and, in some cases, aneurysms or thrombi are formed. The causes of KD are unknown, although the onset of the disease appears related to infection as sup- ported by epidemiological observations of endemic KD (Agarwal and Agrawal 2017). CD180 in autoimmune disorders and other inflammatory pathologies Together, these data point towards a protective effect for CD180 in SLE. Loss of CD180 on APCs in SLE cor- relates with disease activity, and functional studies in mice demonstrate that activation of CD180 can help to ameliorate SLE disease activity (Yang et al. 2018a). Gain- ing a deeper understanding into the function of CD180 CD180 has been extensively studied within the context of autoimmune disease and other non-malignant inflamma- tory disorders where it appears to play a pathophysiologi- cal role. Edwards et al. Molecular Medicine (2023) 29:97 Page 13 of 22 Edwards et al. Molecular Medicine in SLE, and how this is differentially downregulated may aide in the development of new treatment approaches or improve the accuracy of predicting disease behaviour. memory B cells (Good et al. 2009), and therefore upregu- lated expression of CD180 by KD B cells might also affect CD150 function, as mentioned above. Phenotypic and functional analysis of B cells as well as macrophages and DCs may shed further light on the exact role of CD180 in KD pathophysiology. CD180 in rheumatoid arthritis (RA) To date, the authors have found no publications of stud- ies on human rheumatoid arthritis (RA) disease. How- ever, there is a study where the murine collagen-induced arthritis (CIA) model has been used to study the role CD180 in RA. CD180−/− mice demonstrate more severe CIA with an earlier onset, compared to wild type mice (Tada et al. 2008). Further, CD180−/− mice more robustly produce INF-γ and TNF-α, inflammatory mediators that play important roles in the pathophysiology of RA (Wil- liams et al. 1992; Manoury-Schwartz et al. 1997). Recently, SARS-CoV-19 infection has been impli- cated as an etiological factor in the initiation of KD or a KD-like syndrome. Observational studies indicate sig- nificant increases in Kawasaki-like disease in areas with high prevalence of SARS-CoV-19 infection, suggesting a role for the novel Corona virus in the presentation of KD (Verdoni et al. 2020; Sancho-Shimizu et al. 2021). To our knowledge, there are no data on CD180 expression in SARS-COV-19 or related systemic disorders. Given the upregulation of CD180 in both KD and viral infec- tion (Imayoshi et al. 2006), analysing changes in CD180 expression may be useful for predicting which individu- als may present with Kawasaki-like disease or systemic inflammation in SARS-CoV-2 infection. Other autoimmune diseases and chronic inflammatory conditionsf Differential expression of CD180 has also been reported in other autoimmune conditions (Table 2). Kikuchi et al. showed that patients with dermatomyositis present with increased proportions of CD180-negative B cells com- pared to patients with polymyositis and normal controls (Kikuchi et al. 2001). Some patients with IgG4-related disease (IgG4-RD) also present with CD180-negative B cells (Koarada et al. 2013) and loss of CD180 corre- sponded with increased disease activity in IgG4-RD. These studies are, however, limited by the fact they are case reports. Differences in CD180 might also modulate the func- tion of other receptors in B cells in KD. For example, given the known functional interactions with classical members of the TLR family, CD180 could modulate TLR function which could, in turn, lead to the production of pro-inflammatory cytokines and perpetuation of the KD phenotype. Interestingly, CD150 is also upregulated by Edwards et al. Molecular Medicine (2023) 29:97 Page 14 of 22 Table 2 The differential expression of CD180 in various autoimmune and inflammatory pathologies FC flow cytometry, qPCR quantitative polymerase chain reaction Disease Cell or tissue Methodology CD180 expression relative to healthy controls References Neuromyelitis optica spectrum disorder and multiple sclerosis B cells FC ↓ (Hayden et al. 2021) Diffuse cutaneous systemic sclerosis B cells qPCR, FC ↓ (Simon et al. 2021; Koarada et al. 2001; Erdo-Bonyar et al. 2019) Dermatomyositis B cells FC ↓ (Kikuchi et al. 2001; Koarada et al. 2001) Kawasaki disease B cells FC ↑ (Imayoshi et al. 2006) Sjogren’s syndrome B cells FC ↓ (Koarada et al. 2001; Kikuchi et al. 2008) IgG4-related disease B cells FC ↓ (Koarada et al. 2013) Table 2 The differential expression of CD180 in various autoimmune and inflammatory pathologies function is therefore important for elucidating mecha- nisms of atherosclerotic plaque development. CD180 is expressed on murine vascular smooth muscle cells, along with TLR4, indicating a more direct role for CD180 in cardiovascular health (Karper et al. 2013). Neointima formation is a common feature of atherosclerotic plaque development and is characterized by an increase in vas- cular smooth muscle cells within a vessel which can promote atherosclerotic plaque development. Loss of CD180 is associated with increased formation of neoin- tima scar tissue in mice with artery cuffs that are used to induce vascular damage (Karper et al. 2013), suggesting a protective role for CD180. Other autoimmune diseases and chronic inflammatory conditionsf Furthermore, local addi- tion of LPS increased neointima formation, although the increase was more dramatic in CD180−/− mice compared to wild-type animals. Later, however, somewhat conflict- ing evidence emerged: in low density lipoprotein receptor (LDLr)−/− CD180−/− mice, there is marked reduction in plaque formation compared to LDLr−/− animals (Karper et al. 2013). Wezel et al. also demonstrated that CD180- negative mice showed reduced plaque formation, as well as reduced monocyte migration (Wezel et al. 2015). This was accounted for by a greater reduction in the chemokine ligand 2 (CCL2) in CD180−/− mice compared to those with intact CD180. These two studies suggest that CD180 expression in fact promotes plaque forma- tion. Consistently, Wezel et al. later showed that CD180 deficient mice had an increased number of unstable lesions in murine vein graft models, which was attributed to increased CCL2 production and thus mast cell recruit- ment (Wezel et al. 2016). CD180 as a prognostic biomarker It is now well documented that M-CLL is associated with superior prognosis in CLL compared to U-CLL (Agath- angelidis et al. 2022). Now data are accumulating to suggest that CD180 could be useful as a prognostic bio- marker in different diseases. Levels of CD180 mRNA are upregulated in patients with osteosarcoma with superior outcomes (Chen et al. 2020). At the protein level, pro- portions of CD180-negatve B cells correspond with SLE disease activity (Koarada et al. 1999). Similarly, we have found an association with CD180 expression and disease outcomes in CLL. We discovered that increased CD180 expression in LNs was associated with early-stage disease and superior overall survival (Edwards et al. 2021). This is consistent with earlier observations that CD180 is more highly expressed in M-CLL (Porakishvili et al. 2005) and positively correlates with CD150 (Gordiienko et al. 2017a), both of which associate with a positive prognos- tic picture in CLL (Bologna et al. 2016). Together, these findings suggest that prediction of disease severity and progression could be benefitted through study of CD180 expression. The negative effect which CD180 has on classical TLRs suggests that CD180 may become upregulated in response to infection to aid prevention of hyperinflam- mation. Perhaps the most notable interaction is with TLR4, where CD180 negatively regulates TLR4-medi- ated responses to LPS (Divanovic et al. 2005). There- fore, the upregulation of CD180 could serve to prevent hyperinflammation during infection, thereby prevent- ing sepsis. Notably, there is significant upregulation of CD180 by some subsets of immune cells in LPS-chal- lenged mice (Dong et al. 2019). Similarly, in Mycobac- terium tuberculosis infection, human macrophages increase their expression of CD180 (Blumenthal et al. 2009). The diverse molecular nature of CD180 signaling could also be utilized to provide better resolution in CLL disease prediction or even therapy response. Among responder CD180-positive CLL cases, the dichotomous activation of AKT or p38MAPK in CLL cells follow- ing CD180 ligation, respectively, corresponds with sur- vival or apoptosis (Porakishvili et al. 2015). Should this reflect an in vivo occurrence, CLL cases that signal via AKT could represent a subset of patients with increased tumor proliferation and perhaps a more aggressive dis- ease course. Alternatively, p38MAPK-s cases could rep- resent a subset of patients with less aggressive disease. CD180-negative cases and CD180-positive non-respond- ers are also unresponsive to cumulative anti-CD40 and IL-4 simulation (Porakishvili et al. CD180 in cardiac injury and atherosclerosis TLRs play an important role in the pathophysiology of cardiovascular disease, and CD180 has also been inves- tigated within the context of cardiovascular pathologies. WT mice develop significantly worse cardiac damage fol- lowing myocardial infarction compared to mice where CD180 was overexpressed in murine cardiac tissue by adenovirus transfection, suggesting that CD180 plays a protective role in cardiac injury (Louwe et al. 2014). Building on this work, it was shown that CD180 over- expression can ameliorate cardiomyocyte apoptosis via inhibition of TLR4 signaling and p38MAPK phospho- rylation signalin ischemia/reperfusion injury rat models (Li et al. 2016; Yang et al. 2015). Moreover, in cardiomyo- cytes, CD180 was able to effectively reduce TLR4-medi- ated production of pro-inflammatory IL-6 and TNF-α in response to ischemia (Li et al. 2016).hf The protective effect of CD180 in ischemia could be dampened through the interference of microRNAs, which are involved heavily in cardiovascular pathologies. Specifically, miR-327, which is preferentially expressed in myocardium, downregulates CD180 and thus promotes proinflammatory TLR4 activation (Yang et al. 2018b). However, miR-383 was shown to promote myocardial health in ischemia and reperfusion injuries in a CD180- dependent manner (Guo et al. 2021). CD180 therefore is a potential therapeutic target in cardiac injury, perhaps through the indirect amelioration of TLR4-mediated inflammation (Yang et al. 2019). Furthermore, a recent study also implicated CD180-mediated inactivation of TLR2 as a potential pathway which promotes cardiac repair following myocardial infarction (Huang et al. 2020).h Together, these data suggest that whilst CD180 is associated with reduced neointima formation, thus decreasing the risk of plaque development, paradoxically CD180 simultaneously promotes atherosclerotic plaque development. Understanding the mechanistic role of CD180 in modulating the immune cells which promote The pathogenesis of atherosclerosis is largely immu- nological in nature. B cells play a role in atheroscle- rotic plaque formation by producing pro-inflammatory cytokines and through B cell activation by neo-anti- gens (Sage et al. 2019). Understanding changes in B cell Edwards et al. Molecular Medicine (2023) 29:97 Page 15 of 22 Page 15 of 22 Edwards et al. Molecular Medicine atherosclerotic plaque development may provide a rationale for CD180 targeted therapies. that the immunopathology of CD180 is contextual and is dependent on (1) the cell by which CD180 is expressed and (2) the disease in which CD180 is func- tioning. The molecular architecture of CD180 supports this idea; given its short intracytoplasmic tail, CD180 likely requires partner receptors to initiate intracellular signaling. CD180 in cardiac injury and atherosclerosis The unique profile of receptor expression by different cell types, coupled with the differential expres- sion of CD180 (and other TLRs) on APCs in pathology, could serve to modulate the behaviour of CD180 and thus explain the divergent properties observed in stud- ies of CD180 function. The current perspective: a model of CD180 in disease CD180 is highly expressed by APCs from healthy indi- viduals where it plays a role in cell activation and mod- ulation of immunoglobulin production. Moreover, it interacts with various other receptors to regulate the action of classical TLRs. However, there is differential expression and function of CD180 in disease. The literature promotes the overarching idea that CD180 is often differentially downregulated or lost in disease. Notably, CD180 tends to be downregulated in hematological malignancies, autoimmune diseases, and some inflammatory conditions which therefore sug- gests that CD180 promotes health. There are, however, some conditions where CD180 expression becomes upregulated and this corresponds with the aetiology of the disease, rather than being directly disease related. For example, the increased upregulation of CD180 in KD could be due to the causative infectious agent, rather than a manifestation of KD itself. The upregula- tion in the expression of CD180 in KD may serve as a physiological response, perhaps to dampen hyperac- tive immune responses to bacteria or viruses. This con- cept is consistent with findings which show that B cells respond to stimulation with synthetic RNA by upregu- lating CD180 expression (Imayoshi et al. 2006).hf CD180 as a prognostic biomarker 2011) or IgM ligation (unpublished observations) and therefore appear to rep- resent a subset of CLL cases with anergic leukemic cells: CD180 does not, however, always conform to these behaviours. For example, CD180 expression by murine macrophages does not increase in response to Myco- bacterium avium infection (Blumenthal et al. 2005). Furthermore, in cardiovascular pathologies, CD180 appears to play an antagonistic role to promote athero- sclerotic plaque formation. Therefore, it is not entirely possible to conclude a defined function for CD180 that unifies all the current data. Rather, it appears Edwards et al. Molecular Medicine (2023) 29:97 Page 16 of 22 Edwards et al. Molecular Medicine Page 16 of 22 a phenotype typically associated with an indolent disease course (Apollonio et al. 2013). CD180 mRNA levels may be a relevant pharmacody- namic biomarker for BRD4 inhibitors in hematological malignancies. Furthermore, recent studies advocate the rationale of the use of BRD inhibitors in CLL (Ozer et al. 2018; Jiang et al. 2020). BRD inhibitors may also increase the sensitivity to venetoclax, and thus could be employed as an adjuvant therapy in CLL (Carrà et al. 2020). Understanding how CD180 is co-expressed and inter- acts with other receptors could also further improve the prognostic utility of CD180. A significant proportion of AKT-S CLL cases (70%) responded to anti-IgM by activa- tion of the BTK/PI3K/AKT axis, whilst only a small pro- portion of p38MAPK-S cases responded to anti-IgM by increasing the phosphorylation of p38MAPK (Porakish- vili et al. 2015). This presents the idea that AKT-S cases are much more receptive to IgM engagement compared to p38MAPK-S cases which may help to explain why this subset of patients readily recruits BCR machinery, poten- tially helping to promote a survival phenotype by CLL cells. However, it is not clear if these differences were due to differential expression of ZAP-70 and CD38 in this study. The capacity of CD180 to rewire IgM-mediated pro-survival signaling to promote apoptosis (Porakish- vili et al. 2015) and the tendency of CD180 to cooperate with IgD to activate p38MAPK indicates that studies of the different BCR isoforms may be useful for defining the prognostic capacity of CD180 (Porakishvili et al. 2017). y Taken together, these data suggest that through study- ing CD180 expression and activation, it may be possible to better predict and explain the observed heterogene- ity in CLL tumor aggressiveness, progression, and ther- apy responsiveness. CD180 as a therapeutic target Although there are no clinical trials reported to date, the data suggest that CD180 could be a potential therapeutic target. Our group showed that pre-engagement of CD180 with anti-CD180 followed by treatment with anti-IgM in AKT-s CLL cells significantly decreases levels of p-AKT, increases p38MAPK, and increases the number of apop- totic cells (Porakishvili et al. 2015). An earlier study in mice also demonstrated that pre-treatment with anti- CD180 can program B cells to undergo apoptosis follow- ing IgM ligation (Yamashita et al. 1996).hii Similarly, there are data to suggest that CD180 may also be useful as a biomarker to indicate pharmacodynamic responses to bromodomain (BRD) inhibitors, which have shown pre-clinical efficacy in hematological malignancies such as MDS and AML. When a range of hematological cancer cell lines, including ALL, MM, and DLBCL types, were treated with AZD5153, a novel BRD4 inhibitor, all demonstrated considerable reduction in CD180 mRNA levels (Yeh et al. 2017). Significant and robust CD180 mRNA down regulation was also observed in ALL murine xenograft models, and in peripheral blood in vitro studies of CLL and MM patients, as well as healthy con- trols. Whilst further validation is required in larger, pro- spective cohorts and with different BRD4 inhibitors, the consistency in the reduction of CD180 mRNA across the different experimental set-ups is striking. In addition to CD180, chemokine receptor type 2 (CCR2) mRNA levels were downregulated in response to AZD5153 treatment, thus BRD4 inhibition may modulate the transmigration of tumor cells within the microenvironment (Macanas- Pirard et al. 2017). Together, these data suggest that g g g These findings are significant as they demonstrate that CD180 may be a targetable molecule, given that it can reprogram certain populations of CLL clones to undergo apoptosis following BCR engagement. Since CLL and other malignant B cells depend heavily on the BCR for survival signals and evasion of apoptosis, priming of malignant B cells to undergo apoptosis via BCR engage- ment following pre-treatment with a molecule to activate CD180 is an attractive treatment strategy. Notably, the rewiring phenomenon did not occur in control B cells pre-treated with anti-CD180 and then anti-IgM (Porak- ishvili et al. 2015). Therefore, engagement of CD180 may specifically promote the apoptosis of CLL cells, leaving healthy B cells unaffected. CD180 as a prognostic biomarker Moreover, further insights into the behaviour of CLL cells could be achieved by studying the expression and co-engagement of CD180 with other surface receptors, namely the IgD and IgM BCR iso- forms. The true utility of CD180 as a biomarker would, however, need to be fortified by relating signaling activ- ity with longitudinal clinical data on disease progression, survival, and therapy responses. The applicability of these data to other hematological malignancies is also yet to be established. However, it is known that CD180 expression is modulated in various B cell malignancies, other than CLL. Therefore, it is possible that CD180 may also be of value for predicting outcomes in these cancers. Understanding how CD180 activation and corecep- tor interactions affect the CLL signalosome could also be exploited for prediction of therapy responses. Should CLL cells differentially downregulate the use of signaling axes which depend on BKT or PI3K, it is plausible that these patients may be resistant to the BTK and PI3K pro- tein kinase inhibitors ibrutinib or idelalisib. Therefore, since CD180 can modulate the phosphorylation and uti- lization of BTK and PI3K, it may be a strong candidate to predict protein kinase inhibitor responsiveness. CD180 as a therapeutic target Thus, such an approach may be useful only in a fraction of U-CLL cases. of functional intracellular signaling domain mean that CD180 requires signaling partners to propagate extrin- sic cues. Which ligands initiate CD180 signaling, in vivo, remain unknown, and their discovery will aide in a bet- ter understanding of the function of CD180 in physiol- ogy. It appears, however, that even as an orphan receptor, CD180 functions to modulate other receptors, particu- larly TLR4. CD180 expression becomes either upregulated or downregulated on B cells in diseases such as SLE, cardiac pathologies and B cell malignancies. The aberrant expres- sion of CD180 in disease is also accompanied by func- tional differences, particularly in CLL where the receptor has apparently unique signaling properties which can powerfully regulate CLL cell behaviour and the action of the BCR. Expression and functional studies are accompanied by data which indicate a role for CD180 in disease prog- nostication, with increased CD180 expression being generally associated with a less severe disease pheno- type, although this is not always the case. Further stud- ies which define CD180 signaling behaviour at a greater resolution will aid in developing CD180 as a prognostic biomarker and may help to highlight potential therapeu- tic avenues, particularly in CLL, SLE and cardiovascular pathologies. It has also been suggested that rather than engaging CD180, utilizing CD180 expression on B cell subsets could be a way to exploit CD180 for therapy. Koarada and Tada suggested that targeting of the autoantibody producing CD180-negative B cells could be an effective treatment modality of SLE (Koarada and Tada 2012). Targeting of CD180-negative B cell populations may be possible since CD180-negative B cell subsets have an increased expression of certain antigens, such as BCMA in SLE (Koarada et al. 2010). CD180 as a therapeutic target Abbreviations BCR B cell receptor Ag Antigen AKT Protein kinase B AML Acute myeloid leukemia APC Antigen presenting cells ATRA All-trans retinoic acid BAD B cellleukemia/lymphoma 2 protein (BCL2)-associated agonist of cell death BAFF B cell activating factor BCL-XL B cell lymphoma-extra large BCMA B cell maturation antigen BLC6 B cell leukemia/lymphoma 2 protein BTK Bruton’s tyrosine kinase CCL2 Chemokine ligand 2 CCL5 Chemokine ligand 5 CIA Collagen induced arthritis model CLL Chronic lymphocytic leukemia DAMP Damage-associated molecular pattern DCs Dendritic cells FC Flow cytometry FITC Fluorescein isothiocyanate FO Follicular FL Follicular lymphoma HEK Human embryonic kidney cells Ig Immunoglobulin IGVH Immunoglobulin Heavy Chain Gene IL-4 Interleukin-4 IL-6 Interleukin-6 IL-7 Interleukin-7 IRF8 Interferon regulatory factor 8 KD Kawasaki disease KO Knockout LDLr Low density lipopolysaccharide receptor Since it is known that CD180 can negatively regulate both TLR4 and TLR2 signaling (Divanovic et al. 2005), an alternative treatment approach in inflammatory disor- ders may be to increase CD180 expression which could in turn, help to downregulate proinflammatory cytokines and reduce tissue damage. One approach could be to silence microRNAs, which negatively regulate CD180 expression. Multiple microRNAs, including miR-141-p, miR-327, and miR-383, can interfere with CD180 expres- sion and inhibition of these microRNAs can ameliorate cardiac tissue damage in ischemia models (Yang et al. 2018b; Qin et al. 2019; Guo et al. 2021). This modal- ity therefore might represent a target for therapeutic intervention. CD180 as a therapeutic target f If the same program of dichotomous signaling is pre- sent in other types of malignant cells, then the rationale for this treatment approach could be extended. Further, Edwards et al. Molecular Medicine (2023) 29:97 Page 17 of 22 Page 17 of 22 Edwards et al. Molecular Medicine targeting of malignancies which express CD180 highly on the cell surface could also help to improve the efficacy of this potential modality of treatment. The augmenta- tion of BCR signaling to facilitate apoptosis via BCR engagement is also a particularly attractive strategy for the treatment of U-CLL. This is because CLL cells with unmutated IGHV genes are in a heightened state of BCR activation (Burger and Chiorazzi 2013) which could assist CLL clones in rewiring their signals to promote apoptosis more readily. This is supported by an earlier study which demonstrated that U-CLL cells respond more read- ily to IgM cross-linking (Lanham et al. 2003). It is first required, however, to accurately stratify patients by the tumor’s capacity to rewire IgM signals to promote apop- tosis. This approach would obviously not be applicable to cases of CLL or other B cell cancers where both CD180 and IgM are not expressed. Notably, however, CD180 tends to be downregulated in U-CLL, compared to M-CLL (as discussed previously). Thus, such an approach may be useful only in a fraction of U-CLL cases. targeting of malignancies which express CD180 highly on the cell surface could also help to improve the efficacy of this potential modality of treatment. The augmenta- tion of BCR signaling to facilitate apoptosis via BCR engagement is also a particularly attractive strategy for the treatment of U-CLL. This is because CLL cells with unmutated IGHV genes are in a heightened state of BCR activation (Burger and Chiorazzi 2013) which could assist CLL clones in rewiring their signals to promote apoptosis more readily. This is supported by an earlier study which demonstrated that U-CLL cells respond more read- ily to IgM cross-linking (Lanham et al. 2003). It is first required, however, to accurately stratify patients by the tumor’s capacity to rewire IgM signals to promote apop- tosis. This approach would obviously not be applicable to cases of CLL or other B cell cancers where both CD180 and IgM are not expressed. Notably, however, CD180 tends to be downregulated in U-CLL, compared to M-CLL (as discussed previously). Conclusions In physiology, CD180 plays roles in B cell activation and modulation of TLR responses. CD180 provides cellular stimulation and interacts with other receptors to modu- late APC function in a heterogenous way. It appears that the heterogeneity in function is dependent on the avail- ability of other receptors. This is likely due to the molecu- lar nature of CD180. Its short cytoplasmic tail and lack Page 18 of 22 Edwards et al. Molecular Medicine (2023) 29:97 LPS Lipopolysaccharide Lyn Tyrosine-protein kinase Lyn mAb Monoclonal antibody MAMP Microbe associated molecular patterns MCL Mantle cell lymphoma Mcl-1 Induced myeloid leukemia cell differentiation protein Mcl-1 M-CLL Mutated chronic lymphocytic leukemia MD-1 Myeloid differentiation 1 MD-2 Myeloid differentiation 2 MDS Myelodysplastic syndrome MHC II Major histocompatibility complex II miRNA MicroRNA MS Multiple sclerosis MM Multiple myeloma MyD88 Myeloid differentiation primary-response protein 88 MZ Marginal zone MZL Marginal zone lymphoma NF-κB Nuclear factor kappa-light-chain-enhancer of activated B cells NMOSD Neuromyelitis optica spectrum disorder NS Non-switched; non-signaler OxPAPC Oxidised 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine p38MAPK P38 mitogen-activated protein kinase Pam3CSK4 Synthetic bacterial lipopeptide Pam3-Cys-Ser-Lys4 PE Phycoerythrin PI3K Phosphatidylinositol-3 kinase PU.1 Transcription factor PU qPCR Quantitative polymerase chain reaction RA Rheumatoid arthritis RFI Relative fluorescent intensity SAC Staphylococcus aureus Cowan SLE Systemic lupus erythematosus SYK Spleen associated tyrosine kinase TIR toll-like/interleukin-1 receptor TIRAP Toll/Interleukin 1-receptor adaptor protein TLR Toll-like receptor TNF-α Tumor necrosis factor-alpha TP53 Tumor protein 53 TRAM Toll/IL-1R domain-containing adaptor-inducing interferon-BETA related adaptor molecule TRIF Toll-interleukin-1 receptor homology domain (TIR)-domain-con- taining adapter-inducing interferon-BETA U-CLL Unmutated chronic lymphocytic leukemia ULK1 Unc-51 like autophagy activating kinase 1 WT Wild-type Funding Blomhoff HK, Smeland EB, Erikstein B, Rasmussen AM, Skrede B, Skjønsberg C, Blomhoff R. Vitamin A is a key regulator for cell growth, cytokine production, and differentiation in normal B cells. J Biol Chem. 1992;267:23988–92. g KE was supported by a graduate scholarship from the University of Westminster. Declarations Blumenthal A, Kobayashi T, Pierini LM, Banaei N, Ernst JD, Miyake K, Ehrt S. RP105 facilitates macrophage activation by Mycobacterium tubercu- losis lipoproteins. Cell Host Microbe. 2009;5:35–46. https://doi.org/10. 1016/j.chom.2008.12.002. Received: 28 March 2023 Accepted: 8 June 2023 Received: 28 March 2023 Accepted: 8 June 2023 Received: 28 March 2023 Accepted: 8 June 2023 Availability of data and materials Blumenthal A, Lauber J, Hoffmann R, Ernst M, Keller C, Buer J, Ehlers S, Reil- ing N. Common and unique gene expression signatures of human macrophages in response to four strains of Mycobacterium avium that differ in their growth and persistence characteristics. Infect Immun. 2005;73:3330–41. https://doi.org/10.1128/IAI.73.6.3330-3341.2005. The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Author contributions KE, PML, NP and EVV conceived and wrote the manuscript. NK & TT conducted the flow cytometry studies and gave valuable advice. NC gave valuable advice for redrafting and improving the manuscript. All authors approved the final manuscript. Bernasconi NL, Traggiai E, Lanzavecchia A. Maintenance of serological memory by polyclonal activation of human memory B cells. Science (80-). 2002;298:2199–202. https://doi.org/10.1126/science.1076071.f 2002;298:2199–202. https://doi.org/10.1126/science.1076071.f Acknowledgements Bai L-Y, Ma Y, Kulp SK, Wang S-H, Chiu C-F, Frissora F, Mani R, Mo X, Jarjoura D, Byrd JC, Chen C-S, Muthusamy N. OSU-DY7, a novel D-tyrosinol deriva- tive, mediates cytotoxicity in chronic lymphocytic lleukemialeukemia and Burkitt lymphoma through p38 mitogen-activated protein kinase pathway. 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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: ? Publisher’s Note 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. 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https://openalex.org/W2004148529	https://bmcresnotes.biomedcentral.com/track/pdf/10.1186/1756-0500-5-333	English	null	First postoperative day review after uneventful phacoemulsification cataract surgery: Is it necessary?	BMC research notes	2,012	cc-by	3,382	© 2012 Chatziralli 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. RESEARCH ARTICLE Open Access Chatziralli et al. BMC Research Notes 2012, 5:333 http://www.biomedcentral.com/1756-0500/5/333 Chatziralli et al. BMC Research Notes 2012, 5:333 http://www.biomedcentral.com/1756-0500/5/333 Abstract Background: Our purpose was to examine the value of the first postoperative day review after uneventful phacoemulsification cataract surgery. Methods: 291 patients who underwent uneventful phacoemulsification were randomized into two groups (ClinicalTrials.gov Identifier: NCT01247155): i) Next day review (NDR group, n = 146) and ii) No next day review (NNDR group, n = 145). The rate of complications, percentage of patients seeking non-scheduled medical consultation up to postoperative day 14, presence of any inflammation-related sign and best corrected visual acuity (BCVA) on postoperative day 28 were analyzed. Results: In the NDR group, 5.5% of patients developed a postoperative complication, whereas the respective rate was 6.2% in the NNDR group. The difference was not statistically significant (p = 0.791). The most frequent complications were: elevated intraocular pressure, allergy to postoperative treatment, corneal abrasion, punctuate epitheliopathy, iris prolapse and postoperative hyphema, whose rates did not differ between the two groups. The rate of patients seeking non-scheduled medical consultation up to postoperative day 14, presence of any inflammation-related sign, as well as BCVA on day 28 did not exhibit any significant differences between the study groups. Conclusions: First postoperative day review could be omitted in cases of uneventful cataract surge clusions: First postoperative day review could be omitted in cases of uneventful cataract surgery. Keywords: Cataract, Review, Postoperative, First day * Correspondence: eirchat@yahoo.gr 1Department of Ophthalmology, General Hospital of Veroia, Veroia, Greece 328, Papanastasiou street, Agios Dimitrios,, 17342, Athens, Greece Full list of author information is available at the end of the article First postoperative day review after uneventful phacoemulsification cataract surgery: Is it necessary? Irini P Chatziralli1,3, Theodoros N Sergentanis2, Evgenia Kanonidou1 and Leonidas Papazisis1 Methods first postoperative night, while a doctor examined them in the following morning (slit lamp examination, tono- metry). On the other hand, patients allocated to the NNDR group were discharged 3–4 hours after surgery. The patients were recruited from the Department of Ophthalmology, General Hospital of Veroia, Veroia, Greece over a 10-month period. Patients were randomly selected from the grand pool of phacoemulsification pro- cedures taking place in the Department; the random se- lection was based on random numbers allocation, so as to eliminate any selection bias. 311 patients were asked to participate and 304 consented (participation rate: 97.7%). Patients’ recorded data included age, sex, current smoking habits and clinical features, such as presence of hypertension, diabetes mellitus (with or without diabetic retinopathy), pseudoexfoliation, glaucoma and age- related macular degeneration. All patients underwent a full ophthalmologic examination preoperatively i.e., measurement of BCVA (Snellen chart), slit lamp exam- ination, tonometry and fundoscopy in addition to a complete medical history. The study was in accordance with the Declaration of Helsinki and has been approved by the Institutional Review Board of General Hospital of Veroia, Veroia, Greece. Written informed consent was obtained from all patients. Exclusion criteria were the following: i) intra-operative complications, such as posterior capsule rupture, vitre- ous loss, lost nucleus, zonule dehiscence and wound leak, ii) inadequate social support for overnight care at home, iii) severely limited visual potential in the fellow eye, iv) uveitis or ocular trauma, v) severe systemic dis- eases limiting activity, vi) patients with learning disability or dementia. All patients received the same postoperative treatment i.e., combination of tobramycin 0.3% - dexamethasone 0.1% (TobraDexW, Alcon), one drop four times/day, plus ketorolac tromethamine 0.5% (AcularW, Allergan), one drop three times/day. The topical treatment was admi- nistered for 28 days after phacoemulsification. Appropri- ate postoperative instructions were given to all patients in addition to a contact telephone number for emergen- cies. Two follow-up visits were scheduled for all patients: one on postoperative day 14 and one on postoperative day 28. On postoperative day 14 slit lamp examination, tonometry and fundoscopy were performed; special at- tention was paid to record whether the patient had sought non-scheduled medical consultation up to post- operative day 14. On postoperative day 28 slit lamp examination, tonometry, fundoscopy and BCVA meas- urement were conducted. Background complications, reassurance for the patient and training of staff [3]. As a result, there have been studies which stated that first day postoperative review after unevent- ful phacoemulcification cataract surgery is necessary and others that could not make specific recommenda- tions for the necessity of it [7-9]. Cataract surgery is one of the most common surgical procedures worldwide [1,2]. Phacoemulsification and recent advances in cataract surgery techniques lead to improved postoperative outcomes, as well as to reduced intra- and postoperative complications [2,3]. Therefore, there is a tendency in discharging patients on the same day of surgery, without first day post- operative review [3-6], which is considered to be an increasing issue due to economic reasons. Neverthe- less, routine review on the first day postoperatively has several advantages, such as the early detection of Under the light of the above, the purpose of this ran- domized trial was to examine the value of the review on the first-postoperative day after uneventful phacoemulsi- fication cataract surgery. Three outcomes were adopted in this study, so as to yield a global, comprehensive ap- proach: i. percentage of patients seeking non-scheduled medical consultation up to postoperative day 14, ii. pres- ence of any inflammation-related signs (corneal edema, Tyndall effect, conjunctival hyperemia) [10] on post- operative day 28 and iii. best corrected visual acuity (BCVA) on postoperative day 28. Correspondence: eirchat@yahoo.gr 1Department of Ophthalmology, General Hospital of Veroia, Veroia, Greece 328, Papanastasiou street, Agios Dimitrios,, 17342, Athens, Greece Full list of author information is available at the end of the article Chatziralli et al. BMC Research Notes 2012, 5:333 http://www.biomedcentral.com/1756-0500/5/333 Chatziralli et al. BMC Research Notes 2012, 5:333 http://www.biomedcentral.com/1756-0500/5/333 Page 2 of 5 Methods All patients were evaluated by All patients underwent routine phacoemulsification cataract surgery with posterior chamber intraocular lens implantation by the same consultant surgeon (LP) and were randomized to one of the two postoperative follow- up groups: i) Next day review (NDR group, n = 146) and ii) No next day review (NNDR group, n = 145). Patients assigned to the NDR group stayed at the hospital for the Figure 1 Flow chart depicting the exclusion criteria and randomization of patients. NDR: next day review; NNDR: non next day review; PCR: posterior capsule rupture; IOC: intra-operative complications. Figure 1 Flow chart depicting the exclusion criteria and randomization of patients. NDR: next day review; NNDR: non next day review; PCR: posterior capsule rupture; IOC: intra-operative complications. osterior capsule rupture; IOC: intra-operative complications. Chatziralli et al. BMC Research Notes 2012, 5:333 http://www.biomedcentral.com/1756-0500/5/333 Page 3 of 5 Table 2 The profile of complications in the two study groups Complications NDR group NNDR group p* N(%) N(%) Postoperative complications Allergy to postoperative treatment 3 (2.1) 1 (0.7) 0.622 Iris prolapse 0 (0.0) 1 (0.7) 0.498 Corneal abrasion 2 (1.4) 2 (1.4) >0.999 Elevated intraocular pressure (>30 mmHg) 2 (1.4) 4 (2.8) 0.447 Punctuate epitheliopathy 2 (1.4) 1 (0.7) >0.999 Postoperative hyphema 0 (0.0) 1 (0.7) 0.498 *p-values derived from Fisher’s exact test. Table 2 The profile of complications in the two study groups the same team having performed the phacoemulsifica- tion procedures and specifically by two independent examiners. Three outcomes were adopted in this study: i. percent- age of patients seeking non-scheduled medical consult- ation up to postoperative day 14, ii. presence of any inflammation-related signs (corneal edema, Tyndall ef- fect, conjunctival hyperemia) on postoperative day 28 and iii. BCVA on postoperative day 28. The differences in baseline characteristics, incidence of postoperative complications between the two groups, as well as between outcomes were compared by Chi-square test or Fisher’s exact test (concerning categorical vari- ables) or Mann–Whitney-Wilcoxon (MWW) test for in- dependent samples (concerning continuous variables), as appropriate. Concerning BCVA, the descriptive statistics of the log of the minimum angle of resolution (logMAR) were computed as appropriate [11]. Statistical analysis was performed with STATA 8.0 statistical software (StataCorp, College Station, TX, USA). significant (p = 0.791, Chi-square test). Results Table 1 The baseline features of the two study groups Continuous variables NDR group (n = 146) NNDR group (n = 145) p mean ± SD mean ± SD Age (years) 75.4 ± 7.2 75.8 ± 7.0 0.584MWW BCVA prior to phacoemulsification (logMAR) 0.59 ± 0.13 0.63 ± 0.14 0.193MWW Categorical and ordinal variables N (%) N (%) Sex (male) 73 (50.0) 79 (54.5) 0.444C Current smoking (yes) 24 (16.4) 20 (13.8) 0.529C Hypertension (yes) 104 (71.2) 113 (77.9) 0.190C Diabetes mellitus (yes) 24 (16.4) 23 (15.9) 0.894C Diabetic retinopathy (yes) 14 (9.6) 11 (7.5) 0.530C Glaucoma (yes) 13 (8.9) 17 (11.7) 0.429C Pseudoexfoliation (yes) 44 (30.1) 35 (24.1) 0.250C Age-related macular degeneration (yes) 11 (7.5) 10 (6.9) 0.834C MWWp-value derived from Mann–Whitney-Wilcoxon test for independent samples, Cp-value derived from Pearson’s chi-square, Fp-value derived from Table 1 The baseline features of the two study groups Methods Two patients (one in the NDR group and one in the NNDR group) presented with coexistent corneal abrasion and punctu- ate epitheliopathy; as a result, the total number of com- plications in the NDR and NNDR group were nine and ten, respectively. Worthy of note, regarding NDR group, all complica- tions appearing in Table 2 became evident on next-day review, except for allergy to postoperative treatment which prompted patients to seek non-scheduled medical consultation (see below). With respect to NNDR group, all complications had become evident prior to post- operative day 14, either at the scheduled visit or at a non-scheduled consultation (two patients, see below). Results The study design, as well as the randomization of patients in the two groups, is depicted in the respective flow chart (Figure 1). Table 1 represents the baseline demographic, clinical features and lifestyle habits of the study groups. The postoperative complications with their statistical significance are illustrated in Table 2. Of the 146 patients randomized to NDR group, eight (5.5%; 95%CI: 2.4%-10.5%) developed a postoperative complica- tion vs. nine (6.2%; 95%CI: 2.9%-11.5%) of 145 patients in the NNDR group. The difference was not statistically Concerning the three outcomes of the study, no statis- tically significant differences were noted (Table 3). The percentage of patients seeking non-scheduled medical consultation up to postoperative day 14 was minimal in both groups; accordingly, the frequency of any inflammation-related sign was scarce on postoperative day 28. It is worth mentioning that the underlying cause in all three patients seeking non-scheduled medical con- sultation up to postoperative day 14 in NDR group was allergy to postoperative treatment (allergy emerged on postoperative day 2 for two patients and on postoperative day 3 for one patient); on the contrary, the underlying cause for the respective two cases in NNDR group was corneal abrasion (the two patients sought non-scheduled medical consultation due to pain on postoperative day 5 and 9, respectively). BCVA on postoperative day 28 did not exhibit statistically significant difference between the two groups (0.06 ± 0.08 vs. 0.06 ± 0.06 for NDR and NNDR respectively, p = 0.859, MWW). Of note, the agreement between the two examiners was 100%, regard- ing the assessment at day 14 and day 28. Discussion The principal message of this study is that the first post- operative day review could be omitted after uneventful Page 4 of 5 Chatziralli et al. BMC Research Notes 2012, 5:333 http://www.biomedcentral.com/1756-0500/5/333 Table 3 Frequency of the study outcomes in the two study groups Categorical outcomes NDR group NNDR group p N(%) Seeking non-scheduled medical consultation up to postoperative day 14 3 (2.1) 2 (1.4) >0.999F Presence of any inflammation-related sign on postoperative day 28 2 (1.4) 2 (1.4) >0.999F Continuous outcome mean ± SD BCVA on postoperative day 28 8.77 ± 1.27 8.85 ± 1.13 0.859MWW Fp-value derived from Fisher’s exact test, MWWp-value derived from Mann–Whitney-Wilcoxon test for independent samples. Table 3 Frequency of the study outcomes in the two study groups be educated in postoperative care and drop installation [3,8]. Nevertheless, severe complications, such as endophthalmitis and retinal detachment, are rare and not necessarily detected on the first postoperative day [3,4,8], as our study also suggests. As a result, it seems better to have a review on postoperative day 3 to 4 in- stead of first postoperative day review. It is also import- ant to ensure that all patients might easily have access to eye care providers postoperatively at any time, so as to be appropriately advised if they have any discomfort or sight-threatening symptom. A meaningful limitation of this study that should be declared pertains to the design of the study. A blind (masked) assessment at day 14 and 28 was not feasible, as the team of surgeons performing the phacoemulsifica- tion procedure were essentially the same as the evalu- ation team. Nevertheless, it should be stressed that two independent examiners evaluated the patients with 100% agreement, a fact pointing to the rather negligible effect mediated by the non-blind evaluation. phacoemulsification cataract surgery. The frequency of serious complications detected on the first postoperative day was low and there was no statistically significant dif- ference between the two groups concerning non- scheduled consultation, inflammation-related signs or postoperative complications. Moreover, BCVA seems not to be affected by the first day postoperative review, as Tinley et al. have also noted [4]. Author details 1 1Department of Ophthalmology, General Hospital of Veroia, Veroia, Greece. 2Department of Epidemiology and Biostatistics, Medical School, National University of Athens, Athens, Greece. 328, Papanastasiou street, Agios Dimitrios,, 17342, Athens, Greece. Conclusion In conclusion, this study indicates that first postopera- tive day review could be omitted in cases of uneventful phacoemulsification cataract surgery, supplemented by patients-initiated review in the interim. Regarding complications, iris prolapse is rare after small incision cataract surgery and is thought to be asso- ciated with poor wound construction or postoperative manipulation [8]. Allergy to postoperative treatment is not a vision-threatening complication and can be pre- dicted based on a thorough medical history [12]. With respect to corneal abrasion, pain was the symptom that led patients to seek non-scheduled advice, when first postoperative day review had been withdrawn. Pain may be indeed an alarming sign for punctuate epitheliopathy as well [13]. Worthy of note, the meaningful triad of inflammation-related signs i.e., corneal edema, Tyndall effect, conjunctival hyperemia seemed to follow the same trend in both groups [10]. Received: 5 February 2012 Accepted: 27 June 2012 Published: 27 June 2012 Received: 5 February 2012 Accepted: 27 June 2012 Published: 27 June 2012 Abbreviations BCVA: Best corrected visual acuity; NDR: Next day review; NNDR: No next day review; MWW: Mann–Whitney-Wilcoxon. Competing interests Competing interests The authors declare that they have no competing interests. Authors’ contributions Overall, the most frequent postoperative complication in our study was elevated intraocular pressure. This is in agreement with the findings of previous investigations [3,7,8]. Interestingly, Dinakaran et al. highlighted that first postoperative day review is necessary, so as to check intraocular pressure [7]. However, it has been shown that the postoperative rise in intraocular pressure is transient as its peak occurs between 3 and 6 hours post- operatively [14]. Of note, intraocular pressure elevation is more common in the subset of patients with coexist- ing glaucoma and can be prevented by using prophylac- tic topical intraocular pressure lowering agents [3,15,16]. Taken as whole, first postoperative day review may offer little to the reduction of intraocular pressure. IC conceived the idea of the study, designed the study, collected data and drafted the manuscript. TS performed the statistical analysis and drafted the manuscript. EK collected data and revised critically the manuscript. LP conceived the idea of the study and revised critically the manuscript. All authors read and approve the final manuscript. Received: 5 February 2012 Accepted: 27 June 2012 Published: 27 June 2012 References 1. Forooghian F, Agrón E, Clemons TE, Ferris FL 3rd, Chew EY, Age-Related Eye Disease Study Research Group: Visual acuity outcomes after cataract surgery in patients with age-related macular degeneration: age-related eye disease study report no. 27. 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Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission
https://openalex.org/W2250376026	https://aclanthology.org/W14-4202.pdf	English	null	Learning from a Neighbor: Adapting a Japanese Parser for Korean Through Feature Transfer Learning	null	2,014	cc-by	8,194	Abstract minimal human supervision. Dependency parsing can handle long-distance relationships and coor- dination phenomena very well, and has proven to be very effective for parsing free-order languages such as Korean and Japanese (K¨ubler et al., 2009). We present a new dependency parsing method for Korean applying cross-lingual transfer learning and domain adaptation techniques. Unlike existing transfer learn- ing methods relying on aligned corpora or bilingual lexicons, we propose a feature transfer learning method with minimal su- pervision, which adapts an existing parser to the target language by transferring the features for the source language to the tar- get language. Speciﬁcally, we utilize the Triplet/Quadruplet Model, a hybrid pars- ing algorithm for Japanese, and apply a delexicalized feature transfer for Korean. Experiments with Penn Korean Treebank show that even using only the transferred features from Japanese achieves a high accuracy (81.6%) for Korean dependency parsing. Further improvements were ob- tained when a small annotated Korean cor- pus was combined with the Japanese train- ing corpus, conﬁrming that efﬁcient cross- lingual transfer learning can be achieved without expensive linguistic resources. Most statistical parsing methods rely on anno- tated corpora labeled with phrase structures or dependency relationships, but it is very expen- sive to create a large number of consistent anno- tations. Recently, treebanks have become avail- able for many languages such as English, Ger- man, Arabic, and Chinese. However, the pars- ing results on these treebanks vary a lot depend- ing on the size of annotated sentences and the type of annotations (Levy and Manning, 2003; Mc- Donald et al., 2013). Further, many languages lack annotated corpus, or the size of the anno- tated corpus is too small to develop a reliable sta- tistical method. To address these problems, there have been several attempts at unsupervised pars- ing (Seginer, 2007; Spitkovsky et al., 2011), gram- mar induction (Klein and Manning, 2004; Naseem et al., 2010), and cross-lingual transfer learning using annotated corpora of other languages (Mc- Donald et al., 2011). However, the accuracies of unsupervised methods are unacceptably low, and results from cross-lingual transfer learning show different outcomes for different pairs of languages, but, in most cases, the parsing accuracy is still low for practical purposes. A recent study by McDon- ald et al. (2013) concludes that cross-lingual trans- fer learning is beneﬁcial when the source and tar- get languages were similar. Abstract In particular, it reports that Korean is an outlier with the lowest scores (42% or less in UAS) when a model was trained from European languages. Learning from a Neighbor: Adapting a Japanese Parser for Korean through Feature Transfer Learning Youngja Park IBM Research - T.J.Watson Research Center Yorktown Heights, NY, USA young park@us.ibm.com Hiroshi Kanayama IBM Research - Tokyo Koto-ku, Tokyo, Japan hkana@jp.ibm.com Dongmook Yi Korea Software Solutions Laboratory, IBM Korea Gangnam-gu, Seoul, Korea dmyi@kr.ibm.com Dongmook Yi Korea Software Solutions Laboratory, IBM Korea Gangnam-gu, Seoul, Korea dmyi@kr.ibm.com Yuta Tsuboi IBM Research - Tokyo Koto-ku, Tokyo, Japan yutat@jp.ibm.com Yuta Tsuboi IBM Research - Tokyo Koto-ku, Tokyo, Japan yutat@jp.ibm.com Language Technology for Closely Related Languages and Language Variants (LT4CloseLang), pages 2–12, October 29, 2014, Doha, Qatar. c⃝2014 Association for Computational Linguistics 2.1 Parsing for Korean Since Korean is a morphologically-rich language, many efforts for Korean parsing have focused on automatically extracting rich lexical informa- tion such as the use of case frame patterns for the verbs (Lee et al., 2007), the acquisition of case frames and nominal phrases from raw cor- pora (Park et al., 2013), and effective features from phrases and their neighboring contexts (Choi and Palmer, 2011). Recently, Choi et al. (2012) discussed the transformation of eojeol-based Ko- rean treebank to entity-based treebank to effec- tively train probabilistic CFG parsers. We apply similar techniques as in (Choi et al., 2012) to miti- gate the differences between Korean and Japanese syntactic structures. We exploit many similarities between the two languages, such as the head-ﬁnal structure, the noun to verb modiﬁcation via case and topic mark- ers, and the similar word-order constraints. It was reported that the mapping of the grammar formal- ism in the language pair was relatively easy (Kim et al., 2003b; Kim et al., 2003a). However, as the two languages are classiﬁed into independent lan- guage families (Gordon and Grimes, 2005), there are many signiﬁcant differences in their morphol- ogy and grammar (especially in the writing sys- tems), so it is not trivial to handle the two lan- guages in a uniform way. Chung and Rim (2003) applied the Triplet/Quadruplet Model for Korean parsing as done in our work. They reported that the model performed well for long-distance dependencies, but, in their experiments, the number of modi- ﬁcation candidates was not effectively reduced (only 91.5% of phrases were in one of the three positions, while it was 98.6% in Kanayama’s work for Japanese). In this paper, we introduce more sophisticated grammatical knowledge and heuristics to have similar dependency distribu- tions in the two languages. Smith and Smith (2004) attempted a bilingual parsing for English and Korean by combining statistical dependency parsers, probabilistic context-free grammars, and word translation models into a uniﬁed framework that jointly searches for the best English parse, Korean parse and word alignment. However, we utilize an existing parser and align the features from the source language to the features for the target language, and, thus, our method is applicable to situations where there is no aligned corpora or word translation models. 1 Introduction Motivated by increasing demands for advanced natural language processing (NLP) applications such as sentiment analysis (Pang et al., 2002; Nasukawa and Yi, 2003) and question answer- ing (Kwok et al., 2001; Ferrucci et al., 2010), there is a growing need for accurate syntactic parsing and semantic analysis of languages, especially for non-English languages with limited linguistic re- sources. In this paper, we propose a new depen- dency parsing method for Korean which requires In this paper, we present a new cross-lingual 2 the delexicalized feature transfer method enables the algorithm applicable to any two languages that have similar syntactic structures. transfer learning method that learns a new model for the target language by transferring the fea- tures for the source language. Unlike other ap- proaches which rely on aligned corpora or a bilingual lexicon, we learn a parsing model for Korean by reusing the features and annotated data used in the Japanese dependency parsing, the Triplet/Quadruplet Model (Kanayama et al., 2000), which is a hybrid approach utilizing both grammatical knowledge and statistics. 2.1 Parsing for Korean We show the Triplet/Quadruplet Model is suit- able for bilingual transfer learning, because the grammar rules and heuristics reduce the number of modiﬁcation candidates and can mitigate the differences between two languages efﬁciently. In addition, this model can handle the relationships among the candidates as a richer feature space, making the model less dependent upon the lexi- cal features of the content words that are difﬁcult to align between the two languages. Similarly to the delexicalized parsing model in (McDonald et al., 2011), we transfer only part-of-speech infor- mation of the features for the content words. We create new mapping rules to extract syntactic fea- tures for Korean parsing from the Japanese anno- tated corpus and reﬁne the grammar rules to get closer modiﬁcation distributions in two languages. Our experiments with Penn Korean Tree- bank (Han et al., 2002) conﬁrm that the Triplet/Quadruplet Model adapted for Korean out- performs a distance-based dependency parsing method, achieving 81.6% accuracy when no an- notated Korean corpus was used. Further perfor- mance improvements were obtained when a small size of annotated Korean corpus was added, con- ﬁrming that our algorithm can be applied with- out more expensive linguistic resources such as an aligned corpora or bilingual lexicons. Moreover, 2.2 Transfer learning and domain adaptation Figure 1: An example of dependency structures of a Korean sentence “아내가산프랑스의여행가방 을친구에게보이고싶다.” (‘(I) want to show the French travel bag which (my) wife bought to (my) friend’). Each box corresponds to a Korean phrasal unit eojeol. 妻が妻/n が/pc 買った買/v た/aux フランスのフランス/np の/pn 旅行かばんを旅行/n かばん/n を/pc 友達に友達/n に/pc 見せたい。見せ/v たい/aux 。/ ‘wife-NOM’ ‘buy-PAST’ ‘France-GEN’ ‘travel bag-ACC’ ‘friend-DAT’ ‘want to show’ b1 b2 b3 b4 b5 b6 ? ? ? ? ? Figure 2: A dependency structure of a Japanese sentence which corresponds to the Korean sentence in Figure 1, “妻が買ったフランスの旅行かばんを友達に見せたい。”. Each box corresponds to a Japanese phrasal unit bunsetsu. Figure 2: A dependency structure of a Japanese sentence which corresponds to the Korean sentence in Figure 1, “妻が買ったフランスの旅行かばんを友達に見せたい。”. Each box corresponds to a Japanese phrasal unit bunsetsu. of the features trained by source, by target, and by combined domains. syntactic parsing, where a parsing model for a tar- get language is learned from linguistic resources in one or more different languages (Hwa et al., 2005; Zeman and Resnik, 2008; McDonald et al., 2011; Durrett et al., 2012; Georgi et al., 2012; Naseem et al., 2012). McDonald et al. (2011) proposed a delexicalized parsing model for cross- lingual dependency parsing and demonstrated that a high accuracy parsing was achieved for Indo- European languages where signiﬁcant amount of parallel texts exist. However, in more recent work, McDonald et al. (2013) showed that, unlike trans- fer learning within close language families, build- ing a Korean parser from European languages was not successful with a very low accuracy. Durrett et al. (2012) and Georgi et al. (2012) show that transfer parsing can be improved when additional bilingual resources are available, such as bilingual dictionaries and parallel corpora of glossed texts respectively. 2.2 Transfer learning and domain adaptation Recently, transfer learning has attracted much at- tention, as it can overcome the lack of training data for new languages or new domains for both classiﬁcation and regression tasks (Pan and Yang, 2010). Transfer learning has also been applied to 3 아내가 아내/NNC 이/PCA 산 사/VV 은/EAN 프랑스의 프랑스/NPR 의/PAN 여행 여행/NNC 가방을 가방/NNC 을/PCA 친구에게 친구/NNC 에게/PAD 보이고 보이/VV 고/ECS 싶다 싶/VX 다/EFN ． ./SFN ‘wife-NOM’ ‘buy-PAST’ ‘France-GEN’ ‘travel’ ‘bag-ACC’ ‘friend-DAT’ ‘show’ ‘want’ ‘.’ e1 e2 e3 e4 e5 e6 e7 e8 e9 ? ? ? ? ? ? ? ? Figure 1: An example of dependency structures of a Korean sentence “아내가산프랑스의여행가방 을친구에게보이고싶다.” (‘(I) want to show the French travel bag which (my) wife bought to (my) friend’). Each box corresponds to a Korean phrasal unit eojeol. 妻が妻/n が/pc 買った買/v た/aux フランスのフランス/np の/pn 旅行かばんを旅行/n かばん/n を/pc 友達に友達/n に/pc 見せたい。見せ/v たい/aux 。/ ‘wife-NOM’ ‘buy-PAST’ ‘France-GEN’ ‘travel bag-ACC’ ‘friend-DAT’ ‘want to show’ b1 b2 b3 b4 b5 b6 ? ? ? ? ? Figure 2: A dependency structure of a Japanese sentence which corresponds to the Korean sentence in Figure 1, “妻が買ったフランスの旅行かばんを友達に見せたい。”. Each box corresponds to a Japanese phrasal unit bunsetsu. 아내가 아내/NNC 이/PCA 산 사/VV 은/EAN 프랑스의 프랑스/NPR 의/PAN 여행 여행/NNC 가방을 가방/NNC 을/PCA 친구에게 친구/NNC 에게/PAD 보이고 보이/VV 고/ECS 싶다 싶/VX 다/EFN ． ./SFN ‘wife-NOM’ ‘buy-PAST’ ‘France-GEN’ ‘travel’ ‘bag-ACC’ ‘friend-DAT’ ‘show’ ‘want’ ‘.’ e1 e2 e3 e4 e5 e6 e7 e8 e9 ? ? ? ? ? ? ? ? Figure 1: An example of dependency structures of a Korean sentence “아내가산프랑스의여행가방 을친구에게보이고싶다.” (‘(I) want to show the French travel bag which (my) wife bought to (my) friend’). Each box corresponds to a Korean phrasal unit eojeol. 아내가 아내/NNC 이/PCA 산 사/VV 은/EAN 프랑스의 프랑스/NPR 의/PAN 여행 여행/NNC 가방을 가방/NNC 을/PCA 친구에게 친구/NNC 에게/PAD 보이고 보이/VV 고/ECS 싶다 싶/VX 다/EFN ． ./SFN ‘wife-NOM’ ‘buy-PAST’ ‘France-GEN’ ‘travel’ ‘bag-ACC’ ‘friend-DAT’ ‘show’ ‘want’ ‘.’ e1 e2 e3 e4 e5 e6 e7 e8 e9 ? ? ? ? ? ? ? ? Figure 1: An example of dependency structures of a Korean sentence “아내가산프랑스의여행가방 을친구에게보이고싶다.” (‘(I) want to show the French travel bag which (my) wife bought to (my) friend’). Each box corresponds to a Korean phrasal unit eojeol. 3 Dependency Structures of Korean and Japanese A dependency structure in Korean is typically an- alyzed in terms of eojeol units, a basic phrase that consists of a content word agglutinated with optional functional morphemes such as postposi- tional particles or endings for verbs. Figure 1 shows an example Korean sentence with the de- pendencies between the eojeols indicated by ar- rows. Figure 2 illustrates the dependency struc- ture between the bunsetsus in Japanese that cor- responds to the Korean structure in Figure 1. As these ﬁgures show, the syntactic structures are quite similar in these languages: All of the de- pendencies are directed from left to right, and the postpositional particles determine if the content word modiﬁes a verb (“가” in e1 and “が” in b1) or a noun (“의” in e3 and “の” in b3). The eojeols in Korean roughly correspond to the bunsetsus in Japanese. In the remainder of this paper, we de- note both an eojeol or a bunsetsu as a ‘PU’ (phrasal unit) when distinction is not needed. Our method does not require such resources and does not have restrictions on the sentence type that can be parsed. Instead, we use a mixture of a small corpus in a target language (i.e., Korean) and a larger corpus of a source language (Japanese). This task is similar to domain adaptation, and our objective is to outperform the training model built on each language separately. To avoid the loss of accuracy due to the differences between two do- mains, we apply the domain adaptation technique proposed by Daum´e III (2007) which duplicates the feature space into three categories with each While Korean and Japanese have similar syn- tactic structures, the two languages have many dif- ferences. The eojeols in Korean are separated by white space, while the bunsetsus in Japanese are not. Further, the statistics show several differences in the two languages. Table 1 compares a Korean corpus, Penn Korean Treebank (henceforth KTB) 4 Table 1: Statistics of Korean and Japanese corpora. 3 Dependency Structures of Korean and Japanese The Japanese pars- ing method that will be described in the next sec- tion exploits these characteristics, which we apply to Korean parsing. An analysis of the EDR corpus shows that 98.6% of the correct dependencies are either the nearest PU, the second nearest PU, or the farthest PU from the modiﬁer (more details in Table 4(a)). Therefore, the model can be simpliﬁed by restrict- ing the candidates to these three candidates and by ignoring the other PUs with a small sacriﬁce (1.4%) of parsing accuracy. 4 Triplet/Quadruplet Model Let u be a modiﬁer PU in question, cun the u’s n- th modiﬁcation candidate PU, Φu and Ψcun the at- tributes of u and cun, respectively. Then the prob- ability that u modiﬁes its n-th candidate is calcu- lated by the triplet equation (1) or the quadruplet equation (2) when u has two or three candidates, respectively 1. This section describes the Triplet/Quadruplet Model (Kanayama et al., 2000) which was origi- nally designed for Japanese parsing. First, we re- view the two main ideas of the model – restriction of modiﬁcation candidates and feature selection for probability calculation. Then, we describe how we apply the Triplet/Quadruplet Model to Korean parsing in Section 4.3. P(u →cun) = P(n \| Φu, Ψcu1, Ψcu2) (1) P(u →cun) = P(n \| Φu, Ψcu1, Ψcu2, Ψcu3) (2) (1) 3 Dependency Structures of Korean and Japanese KTB (Korean) EDR (Japanese) Average number of characters (except for whitespace) per sentence 73.7 28.0 Average number of PUs per sentence 25.5 8.53 Average number of morphemes per PU 1.83 2.86 Ratio of modiﬁcation to the next PU 70.0% 61.8% KTB (Korean) EDR (Japanese) Average number of characters (except for whitespace) per sentence 73.7 28.0 Average number of PUs per sentence 25.5 8.53 Average number of morphemes per PU 1.83 2.86 Ratio of modiﬁcation to the next PU 70.0% 61.8% Table 2: Simpliﬁed examples of Japanese grammar rules. Rightmost morpheme of the modiﬁer PU Conditions for the modiﬁed PUs postpositional “を” wo (accusative) verb, adjective postpositional “の” no (genitive, nominative) noun, verb, adjective postpositional “と” to (conjunctive) noun, verb, adjective, adverb “一緒に” isshoni (‘together’) adverb verb, adjective, adverb, copula Table 2: Simpliﬁed examples of Japanese grammar rules. that determine whether a PU can modify each PU to its right in a sentence. The main goal of the grammar rules is to maximize the coverage, and the rules are simple describing high-level syntac- tic dependencies, and, thus, the rules can be eas- ily created without worrying about the precision or contradictory rules. The statistical information is later used to select the right rules for a given sentence to produce an accurate parsing result. Ta- ble 2 shows several grammar rules for Japanese, in which the modiﬁed PUs are determined depend- ing on the conditions of the rightmost morpheme in the modiﬁer PU. (Han et al., 2002), and a Japanese corpus, EDR Corpus (EDR, 1996). Both corpora consist of word-level bracketed constituents, so they are con- verted into PU-level dependency structures using the method described in Choi and Palmer (2011). Though both corpora consist mainly of newspaper or magazine articles, the sentences are not aligned with each other, so the statistics show the compar- isons of the two corpora, rather than the theoret- ical comparisons of the two languages. However, we can see that Korean sentences tend to be longer than Japanese sentences both in terms of the num- ber of characters and PUs. More eojeols modify an adjacent eojeol in Korean than in Japanese. For in- stance, e1, e4, e6, e7, and e8 modify the next eojeol in Figure 1, but only b1, b3, and b5 modify the next bunsetsu in Figure 2. Those differences suggest some of the difﬁculties in applying the Japanese dependency model to Korean. 1It is trivial to show that P(u →cu1) = 1, when u has only one candidate. 4.1 Restriction of modiﬁcation candidates The Triplet/Quadruplet Model utilizes a small number (about 50) of hand-crafted grammar rules 1It is trivial to show that P(u →cu1) = 1, when u has only one candidate. 5 Table 3: Simpliﬁed examples of Korean grammar rules. Table 3: Simpliﬁed examples of Korean grammar rules. ab e 3: S p ed e a p es o o ea g a a u es. Rightmost morpheme of the modiﬁer PU Conditions for the modiﬁed PUs PCA,PCJ,PAD,PAU (postpositional particles) V* (verb, adjective or auxiliary), CO (copula) EAN (nominal verb ending e.g. “은” eun) N* (noun) ADV (adverb), ADC (conjunction) N* (noun), V* (verb, adjective or auxiliary), ADV (adverb), ADC (conjunction) postpositional “과” gwa (conjunctive) N* (noun), V* (verb, adjective or aux), adverb “함께” hamkke (‘together’) N* (noun) N* (noun), V* (verb, adjective or auxiliary) Rightmost morpheme of the modiﬁer PU Conditions for the modiﬁed PUs Table 4: Distribution (percentage) of the position of the correct modiﬁed PU among the candidate PUs selected by the initial grammar rules. The column ‘Sum’ shows the coverage of the 1st, 2nd and last PUs. The EDR corpus was used for Japanese, and the KTB was used for Korean in this analysis. Table 4: Distribution (percentage) of the position of the correct modiﬁed PU among the candidate PUs selected by the initial grammar rules. The column ‘Sum’ shows the coverage of the 1st, 2nd and last PUs. The EDR corpus was used for Japanese, and the KTB was used for Korean in this analysis. (b) Korean (with the initial grammar) # of Candidates Ratio 1st 2nd Last Sum 1 10.5 100.0 − − 100.0 2 11.4 85.9 14.1 − 100.0 3 10.4 76.2 13.4 10.4 100.0 4 9.3 74.7 11.3 8.0 93.9 ≥5 58.4 75.5 10.0 4.9 90.5 Total 100 − − − 93.9 (a) Japanese # of Candidates Ratio 1st 2nd Last Sum 1 32.7 100.0 − − 100.0 2 28.1 74.3 26.7 − 100.0 3 17.5 70.6 12.6 16.8 100.0 4 9.9 70.4 11.1 13.8 95.3 ≥5 11.8 70.2 11.1 10.5 91.9 Total 100 − − − 98.6 These probabilities are estimated by the maxi- mum entropy method with a feature set to express Φ and Ψ. 4.1 Restriction of modiﬁcation candidates Assuming the independence of those modiﬁcations, the probability of the dependency tree for an entire sentence P(T) is calculated as the product of the probabilities of all of the depen- dencies in the sentence using beam search to max- imize P(T) under the constraints of the projected structure. the modiﬁcations. Second, the probability of each modiﬁcation is calculated based on the relative po- sitions of the candidates, instead of the distance from the modiﬁer PU in the surface sentence, and, thus, the model is more robust. 4.3 Korean dependency parsing with the Triplet/Quadruplet Model We design the Korean parser by adapting the Triplet/Quadruplet Model based on the analogous characteristics of Japanese and Korean. First, we created the Korean grammar rules for generating candidate modiﬁed PUs by modifying the rules for Japanese shown in Table 2 for Korean. The rule set, containing fewer than 50 rules, is sim- ple enough to be created manually, because the rules simply describe possible dependencies, and Japanese phenomena are good hints for Korean phenomena. Table 3 shows some examples of the rules for Korean based on the POS schema used in the KTB corpus. We did not automatically extract the rules from the annotated corpora so that the rules are general and independent of the training corpus. Nonetheless, 96.6% of the dependencies in KTB are covered by the grammar rules. The re- maining dependencies (3.4%) not covered by the rule set are mainly due to rare modiﬁcations and may indicate inconsistencies in the annotations, so we do not seek any grammar rules to achieve nearly 100%. P(T) ≃ ∏ u P(u →cun) (3) (3) In comparison, a traditional statistical parser (Collins, 1997) uses Equation (4) to calculate the probability of u modifying t. In comparison, a traditional statistical parser (Collins, 1997) uses Equation (4) to calculate the probability of u modifying t. P(u →t) = P(True \| Φu, Ψt, ∆u,t) (4) (4) We call the model based on Equation (4) the Dis- tance Model, since ∆u,t (the distance between u and t) is typically used as the key feature. Though other contextual information, in addition to the at- tributes of u and t, can be added, the model calcu- lates the probabilities of the dependencies between u and t independently and thus often fails to incor- porate appropriate contextual information. We call the model based on Equation (4) the Dis- tance Model, since ∆u,t (the distance between u and t) is typically used as the key feature. Though other contextual information, in addition to the at- tributes of u and t, can be added, the model calcu- lates the probabilities of the dependencies between u and t independently and thus often fails to incor- porate appropriate contextual information. Equations (1) and (2) have two major advan- tages over the Distance Model: First, all the at- tributes of the modiﬁer and its candidates can be handled simultaneously. 4.3 Korean dependency parsing with the Triplet/Quadruplet Model The combination of those attributes helps the model to express the context of 6 아내가(‘wife-NOM’) 산(‘buy’) 보이고(‘show’) 싶다(‘want’) e1 e2 e7 e8 ? ? ? ⟨1⟩NNC (common noun) ⟨2⟩PCA (postpositional) ⟨3⟩“이” (‘-NOM’) ⟨5⟩VV (verb) ⟨7⟩EAN (adnominal ending) ⟨8⟩“은” (past adnominal) ⟨5⟩VV (verb) ⟨7⟩ECS (ending) ⟨8⟩“고” (conjunctive) ⟨5⟩VX (auxiliary) ⟨7⟩EFN (ﬁnal ending) ⟨8⟩“다” (predicative) Figure 3: The features used to select the modiﬁed PU of e1 among its three candidates. The full sentence of this example is shown in Figure 1. The numbers in brackets correspond to the feature IDs in Table 5. Figure 3: The features used to select the modiﬁed PU of e1 among its three candidates. The full sentence of this example is shown in Figure 1. The numbers in brackets correspond to the feature IDs in Table 5. Table 5: The features to express attributes of a modiﬁer and modiﬁcation candidates. Feature set ID Description ⟨1⟩ PoS of the head morpheme of the modiﬁer ⟨2⟩ PoS of the last morpheme of the modiﬁer ⟨3⟩ Lex of the postpositional or endings of the modiﬁer ⟨4⟩ Lex of the adverb of the modiﬁer ⟨5⟩ PoS of the head morpheme of the modiﬁcation candidate ⟨6⟩ Lex of the head morpheme of the modiﬁcation candidate ⟨7⟩ PoS of the last morpheme of the modiﬁcation candidate ⟨8⟩ Lex of the postpositional or endings of the modiﬁcation candidate ⟨9⟩ Existence of a quotation expression “다고” dago or “라고” rago ⟨10⟩ Number of “은” eun (TOPIC marker) between the modiﬁer and modiﬁcation candidate ⟨11⟩ Number of commas between the modiﬁer and modiﬁcation candidate combination ⟨1⟩× ⟨5⟩ / ⟨2⟩× ⟨5⟩ / ⟨2⟩× ⟨7⟩ / ⟨3⟩× ⟨5⟩ / ⟨3⟩× ⟨8⟩ Table 5: The features to express attributes of a modiﬁer and modiﬁcation candidates. Table 5: The features to express attributes of a modiﬁer and modiﬁcation candidates. 4.3 Korean dependency parsing with the Triplet/Quadruplet Model Feature set ID Description ⟨1⟩ PoS of the head morpheme of the modiﬁer ⟨2⟩ PoS of the last morpheme of the modiﬁer ⟨3⟩ Lex of the postpositional or endings of the modiﬁer ⟨4⟩ Lex of the adverb of the modiﬁer ⟨5⟩ PoS of the head morpheme of the modiﬁcation candidate ⟨6⟩ Lex of the head morpheme of the modiﬁcation candidate ⟨7⟩ PoS of the last morpheme of the modiﬁcation candidate ⟨8⟩ Lex of the postpositional or endings of the modiﬁcation candidate ⟨9⟩ Existence of a quotation expression “다고” dago or “라고” rago ⟨10⟩ Number of “은” eun (TOPIC marker) between the modiﬁer and modiﬁcation candidate ⟨11⟩ Number of commas between the modiﬁer and modiﬁcation candidate combination ⟨1⟩× ⟨5⟩ / ⟨2⟩× ⟨5⟩ / ⟨2⟩× ⟨7⟩ / ⟨3⟩× ⟨5⟩ / ⟨3⟩× ⟨8⟩ 5 Adaptation for Bilingual Transfer Learning Table 4(a) and (b) show the distribution of the numbers of candidate PUs and the position of the correct modiﬁed PUs obtained from the analysis of the EDR corpus and the KTB corpus respec- tively. As we can see, the ﬁrst candidate is pre- ferred in both languages, but the preference of the nearer candidate is stronger in Korean. For in- stance, when there are more than one candidates, the probability that the ﬁrst candidate is the cor- rect one is 78% for Korean but 71% for Japanese. Further, when there are more than 2 candidates, Japanese prefers the last candidate, while Korean prefers the second candidate. Based on the analy- sis results, the number of modiﬁcation candidates is restricted to at most three (the ﬁrst, second and last candidates) for Korean as well. In Section 4.3, we explained how the Triplet/Quadruplet Model can be used for Korean. In this section, we describe the feature adaption techniques in more detail and investigate if the new model with transferred features works well when a small amount of annotated corpus for the target language is provided. Further, we study if we can leverage the annotated corpus for the source language in addition to the parsing model and train a model for the target language using the training data for the source language. 5.1 Feature Transfer With the assumption that Korean and Japanese have similar syntactic dependencies, we adopt the delexicalized parsing model presented in Mc- Donald et al. (2011). We transfer the part-of- speech (POS) in the Japanese features to the POS scheme in the KTB corpus, and translate Japanese functional words to the corresponding functional words in Korean. This transfer process is manda- tory because we use the language speciﬁc POS systems to capture language-speciﬁc dependency phenomena, unlike other works using language universal but coarser POS systems. The next step is to design Φu and Ψcun, which are required in Equations (1) and (2) to choose the correct modiﬁed PU. We converted the feature set from the Japanese study to get the Korean features as listed in Table 5. For example, to ﬁnd the mod- iﬁed PU of e1 “아내가” anae-ga (’wife-NOM’) in the sentence shown in Figure 1, the attributes of e1 and the attributes of the three candidates, e2, e7, and e8, are extracted as shown in Figure 3, and their attributes are used to estimate the probability of each candidate in Equation (2). We do not transfer lexical knowledge on con- 7 Table 6: Example of mapping rules for parts-of-speech and functional words. Japanese PoS Korean PoS common noun NNC verb VV adjective VJ nominal sufﬁx XSF case particle “で”,“に”,“へ”,“から” PAD others PCA Japanese particle Korean particle “を” wo (‘-ACC’) “을” eul “より” yori (‘from’) “부터” buteo “は” ha (‘-TOPIC’) “은” eun “も” mo (‘too’) “도” do “が” ga case particle (‘-NOM’) “이” i conjunctive particle (‘but’) “지만” jiman Table 6: Example of mapping rules for parts-of-speech and functional words. Japanese particle Korean particle “を” wo (‘-ACC’) “을” eul “より” yori (‘from’) “부터” buteo “は” ha (‘-TOPIC’) “은” eun “も” mo (‘too’) “도” do “が” ga case particle (‘-NOM’) “이” i conjunctive particle (‘but’) “지만” jiman tent words and exceptional cases, so feature sets ⟨4⟩and ⟨6⟩are not transferred. Table 6 shows some examples of the feature transfer which han- dle POS tags and functional words. We note that the Korean features shown in Figure 3 are directly extracted from Japanese corpus using those rules. and e4 is determined to modify e5 (line 3). Sub- sequently, e3’s modiﬁability to e4 is rejected (line 5), and, thus, the correct modiﬁed PU of e3 is de- termined as e5. 5.2 Adaptation of parsing rules While Japanese and Korean are similar in terms of syntactic dependencies, there are signiﬁcant dif- ferences between the two languages in the distri- bution of modiﬁcation as shown in the Table 4(a) and (b): In Korean, more than half of modiﬁers have 5 or more candidates, while only 12% of Japanese modiﬁers do. In Japanese, 98.6% of cor- rect modiﬁed PUs are located in one of the three positions (1st, 2nd or last), but, in Korean, the ratio falls to 93.9% as shown in Table 4. Another ma- jor difference of the two languages is the different average numbers of PUs per sentence as shown in Table 1. Korean has 25.5 PUs per sentence, while the number is only 8.5 in Japanese. This is mainly caused by the difference between eojeol in Korean and bunsetsu in Japanese. In Japanese, compound nouns and verb phrases with an auxiliary verb are likely to form a single bunsetsu, while, in Korean, they are split into multiple eojeols with a whites- pace in-between. 5.1 Feature Transfer After reﬁning the rules for com- pound nouns and auxiliary verbs, the probability of the correct modiﬁed PU being the 1st, 2nd or last candidate PU increases from 93.9% to 96.3% as shown in Table 7, and the distribution of the candidate’s positions for Korean became closer to the Japanese distribution shown in Table 4(a). 5.3 Learning from heterogeneous bilingual corpora The feature transfer and rule adaptation methods described in previous sections generate a very ac- curate Korean parser using only a Japanese cor- pus as shown in the ﬁrst row in Table 8. The next question is if we can leverage bilingual corpora to further improve the accuracy, when annotated cor- pus for the target language (Korean) is available. We note that the two corpora do not need to be aligned and can come from different domains. To mitigate the side effects of merging heterogeneous training data in different languages, we apply the domain adaptation method proposed by Daum´e III (2007) and augment the feature set to a source language-speciﬁc version, a target-speciﬁc version and a general version. Speciﬁcally, a feature set x in Table 5 is expanded as follows: These differences signiﬁcantly reduce the ef- fect of transfer learning. To address these prob- lems, we further reﬁne the grammar rules as in the following. We added heuristic rules for the Korean model to effectively reduce the number of candidates in compound nouns which consist of a noun sequence in multiple eojeols, and verbs or adjectives followed by auxiliary verbs. Figure 4 shows an algorithm to reduce the number of mod- iﬁed PUs considering the structure of compound nouns. In this example, both PUs e4 (“travel”) and e5 (“bag-ACC”) can be candidate PUs for eojeol e3. However, based on the rule in Figure 4, e4 and e5 are considered as a compound noun (line 1), xK=< x, 0, x > (5) xJ=< 0, x, x > (6) (5) (6) (6) where xK and xJ denote the feature sets extracted from the Korean corpus and the Japanese corpus respectively. Then, the features speciﬁc to Korean and Japanese get higher weights for the ﬁrst part or the second part respectively, and the character- istics existing in both languages inﬂuence the last part. 8 if PoS of ui’s last morpheme is N* and PoS of ui+1’s ﬁrst morpheme is N* then ui must modify ui+1 if ui−1’s last morpheme is not “의” then ui−1 cannot modify ui+1 else ui−1 cannot modify ui u1 to ui−2 cannot modify ui 프랑스의 프랑스/NPR 의/PAN 여행 여행/NNC 가방을 가방/NNC 을/PCA ‘France-GEN’ ‘travel’ ‘bag-ACC’ e3 e4 e5 ? ? ? Figure 4: Heuristic rules to reduce the number of modiﬁcation candidates surrounding compound nouns in Korean. 6 Experiments In this section, we validate the effectiveness of learning a Korean parser using the feature transfer learning from the Japanese parser and compare the Korean model with other baseline cases. We also compare the parsing results when various sizes of bilingual corpora were used to train the Korean model. 5.3 Learning from heterogeneous bilingual corpora The example in the right ﬁgure shows that candidates in the dotted lines are removed by the heuristics. Figure 4: Heuristic rules to reduce the number of modiﬁcation candidates surrounding compound nouns in Korean. The example in the right ﬁgure shows that candidates in the dotted lines are removed by the heuristics. Figure 4: Heuristic rules to reduce the number of modiﬁcation candidates surrounding compound nouns in Korean. The example in the right ﬁgure shows that candidates in the dotted lines are removed by the heuristics. Table 7: Distribution of the position of correct modiﬁed PU for Korean after the reﬁnement of the Korean grammar rules. # of candidates Ratio 1st 2nd Last Sum 1 46.4% 100.0% − − 100.0% 2 9.8% 79.0% 21.0% − 100.0% 3 9.2% 75.5% 12.7% 11.8% 100.0% 4 8.0% 71.0% 11.8% 9.6% 92.4% ≥5 26.6% 70.4% 10.1% 7.8% 88.3% Total 100% − − − 96.3% stribution of the position of correct modiﬁed PU for Korean after the reﬁnement of the Korean les Table 7: Distribution of the position of correct modiﬁed PU for Korean after the reﬁnement of the Korean grammar rules. We compare the Triplet/Quadruplet Model- based models with the Distance Model. For the Distance Model, we used the same feature set as in Table 5, and added the distance feature (∆u,t) by grouping the distance between two PUs into 3 categories (1, 2 to 5, and 6 or more). The perfor- mances are measured by UAS (unlabeled attach- ment score), and the results of the two methods are shown in the second column, where Japanese Corpus Size=0, in Table 8 (a) and (b) respectively. The top leftmost cells (80.61% and 71.63%) show the parsing accuracies without any training cor- pora. In these cases the nearest candidate PU is selected as the modiﬁed PU. The difference be- tween two models suggests the effect of restriction of modiﬁcation candidates by the grammar rules. We note that the Triplet/Quadruplet Model pro- duces more accurate results and outperforms the Distance Model by more than 2 percentage points in all cases. The results conﬁrm that the method for Japanese parsing is suitable for Korean pars- ing. 6.1 Korean parsing using the Triplet/Quadruplet Model First, to validate the effectiveness of the Triplet/Quadruplet Model for parsing Korean, we built eight Korean dependency parsing models us- ing different numbers of training sentences for Ko- rean. The KTB corpus Version 2.0 (Han et al., 2002) containing 5,010 annotated sentences was used in this study. We ﬁrst divide the corpus into 5 subsets by putting each sentence into its (sentence ID mod 5)-th group. We use sentences from the ﬁrst subgroup for estimating the parameters, sen- tences from the second subgroup for testing, and use the remaining three subgroups for training. We built 8 models in total, using from 0 sentence up to 3,006 sentences selected from the training set. The number of training sentences in each model is shown in the ﬁrst column in Table 8. The pa- rameters were estimated by the maximum entropy method, and the most preferable tree is selected using each dependency probability and the beam search. The test data set contains 1,043 sentences. 6.2 Results of bilingual transfer learning Next, we evaluate the transfer learning when anno- tated sentences for Japanese were also added. Ta- ble 8(a) shows the accuracies of our model when various numbers of training sentences from Ko- rean and Japanese are used. The ﬁrst row shows the accuracies of Korean parsing when the models were trained only with the Japanese corpus, and 9 Table 8: The accuracy of Korean dependency parsing with various numbers of annotated sentences in the two languages. † denotes that the mixture of bilingual corpora signiﬁcantly outperformed (p < .05) the parser trained with only the Korean corpus without Japanese corpus. (a) Triplet/Quadruplet Model Japanese Corpus Size 0 2,500 5,000 10,000 Korean Corpus Size 0 80.61% 80.78% 81.23% † 81.58% † 50 82.21% 82.32% 82.40% † 82.43% † 98 82.36% 82.66% † 82.69% † 82.70% † 197 83.13% 83.18% 83.30% † 83.28% 383 83.62% 83.92% † 83.94% † 83.91% † 750 84.03% 84.00% 84.06% 84.06% 1,502 84.41% 84.34% 84.32% 84.28% 3,006 84.77% 84.64% 84.64% 84.65% (b) Distance Model Japanese Corpus Size 0 2,500 5,000 Korean Corpus Size 0 71.63% 62.42% 54.92% 50 79.31% 79.55% † 79.54% † 98 80.53% 80.63% † 80.72% † 197 80.91% 80.84% 80.85% 383 81.86% 81.75% 81.76% 750 82.10% 81.92% 81.94% 1,502 82.50% 82.48% 82.50% 3,006 82.66% 82.57% 82.54% other rows show the results when the Korean and Japanese corpora were mixed using the method described in Section 5.3. proved the feature mapping, and our approach with the domain adaptation technique (Daum´e III, 2007) successfully solved the difﬁculty. Table 8(b) shows the results of the Distance Model. As we can see from the ﬁrst row, using only the Japanese corpus did not help the Dis- tance Model at all in this case. The Distance Model was not able to mitigate the differences be- tween the two languages, because it does not use any grammatical rules to control the modiﬁability. This demonstrates that the hybrid parsing method with the grammar rules makes the transfer learn- ing more effective. On the other hand, the domain adaptation method described in (5) and (6) suc- cessfully counteracted the contradictory phenom- ena in the two languages and increased the accu- racy when the size of the Korean corpus was small (size=50 and 98). This is because the interactions among multiple candidates which cannot be cap- tured from the small Korean corpus were provided by the Japanese corpus. 6.2 Results of bilingual transfer learning As we can see from the results, the bene- ﬁt of transfer learning is larger when the size of the annotated corpus for Korean (i.e., target language) is smaller. In our experiments with Triplet/Quadruplet Model, positive results were obtained by the mixture of the two languages when the Korean corpus is less than 500 sentences, that is, the annotations in the source language success- fully compensated the small corpus of the target language. When the size of the Korean corpus is relatively large (≥1, 500 sentences), adding the Japanese corpus decreased the accuracy slightly, due to syntactic differences between the two lan- guages. Also the effect of the corpus from the source language tends to saturate as the size of the source corpus, when the target corpus is larger. This is mainly because our mapping rules ignore lexical features, so few new features found in the larger corpus were incorrectly processed. Some of previous work reported the parsing accuracy with the same KTB corpus; 81% with trained grammar (Chung et al., 2010) and 83% with Stanford parser after corpus transformation (Choi et al., 2012), but as Choi et al. (2012) noted it is difﬁcult to directly compare the accuracies. When merging the corpus in two languages, if we simply concatenate the transferred features from the source language and the features from the target language (instead of using the dupli- cated features shown in Equations (5) and (6)), the accuracy dropped from 82.70% to 82.26% when the Korean corpus size was 98 and Japanese cor- pus size was 10,000, and from 83.91% to 83.40% when Korean=383. These results support that there are signiﬁcant differences in the dependen- cies between two languages even if we have im- 6.3 Discussion The analysis of e2’s dependency in Figure 1 is a good example to illustrate how the Triplet/Quadruplet Model and the Japanese corpus help Korean parsing. Eojeol e2 has three modiﬁcation candidates, e3, e5, and e6. In the 10 Distance Model, e3 is chosen because the distance between the two eojeols (∆e2,e3) was 1, which is a very strong clue for dependency. Also, in the Triplet/Quadruplet Model trained only with a small Korean corpus, e3 received a higher probability than e5 and e6. However, when a larger Japanese corpus was combined with the Korean corpus, e5 was correctly selected as the Japanese corpus provided more samples of the dependency relation of “verb-PAST” (e2) and “common noun-ACC (을)” (e5) than that of “verb-PAST” and “proper noun-GEN (의)” (e3). References Jinho D. Choi and Martha Palmer. 2011. 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ACM Trans- actions on Information Systems (TOIS), 19(3):242– 262. David A. Smith and Noah A. Smith. 2004. Bilingual parsing with factored estimation: Using English to parse Korean. In Proceedings of the 2004 Confer- ence on Empirical Methods in Natural Language Processing, pages 49–56. Hyeon-Yeong Lee, Yi-Gyu Hwang, and Yong-Seok Lee. 2007. Parsing of Korean based on CFG using sentence pattern information. International Journal of Computer Science and Network Security, 7(7). Valentin I Spitkovsky, Hiyan Alshawi, Angel X Chang, and Daniel Jurafsky. 2011. Unsupervised depen- dency parsing without gold part-of-speech tags. In Proceedings of the Conference on Empirical Meth- ods in Natural Language Processing, pages 1281– 1290. Roger Levy and Christopher Manning. 2003. Is it harder to parse chinese, or the chinese treebank? In Proceedings of the 41st Annual Meeting on Asso- ciation for Computational Linguistics, ACL, pages 439–446. Daniel Zeman and Philip Resnik. 2008. Cross- language parser adaptation between related lan- guages. In IJCNLP, pages 35–42. Ryan McDonald, Slav Petrov, and Keith Hall. 2011. Multi-source transfer of delexicalized dependency parsers. In Proceedings of the Conference on Em- pirical Methods in Natural Language Processing, pages 62–72. Ryan McDonald, Joakim Nivre, Yvonne Quirmbach- Brundage, Yoav Goldberg, Dipanjan Das, Kuzman Ganchev, Keith Hall, Slav Petrov, Hao Zhang, Oscar T¨ackstr¨om, et al. 2013. Universal dependency an- notation for multilingual parsing. In Proceedings of ACL 2013. 12
https://openalex.org/W4384656052	https://zenodo.org/records/8163312/files/921-Article%20Text-2681-1-10-20230719.pdf	English	null	Incisional Hernia in Subxiphoid Trocar Port in Cholecystectomized Patient	Zenodo (CERN European Organization for Nuclear Research)	2,023	cc-by	2,036	INTRODUCTION note that the risk of developing a port hernia can vary according to factors such as the size and number of ports used, the type of surgery performed and the experience of the surgeon. 6 Laparoscopic surgery is a minimal access procedure that maintains the integrity of the abdominal wall avoiding large abdominal incisions, so the possibility of an incisional hernia decreases1-3 . It has also been associated with a decreased risk of surgical site infection, postoperative pain, improved esthetics and early return to work1 . However, as with any new technique, it is not free of complications, the most common being vascular and intestinal lesions2 and trocar port incisional hernia (TIPH), which is the rarest complication with an incidence of 0.65-6.0%4-5 . Risk factors related to the occurrence of trocar port hernias have been described; they are divided into those associated with the surgical technique and the patient's own characteristics3-6 : The risk of hernia at the trocar site is higher in obese and bariatric patients, due to their larger preperitoneal space and elevated intra-abdominal pressure, which makes it difficult to achieve a full-thickness closure, as well as technical difficulties. 6,7 Laparoscopic port hernias are a rare but significant complication associated with the performance of laparoscopic surgical procedures. These hernias are characterized by protrusion of intra-abdominal structures through the insertion sites of the ports used during laparoscopic surgery. Despite advances in surgical technique and port design, laparoscopic port hernias remain a clinical challenge and can result in significant morbidity for patients. 6 Some surgeons performing minimally invasive procedures generally do not attempt to close the fascia in defects smaller than 10 millimeters, due to the technical difficulties associated with closure 2; however, port size is known to be another important risk factor, as hernias in 3 mm ports have been reported in children. 2,6 The pathophysiology of laparoscopic port hernias involves a combination of mechanical and biological factors. During port insertion and manipulation, a defect is created in the abdominal fascia, allowing tissue and organs to pass through that site. In addition, intra-abdominal pressure during insufflation of the gas used to create the working space in the abdominal cavity can contribute to hernia formation. Lack of The exact incidence of laparoscopic port hernias is not clearly defined, due in part to the lack of a universally accepted definition and variability in monitoring and detection methods. ABSTRACT Laparoscopic surgery is a minimal access procedure that maintains the integrity of the abdominal wall, however, it is not exempt from complications, such as vascular and intestinal injuries and the rarest complication: incisional hernia of the trocar port with an incidence of 0.65. -There are risk factors related to the appearance of trocar port hernias, they are divided into those associated with the surgical technique and the characteristics of the patient. We present a clinical case of a patient with an incisional hernia in the laparoscopic trocar port with a subxiphoid location due to cholecystectomy, which was repaired with polypropylene mesh. Published On: 19 July 2023 Available on: https://ijmscr.org/ KEYWORDS: Laparoscopic cholecystectomy; incisional hernia; subxiphoid port, mesh International Journal of Medical Science and Clinical Research Studies ISSN(print): 2767-8326, ISSN(online): 2767-8342 Volume 03 Issue 07 July 2023 Page No: 1347-1349 International Journal of Medical Science and Clinical Research Studies ISSN(print): 2767-8326, ISSN(online): 2767-8342 Volume 03 Issue 07 July 2023 Page No: 1347-1349 International Journal of Medical Science and Clinical Research Studies ISSN(print): 2767-8326, ISSN(online): 2767-8342 Volume 03 Issue 07 July 2023 Page No: 1347-1349 International Journal of Medical Science and Clinical Research Studies ISSN(print): 2767-8326, ISSN(online): 2767-8342 Volume 03 Issue 07 July 2023 Page No: 1347-1349 Volume 03 Issue 07 July 2023 Page No: 1347-1349 DOI: https://doi.org/10.47191/ijmscrs/v3-i7-22, Impact Factor: 6.597 1347 Volume 03 Issue 07 July 2023 Incisional Hernia in Subxiphoid Trocar Port in Cholecystectomized Patient which reported a hernial defect (Figure 1). With this finding, a protocol was established to perform wall plast. which reported a hernial defect (Figure 1). With this finding, a protocol was established to perform wall plast. adequate healing and weakness of the abdominal fascia at the port insertion site may also predispose to hernia formation. 7 The diagnosis of laparoscopic port hernias can be challenging due to the variety of clinical presentations and the possibility of nonspecific symptoms. Patients may experience pain, discomfort, bulging, or changes in the appearance of port insertion sites. Clinical suspicion and careful evaluation of patients are critical for an accurate diagnosis. Diagnostic tests, such as ultrasound, computed tomography and magnetic resonance imaging, can be helpful in the confirmation and characterization of port hernias. Fig. 1 Ultrasound hernia defect. Management of laparoscopic port hernias can vary depending on the severity of the hernia and the patient's symptoms. Therapeutic options include conservative management with observation, use of bandages or external supports, open or laparoscopic surgical repair of the hernia, and in severe cases, removal of the ports and closure of fascial defects. The choice of therapeutic approach depends on factors such as the size of the hernia, the presence of complications and the experience of the surgeon. 5,6,7 Fig. 1 Ultrasound hernia defect. During surgery an aponeurotic defect of 3 x 2 cm was found, with a sac of 3 x 3 cm. Chevrel M1 W1 R0 incisional hernia is diagnosed and the management is with placement of Atramat® PROPYMESH "sublay" mesh; it is fixed with Polypropylene Monofilament American suture 2-0 single stitches The patient is discharged the next day due to clinical improvement. In summary, laparoscopic port hernias are a rare but important complication of laparoscopic surgical procedures. Understanding their pathophysiology, diagnosis and management is essential to minimize associated morbidity and improve clinical outcomes. Careful perioperative surveillance and adequate clinical evaluation are required for early detection and appropriate therapeutic approach to laparoscopic port hernias. 6 During her outpatient evaluation, the patient was asymptomatic and did not present hernia recurrence. Three months later, the patient came for a new reevaluation without presenting a hernia defect. Incisional Hernia in Subxiphoid Trocar Port in Cholecystectomized Patient The prevention of this complication requires knowledge of the risk factors of this condition, so an adequate preoperative assessment should be performed and the risk of this pathology should be widely explained to all patients who present risk factors, in addition to implementing hygienic and dietary measures prior to surgery. 6 DISCUSSION Port closure is performed by different techniques after release of the pneumoperitoneum. Mostly the classic suture method is used due to its simplicity, although it can sometimes be difficult and is associated with the fear of injuring underlying bowel loops, omentum or other abdominal organs with the needle, resulting in complications such as HIPT.5,6,8 . INTRODUCTION However, the incidence is estimated to vary between 0.5% and 3% in different studies. It is important to 1347 Volume 03 Issue 07 July 2023 Corresponding Author: Gabriela Martínez Maya 1348 Volume 03 Issue 07 July 2023 Corresponding Author: Gabriela Martínez Maya VIII. Nicola DL, Giorgio C, Francesca L Achille G. Port- Site Closure: A New Problem, An Old Device. JSLS. 2002(6): 181-183. IX. Sangeetha J, S DR, Case series of Umbilical and Extra-umbilical Port Site Herniae. Indian Journal of Surgery. 2013;75:488-491. Incisional Hernia in Subxiphoid Trocar Port in Cholecystectomized Patient review of the literature. Rev Peru Gynecol Obstet. 2017;63(4):647-650. In relation to the size of the lithium, in lithiums larger than 2 cm, the fascia wound is usually opened wider to facilitate its extraction, favoring eventration3 . III. Lindsay VL, Roxaana GC, Luis BRS. Incidence of umbilical incisional hernia by laparoscopic ports in cholecystectomized patients over a five-year period. Endoscopic Surgery. 2018;19(2):68-71. According to a study of 19 cases of incisional hernia, it was found that total exsufflation of the pneumoperitoneum intraoperatively, total relaxation of the patient until skin closure and avoidance of heavy lifting for at least one month postoperatively can reduce the risk of HIPT11 . IV. H JK, Jun BP, Ji young S. Umbilical port site hernia and diastasis tecti. J Minim Invasive Surg 2020;23(2):80-85. Several risk factors associated with the development of HIPT have been described; in the current case, the surgical risk factors identified were that the subxiphoid port did not close, despite being a trocar of more than 10 mm. The risk factors associated with the patient include grade II obesity and the fact that she is a female patient. V. Lambertz A, Stüben BO, Bock B, Eickhoff R, Kroh A, Klink CD, et al. Port-site incisional hernia - A case series of 54 patients. Annals of Medicine and Surgery. 2017;14:8-11. VI. Abdulzahra H, Hind M, Rarun S, Santosh B, Jackie N, Shamsi E-H. Long-Term Study of Port-Site Incisional Hernia After Laparoscopic Procedures. JSLS. 2009;13:346 -349. ACKNOWLEDGMENTS X. . Chauvet P, Rabischong B, Curinier S, Gremeau AS, Bourdel N, Kaemmerlen AG, et al. Laparoscopy and laparoscopic surgery: general principles and instrumentation. EMC - Gynecology-Obstetrics. 2018;54(2):1-17. I am grateful to Dr. Barrera and Dr. Uribe who advised and supported me in carrying out this work. 1349 Volume 03 Issue 07 July 2023 Presentation of the clinical case This is a 55-year-old female patient with a history of pathology: grade II obesity, cesarean section, appendectomy 18 years ago, right inguinal plasty, umbilical plasty and 5 months prior to her current care, laparoscopic cholecystectomy with 3 ports (10 mm umbilical, 10 mm subxiphoid and 5 mm right subcostal). The cholecystectomy was performed without complications, and only the umbilical port was closed (the subxiphoid port could not be closed due to the thickness of the subcutaneous cellular tissue), and she was discharged home because she presented favorable evolution and was discharged the following day. Previously, the Deschamps needle was used to achieve full- thickness closure of trocar sites8-9 . Other authors such as Morenzo-Sanz, in his pilot study, have expressed the feasibility of using a surgical plug in the muscle layer of the trocar wounds to prevent trocar site hernias9 . Specific clamps are now available to allow closure of the abdominal wall, after the use of 10 mm trocars, to prevent possible cases of eventration or hernias. Reverdin needles or the Berci clamp10 can also be used. In a 2014 study by Comajuncosas et al, they analyzed whether the preexistence of an umbilical hernia could predispose to the development of HIPT, but no association was found in univariate or multivariate analysis. They also analyzed the association between diabetes mellitus and HIPT and conclude that apart from the known effect of diabetes on tissue scarring, it is difficult to explain this association7 . Two days after her discharge, she reported an increase in volume over the subxiphoid port wound, which was only present with Valsalva maneuvers. It progressed without management or visit to the doctor for another 3 months, increasing in size and appearing when standing upright and in supine decubitus, so she was seen in outpatient surgery where an ultrasound of the abdominal wall was requested, Corresponding Author: Gabriela Martínez Maya Corresponding Author: Gabriela Martínez Maya Corresponding Author: Gabriela Martínez Maya CONCLUSION The incidence of incisional hernia in our hospital is unknown, since we do not have studies that report the incidence of HIPT, adding to this the lack of follow-up, late diagnosis and even the fact that the management of the complication is performed in another institution. VII. VII. Jordi C, Judith H, Pere G, Jaime J, Rolando O, Helena V, Jose L. LN, et al. Risk factors for umbilical trocar site incisional hernia in laparoscopic cholecystectomy: a prospective 3-year follow-up study. The American Journal of Surgery. 2014; 2017(1):1-6. Follow-up studies of the cholecystectomized patient should be implemented to verify the real frequency of this complication. Incisional hernia through the subxiphoid trocar postcholecystectomy should not go unnoticed and it should be considered fundamental to diagnose this post-surgical complication. VIII. REFERENCES I. Rose LH, Jeffery SC, Jamies G, Dermot H, Sean M, Daniel M, et al. Incidence of Port-Site Hernias: A Survey and Literature Review. Surg Laparosc Endosc Percutan Tech. 2016;26:425-430. XI. I. Karin N, Mohamed AC, Asma C, Mohamed BK, Mossaab G, Moez B.Trocar site post incisional hernia: About 19 cases. Pan African Medical Journal. 2018;29 II. Marta RT, Eduardo RV, Avelin VG. Bilateral incisional hernia after laparoscopy. Case report and 1349 Volume 03 Issue 07 July 2023 Corresponding Author: Gabriela Martínez Maya
https://openalex.org/W1971366335	https://zenodo.org/records/2376101/files/article.pdf	English	null	New organo‐metallic compounds**Presented before the Ciiiciiinati Branch of the American Pharmaceutical Association.	Journal of the American Pharmaceutical Association/The Journal of the American Pharmaceutical Association	1,917	public-domain	1,247	JOURNAL OF THE JOURNAL OF THE JOURNAL OF THE 24 * Presented before the Ciiiciiinati Branch of the American Pharmaceutical Association HCI-NH~-OH-CGH~-AS = As-C6HTOH-NHz-HCl As to the results obtained by the use of this compound, and the method of adminis- tration, all are familiar, as well as with its newer derivative, Neosalvarsan; the sodium formaldehyde sulphoxylate salt of Salvarsan, mixed with some sodium sulphite : HO-NHzCsH3-As = As-CeH3-OH-N€I-CH2SO2Na NEW ORGANO-ME'I'ALLIC COMPOUNDS* BY LOUIS F. WERNER, A.M. BY LOUIS F. WERNER, A.M. Inorganic and organic compounds of mercury and arsenic have long been used in the treatment of disease, due to pathological conditions of the blood. Most of these diseases are caused by the existence of bacteria in the blood stream, chiefly of the family known as the Protozoa. To this type of maladies belong sleeping sickness, syphilis, and malaria; in animals, surra, dourine, tsetse fly dis- ease, etc. Of the several diseases mentioned above, there is no doubt but that syphilis has been the most dangerous, and far-reaching in its effect on mankind, and the one giving the chief incentive to the study of compounds useful in the treatment of diseases of this type. It must be remembered, however, that the compounds to be spoken of in this paper, were not always tried out on animals and human subjects infected with syphilis, but in many cases other members of the Protozoa group were the para- sites, notably those causing sleeping sickness. The pharmacology of these drugs is, therefore, not in an altogether satisfactorv condition to make any great general- izations with accuracy as yet. y y One of the earliest derivatives of arsenic or organic nature to be used was Atoxyl, para-aminophenyl arsenic acid (sodium salt) : NH1- C&-AsO(ONa)z NH1- C&-AsO(ONa)z This compound was used with some success in the treatment of sleeping sick- ness and syphilis, but was found very toxic to the host, and often led to blindness, so that it is very little used at present. Salvarsan, 606, or chemically p,p'-dihydroxy-un,m'-diamino-arseno benzene hydrochloride, a derivative of Atoxyl, has been the most successful in the treat- ment of syphilis, of the many organic derivatives of arsenic prepared to date: HCI-NH~-OH-CGH~-AS = As-C6HTOH-NHz-HCl HO-NHzCsH3-As = As-CeH3-OH-N€I-CH2SO2Na It is of interest i o know of some of the compounds probably investigated by Ehrlich in his search which resulted in the discovery of Salvarsan: Para-amino-phenyl arsenic acid Para-hydrouy-phenyl arsenic acid Ortho, meta, and para toluidine arsenic acids Ortho, meta, and para cresol arsenic acids Naphthyl arsenic acids AMERICAN PHARMACEUTICAL ASSOCIATION AMERICAN PHARMACEUTICAL ASSOCIATION 25 25 Naphthol arsenic acids Naphthylamine arsenic acids Amino phenyl arsenoxids Hydroxy phenyl arsenoxids Para-hydroxy-meta-aminophenyl arsenic acid Meta-dihydroxy arsenic acids Meta-para-dihydroxy arsenic acid Meta-para-diarnino arsenic acid Meta-para-diamino arsenoxids Meta-para-dihydroxy arsenoxides p,p‘-dihydroxy-arseno benzol p,p’-diamino-arseno hcnzol Di-acetyl p,p’-diamino phenol Besides the above mentioned compounds, many of their gold mercury, plat- inum and other metallic salts have been made. Furthermore, it should be remembered, that in the arrangement of the ele- ments according to MendelejeiT, in what is known as the periodic system, the fol- lowing elements occur in the same column as arsenic: Nitrogen, Phosphorus, Vanadium, Columbium, Antimony, Tantalum, and Bismuth. Compounds of these elements would, therefore, be expected to have many of the general chem- ical and physiological properties of those of arsenic. Compounds of these elements have been made of analogous structure to those of arsenic, also mixed compounds of them with arsenic: CoIIsSb As-CoH3-NHZ-OII CsHs-Bi = As-C6H3-NH2-OH CsH6-P = AS-CeHa-NHz-OH CaH6-N =z AS-CsHa-NHz-OH Nitro-phenyl stibinic acid Amino-phenyl stibinic acid Hydroxy-phenyl stibinic acid Hydroxy-phenyl stibinoxide Amino-phenyl stibinoxide Nitro-phenyl stihinoxide Compounds oi the sulphur group and arsenic have also been prepared Compounds oi the sulphur group and arsenic have also been prepared Se = As-CsHs-OH-NHp Te = As-CoHs-OH-NHt S = As-CsEI3-OH -NHs Many of these compounds have been found useful, but as they do not excel Sal- varsan, and are often very difficult to make, there is very little probability of them ever being used to any great extent. As might be expected, the commercial success of Salvarsan and Neosalvarsan has greatly stimulatedlthe study of the organic mercury compounds. The organic compounds of mercury may be placed in two classes, Type R-Hg-R Type R-Hg-X Type R-Hg-R Type R-Hg-X where R is an organic radical, either aliphatic or aromatic, and X is a negative radical or atom as the halogens, nitric acid or sulphate radical, the acetyl radical, etc. where R is an organic radical, either aliphatic or aromatic, and X is a negative radical or atom as the halogens, nitric acid or sulphate radical, the acetyl radical, etc. AMERICAN PHARMACEUTICAL ASSOCIATION It has been found that, as a general rule, the compounds of the second type are five times as toxic as those of the first class, and it may be said that the toxicity of the compounds of mercury increases with the solubility in the body fluids, and JOURNAL OF THa 26 the ease with which ionic mercury can be split off, and is lowered by the ease in which it can be eliminated. The organic compounds of mercury have not come up to expectations, as they are slow of action, and have been found in most cases to be more poisonous to the host than to the bacteria they are meant to kill. The following are a few of the two classes of compounds prepared: Type R-Hg-R Mercury diphenyl CsHsHg-CsHs Mercury diphenol Mercury dibenzoyl Mercury dinaphthyl CIOH~H~T-CIOH? Mercury dinaphthol Diamino-dibenzoyl mercury Dibenzyl mercury Diamino-dihydroxy-diphenyl mercury HO-CaHrHg-CsHrOH COOH-CsH4-Hg-CeH4COOH OH-CioHsHg-CioHe,OH NHrCOOH-CsH~Hg-CsHhCOOH-N& C~H~-CH~-H~-CH~-CF,H~ OH-NHz-CsHa-Hg-CsH3-NHz-OH Type R-Hg-X Phenyl mercuric chloride Phenol mercuric chloride Benzoyl mercuric chloride CeHsHg-CI HO-CsH4-Hg-Cl COOH-CsH4-Hg-Cl Naphthyl mercuric chloride CioH7-Hg-CI Naphthol mercuric chloride Aminobenzoyl mercuric chloride Benzyl mercuric chloride Amino-hydroxy-phenyl mercury OH-CioHs-Hg-Cl NHz-COOH-CsHa-Hg-Cl CsHs-CHa-Hg-Cl OH-NHrCsHsHgCl It should be noted that this last compound of mercury is the analogue of It should be noted that this last compound of mercury is the analogue of Salvarsan. It should be noted that this last compound of mercury is the analogue of Salvarsan. Besides the typical compounds listed above, there are a myriad of others; derivatives of the aliphatic hydrocarbons, of the dyes, of the alkaloids, quinoline, the pyrazolones, etc. The ones, however, that apparently are the most effective are aromatic derivatives, but even these are too poisonous to be of value in the treatment of syphilis, so that there is veryjittle probability of them ever becoming of equal importance, in this field, to the arsenic compounds. LABORATORY WERNER DRUG & C ~ M I C A L Co., CINCINNATI, 0. CHANGE OE’ COLOR IN GREEN EXTRACTS. Green extracts hom the leaves of plants very rapidly lose their color when exposed to light and air, the chlorophyll being converted into chlorophyllan. Yet in living plants exposure to light and air has no effect on the green tint. The author assumes that there is a special enzyme in the plant-an “anti-oxidasel’- which protects chlorophyll from the action of light and oxygen, and also from the peroxidase of the tissues, which readily destroys it. In the living plant anti- oxidase accumulates in the plastids in sufficient quantity to counteract the peroxi- dase. In extracts the equilibrium between the enzymes is disturbed and the pig- ments are quickly destroyed. During the period of assimilation of the plastids in the living plant the chlorophyll is constantly used up and constantly replaced. The function of the anti-oxidase is to regulate this process, and thus prevent too rapid oxidation.-V. Lyubimenko, Bull. Acad. Sci. Petrog. ; C h m. Abstr., 1916, 10, 2467; through Pharmaceutical Journal.
https://openalex.org/W1602376655	https://bioone.org/journals/marine-and-coastal-fisheries/volume-7/issue-7/19425120.2015.1037473/Population-Genetic-Structure-of-Southern-Flounder-Inferred-from-Multilocus-DNA/10.1080/19425120.2015.1037473.pdf	English	null	Population Genetic Structure of Southern Flounder Inferred from Multilocus DNA Profiles	Marine and coastal fisheries	2,015	cc-by	11,208	Downloaded From: https://bioone.org/journals/Marine-and-Coastal-Fisheries:-Dynamics,-Management,-and-Ecosystem-Science on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use Verena H. Wang, Michael A. McCartney, and Frederick S. Scharf This is an Open Access article 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. The moral rights of the named author(s) have been asserted. Corresponding author: vhw6375@uncw.edu 1Present address: Minnesota Aquatic Invasive Species Research Center, Department of Fisheries, Wildlife, and Conservation Biology, University of Minnesota, 2003 Upper Buford Circle, St. Paul, Minnesota 55108, USA. Received September 26, 2014; accepted March 27, 2015 Population Genetic Structure of Southern Flounder Inferred from Multilocus DNA Proﬁles Verena H. Wang, Michael A. McCartney,1 and Frederick S. Scharf Department of Biology and Marine Biology, Center for Marine Science, Verena H. Wang,* Michael A. McCartney,1 and Frederick S. Scharf Department of Biology and Marine Biology, Center for Marine Science, University of North Carolina Wilmington, 601 South College Road, Wilmington, North Carolina 28403, USA Verena H. Wang,* Michael A. McCartney,1 and Frederick S. Scharf Department of Biology and Marine Biology, Center for Marine Science, University of North Carolina Wilmington, 601 South College Road, Wilmington, North Carolina 28403, USA Population Genetic Structure of Southern Flounder Inferred from Multilocus DNA Profiles Population Genetic Structure of Southern Flounder Inferred from Multilocus DNA Profiles Authors: Wang, Verena H., McCartney, Michael A., and Scharf, Frederick S. Source: Marine and Coastal Fisheries: Dynamics, Management, and Ecosystem Science, 7(7) : 220-232 Published By: American Fisheries Society URL: https://doi.org/10.1080/19425120.2015.1037473 Source: Marine and Coastal Fisheries: Dynamics, Management, and Ecosystem Science, 7(7) : 220-232 BioOne Complete (complete.BioOne.org) is a full-text database of 200 subscribed and open-access titles in the biological, ecological, and environmental sciences published by nonprofit societies, associations, museums, institutions, and presses. 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BioOne sees sustainable scholarly publishing as an inherently collaborative enterprise connecting authors, nonprofit publishers, academic institutions, research libraries, and research funders in the common goal of maximizing access to critical research. m: https://bioone.org/journals/Marine-and-Coastal-Fisheries:-Dynamics,-Management,-and-Ecosystem-Science on 23 Oct 2024 tps://bioone.org/terms-of-use DOI: 10.1080/19425120.2015.1037473 Downloaded From: https://bioone.org/journals/Marine-and-Coastal-Fisheries:-Dynamics,-Management,-and-Ecosystem-Science on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use Subject editor: Kristina Miller, Fisheries and Oceans Canada, Paciﬁc Biological Station, Nanaimo, British Columbia, Canada Abstract Marine species were once assumed to be panmictic, with long-distance dispersal capability and a lack 220 Downloaded From: https://bioone.org/journals/Marine-and-Coastal-Fisheries:-Dynamics,-Management,-and-Ecosystem-Science on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use one.org/journals/Marine-and-Coastal-Fisheries:-Dynamics,-Management,-and-Ecosystem-Science on 23 Oct 2024 org/terms-of-use m: https://bioone.org/journals/Marine-and-Coastal-Fisheries:-Dynamics,-Management,-and-Ecosystem-Science on 23 Oct 2024 tps://bioone.org/terms-of-use GENETIC STRUCTURE OF SOUTHERN FLOUNDER 221 Atlantic states (Monaghan 1996; Craig and Rice 2008). Prior to estuarine recruitment, Southern Flounder remain in a pelagic larval stage for a moderately long duration (»45 d), which increases the potential for long-range dispersal. The unknown level of adult mixing in the offshore environment combined with the long duration of the larval stage implies that the genetic population structure of Southern Flounder could be more homogeneous than currently assumed. The use of molecular methods should provide increased knowledge of Southern Flounder population structure and connectivity, thereby contributing to effective management decisions for the long-term sustainability of the species. of obvious barriers to gene ﬂow in the ocean resulting in low genetic differentiation among putative populations (Ward et al. 1994; Waples 1998). However, highly polymorphic molecular markers have revealed signiﬁcant genetic structure in marine ﬁsh at multiple geographic scales (Ruzzante et al. 1999; Knutsen et al. 2003; O’Reilly et al. 2004; McCairns and Bernatchez 2008), and Knutsen et al. (2011) suggested that even low levels of population subdivision in marine organisms can be biologically meaningful. Maintenance of subtle genetic stock differentiation may be important in conserving local diversity and adaptive variation. Consequently, exploitation of ﬁsh populations can lead to reductions in genetic diversity, which may impact the ability of depleted stocks to recover (Hutchinson et al. 2003). Incorporating population genetic analyses into ﬁsheries management and considering both his- torical and contemporary patterns of molecular diversity can help to promote sustainability and preserve evolutionary pro- cesses in the face of increasing anthropogenic impacts (Avise 1992; Crandall et al. 2000; Conover et al. 2006; Hauser and Carvalho 2008). Previous population genetics studies of Southern Flounder have been focused on the Gulf, but there has been no compre- hensive study spanning the species’ range within the Atlantic. When examining allozyme variation in Southern Flounder, Blandon et al. (2001) found that Gulf populations were signiﬁ- cantly differentiated from Atlantic populations, and a break in allele frequencies on the coast of Texas between Galveston Bay and Matagorda Bay indicated the presence of population structure within the Gulf. Abstract Updated studies using mitochondrial DNA (mtDNA) and microsatellites again revealed high diver- gence between Gulf and Atlantic populations of Southern Flounder, but in contrast to the allozyme survey, little differen- tiation was detected within the Gulf (Anderson and Karel 2012; Anderson et al. 2012). Given the low levels of within- basin variability detected in previous studies, we applied a powerful genetic tool in an attempt to better characterize diversity and stock structure of Southern Flounder in the Atlantic basin. The Southern Flounder Paralichthys lethostigma is an eco- nomically important ﬂatﬁsh that occurs throughout estuarine and coastal ocean waters in the northern Gulf of Mexico (here- after, Gulf) from northern Mexico to Florida (FL) and in the U.S. South Atlantic (hereafter, Atlantic) from FL to North Car- olina (NC). The species supports a thriving recreational ﬁshery across its range and is also a valuable commercial ﬁnﬁsh resource in some areas, particularly in NC, where commercial landings averaged 1.5 million kg (3.3 million lb) annually between 1991 and 2007 (NCDMF 2013). In fact, the NC stock has experienced elevated commercial harvest rates since at least 1991; although a ﬁshery management plan (NCDMF 2005, 2013) was ﬁrst established in 2005, the most recent stock assessment concluded that NC Southern Flounder remain overﬁshed and are still undergoing overﬁshing (Takade-Heumacher and Batsavage 2009). We examined Southern Flounder populations by using the ampliﬁed fragment length polymorphism (AFLP) technique (Vos et al. 1995). This method rapidly generates a genetic ﬁn- gerprint consisting of hundreds of highly reproducible DNA fragments that are scattered across the genome and that can be compared among individuals and populations without the need for prior characterization of an organism’s genome (Bensch and A kesson 2005). The large number of AFLPs pro- duced allows for assessment of genome-wide variation when analyzing population structure, which can be more informative than targeting mtDNA markers (which are inherited as a single haploid locus). It has been claimed that the information con- tent of an AFLP study is comparable to that obtained from a small number of highly informative microsatellite loci, which are commonly used but require substantial development time and cost (Campbell et al. 2003; Meudt and Clarke 2007; Sønstebø et al. 2007). Abstract Determination of stock structure is an important component of ﬁsheries management; incorporation of molecular genetic data is an effective method for assessing differentiation among putative populations. We examined genetic variation in Southern Flounder Paralichthys lethostigma within and between the U.S. South Atlantic and Gulf of Mexico basins to improve our understanding of the scale of population structure in this wide- ranging species. Analysis of ampliﬁed fragment length polymorphism (AFLP) ﬁngerprints and analysis of mitochondrial DNA (mtDNA) control region sequences found clear divergence between ocean basins. Based on mtDNA sequences, no genetic differentiation was detected within the U.S. South Atlantic at spatial scales that were broad (among states: North Carolina, South Carolina, Georgia, and Florida) or ﬁne (among estuarine regions within North Carolina). Increased genetic resolution was observed with AFLP ﬁngerprint data, and we found signiﬁcant subdivision between nearly all Southern Flounder geographic populations, suggesting the presence of ﬁner-scale genetic population structure within the U.S. South Atlantic. However, AFLP genetic cluster analysis also revealed evidence for a high degree of mixing within the Atlantic basin; patterns of variation, which included genetic similarity between South Carolina and Gulf of Mexico samples, were not aligned closely with geography. We examined the partitioning of genetic variation among groups by using analyses of molecular variance and found no evidence that North Carolina Southern Flounder, which are managed on the state level as a unit stock, are differentiated from the remainder of U.S. South Atlantic Southern Flounder. Our ﬁndings indicate only weak structure and the potential for basinwide mixing among Atlantic Southern Flounder, suggesting that cooperation among U.S. South Atlantic states will be essential for the effective assessment of stock dynamics and future management plans. Accurate identiﬁcation of stock structure is essential for effective ﬁsheries management but remains a challenge for both managers and scientists (Begg et al. 1999; NCDMF 2005, 2013). Examination of genetic variation across a spe- cies’ range is a commonly used approach for deﬁning interbreeding populations, and recent advances in molecular techniques have contributed to an expanded interest in the ﬁeld of ﬁsheries genetics (Ward 2000; Hauser and Carvalho 2008; Waples et al. 2008). Downloaded From: https://bioone.org/journals/Marine-and-Coastal-Fisheries:-Dynamics,-Management,-and-Ecosystem-Science on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use METHODS et al. 2007; Striped Mullet Mugil cephalus: Liu et al. 2009). For comparison with a more established method, we also sequenced a fragment of the mtDNA control region, which is a commonly used molecular marker. The control region has been identiﬁed as highly variable in teleosts (Lee et al. 1995) and was previously used by Anderson et al. (2012) to examine Southern Flounder population structure. et al. 2007; Striped Mullet Mugil cephalus: Liu et al. 2009). For comparison with a more established method, we also sequenced a fragment of the mtDNA control region, which is a commonly used molecular marker. The control region has been identiﬁed as highly variable in teleosts (Lee et al. 1995) and was previously used by Anderson et al. (2012) to examine Southern Flounder population structure. Southern Flounder tissue samples were collected from mul- tiple estuarine sites across four states (NC, South Carolina [SC], Georgia [GA], and FL) spanning the current range of the species in the Atlantic (Figure 1). North Carolina was subdi- vided into three regions (north [NCN], central [NCC], and south [NCS]) to examine genetic population structure within state waters. Samples were obtained from both ﬁshery-inde- pendent and ﬁshery-dependent sources in partnership with state ﬁshery management agencies and commercial ﬁshers within each state and were also obtained from licensed seafood dealers. Sampling was conducted from 2010 to 2013, and sam- pled ﬁsh ranged from age 0 to age 3 (96% were age 0–1), with an average TL of 360 mm (range D 166–622 mm). Additional tissue samples, provided by J. D. Anderson (Texas Parks and Wildlife Department), were collected from two sites (Fig- ure 1) in the Gulf (Apalachicola, FL [APFL]; and San Antonio Bay, Texas [SBTX]). Sampling methods for Gulf collections are described by Anderson et al. (2012). Fin clips and gill tis- sue were excised and preserved in either a salt-saturated dimethyl sulfoxide solution (Atlantic samples) or a 70% In this study, our main objective was to genotype Southern Flounder individuals from across the species’ range by using both AFLPs and mtDNA to address whether distinct genetic populations are present within the Atlantic, particularly whether Southern Flounder in NC represent a unique stock. Abstract Although previously underutilized in studies of animals (reviewed by Bensch and A kesson 2005), AFLPs have become increasingly common in studies of aquatic populations, including commercially important ﬁsh and invertebrate species both in aquaculture (e.g., Channel Catﬁsh Ictalurus punctatus: Mickett et al. 2003; Olive Floun- der Paralichthys olivaceus: Liu et al. 2005) and in natural pop- ulations (e.g., common shrimp Crangon crangon: Weetman Mature Southern Flounder inhabiting estuarine waters migrate offshore annually to spawn during the late-fall and winter months, with recruitment of larvae into estuarine nurs- ery grounds occurring in late winter and early spring (Wenner et al. 1990). Adults typically return to estuarine and nearshore coastal waters after spawning (Monaghan 1996), but recent studies have found that Southern Flounder migration patterns are more variable, as some adults remain offshore through the late-spring and summer months (Watterson and Alexander 2004; Taylor et al. 2008). In addition, previous tagging studies indicate that most individuals display limited movement when occupying estuarine habitats and remain in close proximity to their tagging sites (Wenner et al. 1990; Monaghan 1996; Craig and Rice 2008). Based mainly on the estuarine tagging results, the current ﬁsheries management plan considers Southern Flounder in NC to be a unit stock (NCDMF 2013). However, there is also evidence of extensive southward movement, as some individuals tagged in NC estuaries were found in other 222 WANG ET AL. Downloaded From: https://bioone.org/journals/Marine-and-Coastal-Fisheries:-Dynamics,-Management,-and-Ecosystem-Science on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use METHODS In this AFLP proﬁle optimi- zation procedure, loci with an average peak height below 200 rfu are eliminated from analysis, and presence/absence scoring is based on a threshold of 20% of the average peak height for each locus. The complete AFLP proﬁles (i.e., prior to optimi- zation) were also considered in this analysis but provided simi- lar results and are not reported here. STRUCTURE version 2.3.4 (Pritchard et al. 2000) was used to assign individual Southern Flounder to an inferred number of genetically distinct clusters (K) present in the AFLP data set. Because AFLPs are dominant markers that are scored as the presence or absence of peaks, there is no distinc- tion between homozygous and heterozygous states. To account for this ambiguity, we implemented the model by Falush et al. (2007) for analyzing dominant markers in STRUCTURE. A recessive AFLP proﬁle (consisting of all peak absences) was input into the data set, and the model evaluated all possible genotypes (i.e., for a peak presence phenotype, the genotypes presence/presence and presence/absence are both considered). Based on previous studies that demonstrated a lack of structure among Southern Flounder populations within ocean basins (Anderson and Karel 2012; Anderson et al. 2012), we assumed that populations were admixed and that allele frequencies across populations were correlated. We did not provide the geographic origin of samples a priori. Five replicates for each K-value from 1 to 8 were tested, with 10,000 burn-in iterations followed by 50,000 Markov-chain Monte Carlo iterations. STRUCTURE HARVESTER (Earl and vonHoldt 2012) was used to implement the Evanno et al. (2005) method for identi- fying the most likely number of clusters (i.e., K) in a data set. All replicates from each K-value identiﬁed by this method were then combined using the “FullSearch” algorithm in CLUMPP version 1.1.2 (Jakobsson and Rosenberg 2007). DISTRUCT version 1.1 (Rosenberg 2004) was used to convert CLUMPP outputs into bar plots that showed each individual’s probability of assignment to the genetic clusters. We used Arlequin version 3.5.1.2 (Excofﬁer and Lischer 2010) to compute the genetic differentiation index FST between all pairs of geographic populations for both AFLP and mtDNA data sets. We applied a Bonferroni adjustment to correct for multiple FST comparisons by using software from Lesack and Naugler (2011). Arlequin was also used to conduct analyses of molecular variance (AMOVAs; Excofﬁer et al. 1992). METHODS Control region sequences were deposited in GenBank under accession numbers KP331548–KP331677. forward direction (L-PROF) with ABI BigDye Terminator version 3.1. Sequencing was conducted on the same equip- ment as described for AFLPs. Sequences were edited, trimmed, and aligned manually in Sequencher version 4.10.1 (Gene Codes Corporation, Ann Arbor, Michigan). Control region sequences were deposited in GenBank under accession numbers KP331548–KP331677. forward direction (L-PROF) with ABI BigDye Terminator version 3.1. Sequencing was conducted on the same equip- ment as described for AFLPs. Sequences were edited, trimmed, and aligned manually in Sequencher version 4.10.1 (Gene Codes Corporation, Ann Arbor, Michigan). Control region sequences were deposited in GenBank under accession numbers KP331548–KP331677. solution of ethanol (Gulf samples) and were stored at 4C. Genomic DNA was extracted from ﬁn clips by using the DNeasy Blood and Tissue Kit (Qiagen, Valencia, California) in accordance with the manufacturer’s protocols. Gill tissue was extracted only occasionally, when ﬁn clips were damaged or not readily available. y The AFLPs were generated by using the AFLP Plant Map- ping Protocol (Applied Biosystems, Inc. [ABI], Foster City, California) for regular plant genomes. We produced DNA ﬁn- gerprints for 12 selective primer pairs (chosen from an earlier screen of 24 primer pairs): EcoRI-ACA and MseI-CAA; EcoRI-ACC and MseI-CAC; EcoRI-ACC and MseI-CAG; EcoRI-ACC and MseI-CAT; EcoRI-ACT and MseI-CAA; EcoRI-ACT and MseI-CAC; EcoRI-ACT and MseI-CAT; EcoRI-ACT and MseI-CTC; EcoRI-AGC and MseI-CAA; EcoRI-AGC and MseI-CTT; EcoRI-AGG and MseI-CTA; and EcoRI-AGG and MseI-CTG. Fragments were sized along with GeneScan 500 ROX size standard (Life Technologies, Carls- bad, California) on an ABI 3130XL Genetic Analyzer at the DNA Analysis Core Facility (Center for Marine Science, Uni- versity of North Carolina, Wilmington). To test for reproduc- ibility of AFLP ﬁngerprints, one individual from each sampling location was subjected to repeat analysis across all 12 selective primer pairs beginning from genomic DNA. Frag- ments between 75–500 bp were scored automatically for pres- ence/absence with a default peak calling threshold of 50 relative ﬂuorescence units (rfu) by using ABI GeneMapper version 4.0. Peak heights were normalized across all samples for each primer pair, with only polymorphic loci considered. To further reduce noise and genotyping error, the individual loci in the AFLP proﬁles were ﬁltered to a locus selection threshold of 200 rfu and a phenotype calling threshold of 20% following Whitlock et al. (2008). Downloaded From: https://bioone.org/journals/Marine-and-Coastal-Fisheries:-Dynamics,-Management,-and-Ecosystem-Science on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use METHODS As evidenced by previous studies, we expected divergence between Gulf and Atlantic populations, but we attempted to determine whether ﬁner structure exists within ocean basins and whether this structure alters our assumptions regarding the level of mixing and warrants a change in the spatial scale of management. In addition, we assessed the viability of the AFLP method in comparison with more traditional mtDNA sequencing for determining stock structure in a widely distrib- uted marine species. FIGURE 1. Map of Southern Flounder sampling locations in the U.S. South Atlantic and the Gulf of Mexico. Atlantic locations are represented here on a state scale, although multiple sites were sampled within each state (NC D North Carolina; SC D South Carolina; GA D Georgia; FL D Florida). North Carolina was subdivided into three regions (separated by dashed lines; NCN D NC north [Albemarle Sound]; NCC D NC central [Neuse River–Pamlico River estuary]; NCS D NC south [New River and Cape Fear River estuaries]). Gulf of Mexico samples originated from two speciﬁc locations (black circles; APFL D Apalachi- cola, Florida; SBTX D San Antonio Bay, Texas). Sample size (n) for ampliﬁed fragment length polymorphisms is shown in bold; n for mitochondrial DNA con- trol region sequences is shown in italics. FIGURE 1. Map of Southern Flounder sampling locations in the U.S. South Atlantic and the Gulf of Mexico. Atlantic locations are represented here on a state scale, although multiple sites were sampled within each state (NC D North Carolina; SC D South Carolina; GA D Georgia; FL D Florida). North Carolina was subdivided into three regions (separated by dashed lines; NCN D NC north [Albemarle Sound]; NCC D NC central [Neuse River–Pamlico River estuary]; NCS D NC south [New River and Cape Fear River estuaries]). Gulf of Mexico samples originated from two speciﬁc locations (black circles; APFL D Apalachi- cola, Florida; SBTX D San Antonio Bay, Texas). Sample size (n) for ampliﬁed fragment length polymorphisms is shown in bold; n for mitochondrial DNA con- trol region sequences is shown in italics. one.org/journals/Marine-and-Coastal-Fisheries:-Dynamics,-Management,-and-Ecosystem-Science on 23 Oct 2024 org/terms-of-use m: https://bioone.org/journals/Marine-and-Coastal-Fisheries:-Dynamics,-Management,-and-Ecosystem-Science on 23 Oct 2024 tps://bioone.org/terms-of-use GENETIC STRUCTURE OF SOUTHERN FLOUNDER 223 forward direction (L-PROF) with ABI BigDye Terminator version 3.1. Sequencing was conducted on the same equip- ment as described for AFLPs. Sequences were edited, trimmed, and aligned manually in Sequencher version 4.10.1 (Gene Codes Corporation, Ann Arbor, Michigan). RESULTS When scored for all 201 samples across the 12 primer pair combinations, 671 polymorphic AFLP loci were identiﬁed. In the eight individuals for which the AFLP procedure was repeated, the AFLP proﬁles were 98.8% reproducible across all loci. Using the method of Evanno et al. (2005) to interpret the AFLP results from STRUCTURE, we identiﬁed a strong peak in DK at a K-value of 2 and a smaller but distinct second- ary peak in DK at a K-value of 5. At a K of 2, we observed that each geographic population showed strong membership to one of two distinct genetic clusters representing either the Gulf or the Atlantic (Figure 2). In addition, the Atlantic samples from NCS and southward, particularly SC, displayed partial assign- ment to the genetic cluster dominating the Gulf locales. At a K of 5, the assignments remained similar to those produced at a K-value of 2, but SC ancestry was instead shared speciﬁcally with the western Gulf, and NCS samples displayed some shared assignment with the major genetic cluster found in the Gulf. Mitochondrial DNA control region phylogeny in Southern Flounder was inferred with NETWORK version 4.6.1.2 (Fluxus Engineering; www.ﬂuxus-engineering.com) by using the median joining method (Bandelt et al. 1999) to generate a haplotype network. Network branching patterns were postpro- cessed with maximum parsimony (Polzin and Daneshmand 2003). The mtDNA control region nucleotide composition and pairwise differences were calculated using MEGA version 6 (Tamura et al. 2013). Pairwise differences for the AFLP data set were also determined with MEGA. DnaSP version 5.10 (Librado and Rozas 2009) was used to assign mtDNA control region haplotypes and to calculate molecular diversity indices and Tajima’s D-statistic (Tajima 1989). Sites with gaps were not considered for diversity calculations but were included in all other analyses. Because Southern Flounder from SC unexpectedly showed common ancestry with those sampled in the western Gulf (SBTX), we examined population pairwise distances for indi- vidual AFLP primer pairs in an attempt to identify the source of these similarities. For each primer pair, we compared FIGURE 2. Population structure for Southern Flounder as estimated with ampliﬁed fragment length polymorphisms (AFLPs). Each individual is represented as a vertical line along the x-axis, and individuals are grouped into geographic populations. METHODS An AMOVA examines the partitioning of genetic dif- ferentiation within and among geographical subpopulations. We used this framework to test two different regional group- ings for both AFLPs and mtDNA: (1) ocean-basin-scale struc- ture (Gulf versus Atlantic) and (2) structure within the Atlantic (NCN, NCC, and NCS versus SC, GA, and FL). To further investigate the patterns observed from our STRUCTURE results, we also considered a third grouping for AMOVA: subdivision within NC (NCN and NCC versus NCS, SC, GA, and FL). For all analyses conducted in Arle- quin, AFLPs were coded as haplotypic data. Arlequin does not A fragment of the mtDNA control region was ampliﬁed by using the primers L-PROF (50-AACTCTCACCCC- TAGCTCCCAAAG-30; Meyer et al. 1994) and PL526H (50- AAAAGAGAACCCCTTACCCG-30), the latter of which is a Southern Flounder-speciﬁc primer designed by one of us (M. A. McCartney, unpublished). The PCR was conducted as fol- lows: an initial denaturation step of 94C for 1 min, followed by 35 ampliﬁcation cycles (94C for 1 min; 58C for 30 s; and 72C for 2 min) and a ﬁnal extension of 72C for 5 min. Each 25-mL reaction volume contained 1.25 units of MyTaq DNA Polymerase (Bioline, London, UK), 1 £ MyTaq Red Reaction Buffer (Bioline), 0.5 mM of each primer, and 1 mL of genomic DNA (5–900 ng). The PCR products were puriﬁed by using a StratPrep PCR Puriﬁcation Kit (Agilent Technolo- gies, Santa Clara, California) and were cycle sequenced in the Downloaded From: https://bioone.org/journals/Marine-and-Coastal-Fisheries:-Dynamics,-Management,-and-Ecosystem-Science on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use 224 WANG ET AL. account for dominant marker systems, so the calculated ﬁxa- tion indices for AFLP data are based on peak phenotype (pres- ence/absence) frequencies rather than allele frequencies. Despite this, Arlequin is often used for analysis of AFLPs (Svensson et al. 2004; Bensch and A kesson 2005; Timmer- mans et al. 2005; Parchman et al. 2006; Toews and Irwin 2008); the degree of differentiation and their signiﬁcance val- ues are meaningful, but direct comparisons with FST values from co-dominant data sets should be avoided. RESULTS Subsequently, we conducted a second STRUCTURE analysis of the AFLP data set by using the same methods as previously described, except that we excluded the 38 loci from the primer pair EcoRI-AGC–MseI-CTT. A strong peak in DK was identiﬁed at a K of 2, and a smaller but distinct peak was observed at a K of 4. At the K-value of 2, the pronounced simi- larity between SC and SBTX was no longer present, but at the K of 4, a shared partial assignment among SC samples and Gulf samples was again detected (Figure 2). Overall patterns of genetic structure were similar to those observed prior to removal of the EcoRI-AGC–MseI-CTT primer pair from the AFLP data set. (1) the average pairwise distance among geographic popula- tion pairs that included either SC or SBTX to (2) the average pairwise distance among all other geographic population pairs (i.e., those without SC or SBTX). The percent difference between these two metrics was used to assess the unusual lev- els of genetic differentiation in SC and the western Gulf. The average percent difference was 6.09% (SD D 5.93) across 11 of the 12 primer pairs. The remaining primer pair (EcoRI- AGC–MseI-CTT) displayed higher levels of differentiation, with a percent difference of 56.77%, and was likely responsi- ble for the similarities observed between SC and SBTX sam- ples. Subsequently, we conducted a second STRUCTURE analysis of the AFLP data set by using the same methods as previously described, except that we excluded the 38 loci from the primer pair EcoRI-AGC–MseI-CTT. A strong peak in DK was identiﬁed at a K of 2, and a smaller but distinct peak was observed at a K of 4. At the K-value of 2, the pronounced simi- larity between SC and SBTX was no longer present, but at the K of 4, a shared partial assignment among SC samples and Gulf samples was again detected (Figure 2). Overall patterns of genetic structure were similar to those observed prior to removal of the EcoRI-AGC–MseI-CTT primer pair from the AFLP data set. Across 260 samples, the ﬁnal mtDNA control region align- ment for Southern Flounder was 480 bp, with 88 segregating sites and a total of 130 unique haplotypes. This included a sin- gle-base-pair insertion/deletion that was present in two haplo- types, each composed of one individual from different sites. RESULTS Excluding sites with gaps, overall haplotype diversity was 0.977 (SD D 0.0036) and nucleotide diversity was 0.011 (SD D 0.0003), with an average of 5.06 nucleotide changes between individuals (Table 3). Tajima’s D-statistic was esti- mated to test for neutrality of nucleotide substitutions; the val- ues were signiﬁcantly negative for both ocean basins (Atlantic: D D ¡2.08, P < 0.05; Gulf: D D ¡1.97, P < 0.05), which may be indicative of Southern Flounder population expansion (Fu 1997). Comparisons of FST for Southern Flounder AFLPs showed signiﬁcant differentiation between all but four pairs of geo- graphic populations (FST D 0.020–0.133, P < 0.01; Table 1). The four population pairs that were not signiﬁcantly differenti- ated after Bonferroni adjustment were all within-Atlantic com- parisons (NCN versus GA; NCN versus FL; NCC versus FL; and GA versus FL). In general, FST values for Atlantic versus Gulf population pairs were larger than within-basin compari- sons, although FST values for SC versus NCC and for SC ver- sus NCS were comparable to between-basin values. When comparing Southern Flounder from the Gulf and Atlantic basins, AMOVA identiﬁed signiﬁcant differences at all levels Twenty-nine mtDNA control region haplotypes were com- mon among individual Southern Flounder, and three of the haplotypes were shared between the Gulf and Atlantic. The majority of the haplotypes were singletons, most of which dif- fered from common haplotypes by only one or two nucleotide substitutions. The control region haplotype network showed a pattern of divergence between Southern Flounder in the two ocean basins, although separation was not complete (Figure 3). The majority of Gulf haplotypes were separated from Atlantic haplotypes by at least three nucleotide changes, but there were Atlantic haplotypes present in the Gulf cluster TABLE 1. Population pairwise FST comparisons based on ampliﬁed fragment length polymorphism proﬁles (above diagonal) and mitochondrial DNA control region haplotypes (below diagonal) from Southern Flounder sampled in the U.S. South Atlantic (NCN D North Carolina north; NCC D North Carolina central; NCS D North Carolina south; SC D South Carolina; GA D Georgia; FL D Florida) and in the Gulf of Mexico (APFL D Apalachicola, Florida; SBTX D San Antonio Bay, Texas). Values without superscript letters were not signiﬁcant. TABLE 1. Population pairwise FST comparisons based on ampliﬁed fragment length polymorphism proﬁles (above diagonal) and mitochondrial DNA control region haplotypes (below diagonal) from Southern Flounder sampled in the U.S. South Atlantic (NCN D North Carolina north; NCC D North Carolina central; NCS D North Carolina south; SC D South Carolina; GA D Georgia; FL D Florida) and in the Gulf of Mexico (APFL D Apalachicola, Florida; SBTX D San Antonio Bay, Texas). Values without superscript letters were not signiﬁcant. RESULTS Each color represents a different inferred genetic cluster, and the proba- bility of assignment to each of the K genetic clusters is indicated by the proportion of color along the y-axis: (A) K-value of 2 and (B) K-value of 5. Results for the AFLP data set after removing the primer pair EcoRI-AGC–MseI-CTT are also presented: (C) K-value of 2 and (D) K-value of 4. Location codes are deﬁned in Figure 1. FIGURE 2. Population structure for Southern Flounder as estimated with ampliﬁed fragment length polymorphisms (AFLPs). Each individual is represented as a vertical line along the x-axis, and individuals are grouped into geographic populations. Each color represents a different inferred genetic cluster, and the proba- bility of assignment to each of the K genetic clusters is indicated by the proportion of color along the y-axis: (A) K-value of 2 and (B) K-value of 5. Results for the AFLP data set after removing the primer pair EcoRI-AGC–MseI-CTT are also presented: (C) K-value of 2 and (D) K-value of 4. Location codes are deﬁned in Figure 1. m: https://bioone.org/journals/Marine-and-Coastal-Fisheries:-Dynamics,-Management,-and-Ecosystem-Science on 23 Oct 2024 tps://bioone.org/terms-of-use GENETIC STRUCTURE OF SOUTHERN FLOUNDER 225 of hierarchical population structure (Table 2). However, within-population variation accounted for 89.9% of the differ- ences (FST D 0.101, P < 0.0001). When partitioning differen- tiation in the Atlantic alone (NCN, NCC, and NCS versus SC, GA, and FL), 96.6% of the variation was due to within-popula- tion differences (FST D 0.034, P < 0.0001; Table 2). There was no differentiation between these two Atlantic regions. There was also no differentiation between regions when con- sidering NCN and NCC as a separate group relative to NCS, SC, GA, and FL. (1) the average pairwise distance among geographic popula- tion pairs that included either SC or SBTX to (2) the average pairwise distance among all other geographic population pairs (i.e., those without SC or SBTX). The percent difference between these two metrics was used to assess the unusual lev- els of genetic differentiation in SC and the western Gulf. The average percent difference was 6.09% (SD D 5.93) across 11 of the 12 primer pairs. The remaining primer pair (EcoRI- AGC–MseI-CTT) displayed higher levels of differentiation, with a percent difference of 56.77%, and was likely responsi- ble for the similarities observed between SC and SBTX sam- ples. Downloaded From: https://bioone.org/journals/Marine-and-Coastal-Fisheries:-Dynamics,-Management,-and-Ecosystem-Science on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use dNot signiﬁcant after Bonferroni correction. RESULTS Sample NCN NCC NCS SC GA FL APFL SBTX NCN 0.0218c 0.0436c 0.0506c 0.0122bd 0.0062 0.0969c 0.1133c NCC 0.0049 0.0519c 0.0724c 0.0199b 0.0133bd 0.1119c 0.1330c NCS 0.0052 ¡0.0002 0.0722c 0.0215c 0.0314c 0.0652c 0.1054c SC 0.0369ad ¡0.0079 0.0231 0.0526c 0.0420c 0.1037c 0.0694c GA 0.0521ad 0.0102 0.0367ad ¡0.0074 0.0099bd 0.0771c 0.1105c FL 0.0215 ¡0.0095 ¡0.0029 ¡0.0036 0.0003 0.0869c 0.1100c APFL 0.4234c 0.3993c 0.4558c 0.3841c 0.3962c 0.4165c 0.0304c SBTX 0.3993c 0.3736c 0.4368c 0.3582c 0.3715c 0.3949c 0.0058 aP < 0.05. bP < 0.01. cP < 0.0001. dNot signiﬁcant after Bonferroni correction. Downloaded From: https://bioone.org/journals/Marine-and-Coastal-Fisheries:-Dynamics,-Management,-and-Ecosystem-Science on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use one.org/journals/Marine-and-Coastal-Fisheries:-Dynamics,-Management,-and-Ecosystem-Science on 23 Oct 2024 org/terms-of-use and vice versa. There was no clear pattern o ture within the Atlantic cluster based o network. Pairwise population comparisons using th region showed strong differentiation (FST D 0.0001) between all pairs of populations basins (Table 1). Three pairs of populations (GA and NCS; GA and NCN; and SC and N niﬁcantly differentiated, although FST va smaller (FST D 0.037–0.052, P < 0.05). Non Atlantic comparisons remained signiﬁcant correction was applied, and no other pairs of TABLE 3. Molecular diversity measures for populati der sequenced at the mitochondrial DNA control regio gaps). Number of samples (n), number of polymorphi (Np), number of haplotypes (Nh), haplotype diversity diversity (p) are given for each site. Location codes are Location n Np Nh U.S. South Atlantic NCN 30 28 19 NCC 33 30 25 NCS 34 26 22 SC 31 31 25 GA 31 28 23 FL 30 28 23 Overall (Atlantic) 189 70 93 Gulf of Mexico APFL 36 35 25 SBTX 35 28 19 Overall (Gulf) 71 47 39 Total (Atlantic plus Gulf) 260 87 129 TABLE 2. Results from analyses of molecular varian two different geographic scales: (1) ocean basin (Gulf o Georgia, and Florida; P < 0.05; P < 0.0001). Source of variation df S Among groups 1 Among populations within groups 6 Within populations 193 Total 200 Among groups 1 Among populations within groups 4 Within populations 145 Total 150 226 226 WANG ET AL. TABLE 2. Results from analyses of molecular variance based on ampliﬁed fragment length polymorphisms in Southern Flounder. Populations are grouped at two different geographic scales: (1) ocean basin (Gulf of Mexico versus U.S. South Atlantic) and (2) U.S. South Atlantic (North Carolina versus South Carolina, Georgia, and Florida; P < 0.05; *P < 0.0001). TABLE 2. RESULTS Within the Atlantic, almost all of the variation was explained by differences within populations (98.2%; FST D 0.018, P > 0.05), and only a small portion of the variation was explained by differences between NC and the other Atlantic populations (1.8%; FCT D 0.018, P > 0.05), but neither of these differences was signiﬁcant (Table 4). and vice versa. There was no clear pattern of ﬁner-scale struc- ture within the Atlantic cluster based on the haplotype network. Pairwise population comparisons using the mtDNA control region showed strong differentiation (FST D 0.37–0.45, P < 0.0001) between all pairs of populations compared across basins (Table 1). Three pairs of populations within the Atlantic (GA and NCS; GA and NCN; and SC and NCN) were also sig- niﬁcantly differentiated, although FST values were much smaller (FST D 0.037–0.052, P < 0.05). None of these within- Atlantic comparisons remained signiﬁcant after a Bonferroni correction was applied, and no other pairs of populations within TABLE 3. Molecular diversity measures for populations of Southern Floun- der sequenced at the mitochondrial DNA control region (excluding sites with gaps). Number of samples (n), number of polymorphic sites excluding gaps (Np), number of haplotypes (Nh), haplotype diversity (Hd), and nucleotide diversity (p) are given for each site. Location codes are deﬁned in Table 1. RESULTS Results from analyses of molecular variance based on ampliﬁed fragment length polymorphisms in Southern Flounder. Populations are grouped at two different geographic scales: (1) ocean basin (Gulf of Mexico versus U.S. South Atlantic) and (2) U.S. South Atlantic (North Carolina versus South Carolina, Georgia, and Florida; P < 0.05; *P < 0.0001). Source of variation df Sum of squares Variance component Percentage of variation Fixation index Ocean basin scale Among groups 1 401.326 4.042 6.98 FCT D 0.0698 Among populations within groups 6 587.121 1.822 3.15 FSC D 0.0338 Within populations 193 10,045.97 52.052 89.88 FST D 0.1013 Total 200 11,034.42 57.916 U.S. South Atlantic Among groups 1 87.036 ¡0.179 ¡0.34 FCT D ¡0.0034 Among populations within groups 4 402.025 1.964 3.71 FSC D 0.0370 Within populations 145 7,407.171 51.084 96.62 FST D 0.0338 Total 150 7,896.232 52.869 df Sum of squares Variance component Percentage of variation Fixation index the Atlantic or Gulf were found to be signiﬁcantly differentiated with any test. When comparing the Gulf and Atlantic, the AMOVA for the mtDNA control region showed that the major- ity of variation was explained by differences within populations (59.3%; FST D 0.407, P < 0.0001), although a large portion of the differentiation was due to differences between ocean basins (40.2%; FCT [genetic differences among groups] D 0.402, P < 0.05; Table 4). Within the Atlantic, almost all of the variation was explained by differences within populations (98.2%; FST D 0.018, P > 0.05), and only a small portion of the variation was explained by differences between NC and the other Atlantic populations (1.8%; FCT D 0.018, P > 0.05), but neither of these differences was signiﬁcant (Table 4). the Atlantic or Gulf were found to be signiﬁcantly differentiated with any test. When comparing the Gulf and Atlantic, the AMOVA for the mtDNA control region showed that the major- ity of variation was explained by differences within populations (59.3%; FST D 0.407, P < 0.0001), although a large portion of the differentiation was due to differences between ocean basins (40.2%; FCT [genetic differences among groups] D 0.402, P < 0.05; Table 4). DISCUSSION We observed divergence between Gulf and Atlantic South- ern Flounder populations based on both mtDNA control region sequences and AFLP ﬁngerprints. All population pairwise FST and AMOVA FCT values were signiﬁcant when comparing between ocean basins, regardless of the molecular marker used (Tables 1, 2, 4). The same result was observed with AFLP population assignments (Figure 2) and in the mtDNA haplotype network (Figure 3). These ﬁndings are in agreement with previous Southern Flounder studies that examined allo- zymes (Blandon et al. 2001), mtDNA (Anderson et al. 2012), and microsatellites (Anderson and Karel 2012), and they cor- respond with a well-known biogeographic break in marine fauna between the Gulf and Atlantic (Avise 1992). Southern Flounder are absent from the southern tip of Florida (Ginsburg 1952; Gilbert 1986), so a transition in genetic composition between ocean basins appears to correspond with the habitat discontinuity that is responsible for the disjunct distribution of Southern Flounder across south Florida. Location n Np Nh Hd p U.S. South Atlantic NCN 30 28 19 0.910 0.0082 NCC 33 30 25 0.981 0.0087 NCS 34 26 22 0.934 0.0073 SC 31 31 25 0.981 0.0085 GA 31 28 23 0.974 0.0079 FL 30 28 23 0.972 0.0080 Overall (Atlantic) 189 70 93 0.967 0.0082 Gulf of Mexico APFL 36 35 25 0.946 0.0094 SBTX 35 28 19 0.909 0.0080 Overall (Gulf) 71 47 39 0.931 0.0087 Total (Atlantic plus Gulf) 260 87 129 0.977 0.0106 We predicted that both markers would perform well for evaluating deep divergence, but we also wanted to assess Downloaded From: https://bioone.org/journals/Marine-and-Coastal-Fisheries:-Dynamics,-Management,-and-Ecosystem-Science on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use GENETIC STRUCTURE OF SOUTHERN FLOUNDER 227 FIGURE 3. Median joining network of mitochondrial DNA control region haplotypes identiﬁed in Southern Flounder. Circles represent haplotypes; colored wedges indicate geographic populations. Circles and wedges are proportional to the sample size, and the length of the connection is proportional to the number of nucleotide changes between haplotypes. Location codes are deﬁned in Figure 1. FIGURE 3. Median joining network of mitochondrial DNA control region haplotypes identiﬁed in Southern Flounder. Circles represent haplotypes; colored wedges indicate geographic populations. Circles and wedges are proportional to the sample size, and the length of the connection is proportional to the number of nucleotide changes between haplotypes. Location codes are deﬁned in Figure 1. Whitlock 2004). Downloaded From: https://bioone.org/journals/Marine-and-Coastal-Fisheries:-Dynamics,-Management,-and-Ecosystem-Science on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use DISCUSSION The opposite was seen for population comparisons within the Atlantic, as mtDNA differentiation values (FST D ¡0.010 to 0.052) were nonsigniﬁcant and lower than AFLP values (FST D 0.006–0.072), which showed signiﬁcant differ- entiation for nearly all within-Atlantic comparisons. If we were to interpret the degree of population structure as pro- posed by Wright (1978), genetic differentiation among South- ern Flounder populations when comparing ocean basins would be characterized as “very great” (mtDNA) or “moderate” (AFLPs), whereas comparisons within the Atlantic basin showed “little” to “moderate” genetic differentiation based on AFLPs. In this study, nearly all Southern Flounder geographic pop- ulations within the Atlantic were signiﬁcantly differentiated (after Bonferroni correction for multiple comparisons) when using the AFLP data set (Table 1), thus contradicting the mtDNA results. The power to detect differentiation increases with the number of loci, and even very small values of FST can become statistically signiﬁcant when enough markers are used (Ryman et al. 2006). We compared a single mtDNA marker to an AFLP proﬁle comprising 671 loci, so we expected increased resolution. However, the levels of within-Atlantic AFLP differentiation we detected are not considered low for marine populations. Even very weak but signiﬁcant levels of genetic differentiation can be biologically relevant; for exam- ple, Knutsen et al. (2011) used empirical mark–recapture data to conﬁrm the existence of distinct, localized populations of coastal Atlantic Cod Gadus morhua that produced low levels of genetic differentiation. Based on AFLP differentiation, genetic structure appears to be present among geographic pop- ulations of Southern Flounder in the Atlantic, although this does not necessarily mean that each locale represents a dis- crete stock. The levels of genetic differentiation detected in this study were consistent with previously reported FST values for South- ern Flounder mtDNA (Anderson et al. 2012), but we observed markedly higher differentiation using AFLPs than was previ- ously found with microsatellites (within-basin FST D ¡0.002 to 0.007; between-basin FST D 0.025–0.046; Anderson and Karel 2012). Similar discrepancies between markers have also been observed in recent studies of other species, for which dif- ferentiation was both higher (Mock et al. 2002; Whitehead et al. 2003; Gruenthal et al. 2007) and lower (Maguire et al. 2002) when comparing AFLPs to microsatellites. For analyses of highly variable multi-allelic loci, as in the Anderson et al. DISCUSSION Ampliﬁed fragment length polymorphism loci are scattered across the genome and may better reﬂect demographic, historical, and selective events for which inﬂuences on diversity patterns can vary across loci, so we expected to obtain increased genetic resolution with AFLP analysis. how AFLPs and mtDNA would compare when examining subtle structure, as we expected to see in the Atlantic Southern Flounder populations. Although mtDNA markers are commonly used in population genetic studies because of their high mutation rates and purportedly neutral evolu- tion, the mitochondrial genome is also haploid (maternally inherited, effectively as a single locus) and is very small in comparison with the nuclear genome. Thus, studies that rely solely on mtDNA markers may draw incomplete con- clusions when attempting to make inferences about organ- isms and populations as a whole (reviewed by Ballard and The AFLP and mtDNA results were generally in agreement when assessing Southern Flounder differentiation between the two ocean basins, but the results began to diverge when we examined structure within ocean basins. The magnitude of dif- ferentiation for pairwise comparisons between ocean basins Downloaded From: https://bioone.org/journals/Marine-and-Coastal-Fisheries:-Dynamics,-Management,-and-Ecosystem-Science on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use 228 WANG ET AL. TABLE 4. Results from analyses of molecular variance based on mitochondrial DNA control region sequences in Southern Flounder. Populations are grouped at two different geographic scales: (1) ocean basin (Gulf of Mexico versus U.S. South Atlantic) and (2) U.S. South Atlantic (North Carolina versus South Caro- lina, Georgia, and Florida; P < 0.05; P < 0.0001). lina, Georgia, and Florida; P < 0.05; *P < 0.0001). Source of variation df Sum of squares Variance component Percentage of variation Fixation index Ocean basin scale Among groups 1 141.313 1.344 40.15 FCT D 0.4016 Among populations within groups 6 15.532 0.019 0.56 FSC D 0.0094 Within populations 252 499.841 1.983 59.28 FST D 0.4072** Total 259 656.685 3.346 U.S. South Atlantic Among groups 1 5.263 0.035 1.78 FCT D 0.0178 Among populations within groups 4 7.755 0.000 ¡0.01 FSC D ¡0.0001 Within populations 183 356.284 1.947 98.24 FST D 0.0176 Total 188 369.302 1.982 df Sum of squares Variance component Percentage of variation Fixation index was much higher with mtDNA (FST D 0.358–0.456) than with AFLPs (FST D 0.065–0.133), although both were highly sig- niﬁcant. Downloaded From: https://bioone.org/journals/Marine-and-Coastal-Fisheries:-Dynamics,-Management,-and-Ecosystem-Science on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use DISCUSSION In contrast, SC was always highly signiﬁcantly differentiated from both Gulf locales in pairwise FST comparisons, whether based on mtDNA or AFLPs. With these varying results, the potential distinction of SC genotypes from other Atlantic samples remains inconclusive. The results of this study conﬁrmed a clear divergence between Gulf and Atlantic Southern Flounder. There was also evidence of genetic structure among geographic populations of Southern Flounder at a ﬁner spatial scale, which was only revealed upon analysis of AFLPs. We found intriguing pat- terns of differentiation and genetic clustering that did not cor- respond with obvious geographic patterns. However, there was no differentiation between samples from NC and those from the other Atlantic states when examining hierarchical population structure. Southern Flounder in NC are currently managed as a unit stock. The present molecular analysis sug- gests that a single-stock designation within NC should be ade- quate, as there is no clear evidence for additional subdivisions at a ﬁner spatial scale (i.e., between regions within the state). However, the potential for high levels of mixing throughout the Atlantic basin, as evidenced by the presence of only weak genetic structure within the basin, suggests that interstate cooperation will be necessary to achieve a comprehensive population assessment of Southern Flounder at an appropriate spatial scale. To sustain ﬁshery yields and meet conservation objectives for Southern Flounder, the spatial scale of stock assessment will need to be more closely aligned with the spa- tial scale of important ecological processes that determine stock dynamics. Our present ﬁndings suggest that these pro- cesses likely operate over broad spatial scales and that future By examining pairwise differences in AFLP proﬁles between geographic populations, we were able to identify a single primer pair (EcoRI-AGC–MseI-CTT) as the likely cause for the assignment of SBTX and SC individuals to the same genetic cluster. This was conﬁrmed by a genetic cluster analysis that excluded loci from the primer pair in question, although the similarity between SC and Gulf samples was still present to a smaller degree when the number of assumed genetic clusters was increased (Figure 2). Because one primer pair appears to be responsible for this grouping, it is possible that the pattern may be an artifact of the AFLP procedure. Errors in peak scoring are a common source of AFLP genotyp- ing error (Bonin et al. DISCUSSION The potential inﬂuence of AFLP homoplasy on genetic diversity estimates must be considered, but experimental quantiﬁcation of the degree of homoplasy is not within the scope of most AFLP studies (Meudt and Clarke 2007). In addition to evaluating patterns of differentiation, we also assessed the genetic composition of each Southern Flounder population. When sufﬁcient numbers of loci are used, AFLPs are highly accurate for population assignment tests and can be particularly informative for systems with weak genetic struc- ture (Campbell et al. 2003). We saw similar patterns in indi- vidual assignments to inferred genetic clusters (i.e., K) across the most likely values of K for the AFLP proﬁles (Figure 2). Although we examined patterns of variation at K-values of 2 and 5, the major genetic patterns remained the same. The two summary plots both showed a strong genetic distinction between Gulf and Atlantic Southern Flounder. Within NC, the NCN and NCC samples appeared to be homogeneous, whereas NCS displayed a small difference in assignment probabilities, suggesting that a mild barrier to gene ﬂow may exist between these regions. The most unexpected observation was the shared clustering of SC samples—obtained near the center of the Atlantic populations—with the Gulf samples, particularly those from SBTX. Alternatively, it may be possible for the geographically dis- junct individuals from SC and the Gulf to have similar molec- ular proﬁles based on shared adaptive traits. Like other genome-scale molecular data sets, AFLP loci are anonymous and consist mostly of neutral loci, but they often include loci that are linked to genes under selection (Luikart et al. 2003). To investigate any patterns of distribution that may be related to adaptive evolution, future studies would beneﬁt from a genome scan, which detects AFLP loci that show abnormally high among-population differentiation; such outliers are a sig- nature of selection (Beaumont and Balding 2004). Overall, the AFLP genetic cluster analyses suggested high gene ﬂow within the Atlantic, with the possible exception of SC. Of the three mtDNA haplotypes shared between ocean basins, one consisted of individuals from SC and both Gulf locales, although this was a low-frequency haplotype (Fig- ure 3). In addition, the SC samples were differentiated from one of the NC geographic populations in the mtDNA FST anal- ysis, but this subdivision was no longer signiﬁcant after Bon- ferroni adjustment. DISCUSSION (2012) study where there were up to 53 alleles per microsatel- lite locus, very high levels of within-population heterozygosity can result in reduced magnitudes of differentiation between populations (Hedrick 1999). Different molecular markers are subject to different evolutionary processes, which can affect levels of diversity within and between populations (reviewed by Zink and Barrowclough 2008; Toews and Brelsford 2012). Therefore, the lack of correspondence in the magnitude of dif- ferentiation estimated with the different markers used to assess Southern Flounder genetic structure thus far is not surprising. Waples et al. (2008) cautioned that statistical signiﬁcance is not always correlated with biological signiﬁcance and that the life history and biology of an organism must be taken into consideration when determining stocks. Thus far, we have no evidence that Southern Flounder from different locales within the Atlantic are biologically distinct. Based on otolith morpho- metric analysis, Midway et al. (2014) also detected only weak evidence for differentiation among Southern Flounder popula- tions within the Atlantic, including within NC. These results are consistent with our ﬁndings. Despite the possibility that many AFLP loci should allow for greater power in the detec- tion of population subdivision, AMOVA did not ﬁnd signiﬁ- cant genetic structure when comparing NC (or portions of NC) to the remaining Atlantic locales. The same lack of NC regional structure was found with AMOVA based on mtDNA. GENETIC STRUCTURE OF SOUTHERN FLOUNDER 229 These results do not support the consideration of NC Southern Flounder as a distinct stock within the Atlantic. AFLP loci. Equal-length DNA fragments are scored as a pres- ence on the same locus, regardless of whether the fragments are homologous in genomic origin. 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https://openalex.org/W2614798846	http://science-gate.com/IJAAS/Articles/2017-4-6/06 2017-4-6-pp.43-49.pdf	English	null	Numerical modeling of defective hybrid composite plates	International journal of advanced and applied sciences	2,017	cc-by	4,991	* Corresponding Author. Email Address: b.achour@uoh.edu.sa (B. Achour) https://doi.org/10.21833/ijaas.2017.06.006 2313-626X/© 2017 The Authors. Published by IASE. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) Belkacem Achour 1, , Djamel Ouinas 2, Mabrouk Touahmia 1, Mustapha Boukendakdji 1 1Department of Civil Engineering, College of Engineering, University of Hail, Hail, Saudi Arabia 2Department of Mechanical Engineering, Université Abdelhamid Ibn Badis de Mostaganem, Mostaganem, Algeria A B S T R A C T It is known that the optimization of the positioning of reinforcements within a composite material is likely to increase significantly the performance of a structure. Accordingly, buckling is the most critical failure mode for notched and non-stiffened plates under axial compressive stress. In this study, the buckling response of laminated plates made of hybrid composite materials with and without elliptical notches is analyzed using finite element methods. The plates are made of carbon/epoxy/aluminum and arranged in following ordered manner [Al/ (θ/-θ) /Al]. The resistance to buckling of the hybrid plates under uniaxial compression is examined according to fiber orientations, the orientation of the notch and finally the thickness of the aluminum layer in the composite material. The results showed that when the fibers are at 𝜃= 900 the amplification of the critical load of buckling in the hybrid notchless plate is of the order of 59% and 27.66% for a thickness of the aluminum layer tAl= 0.2mm and 0.127mm respectively. The elliptical notch oriented at 0° reduces the maximum load four times more than when oriented at 90o. Article history: Received 11 February 2017 Received in revised form 11 April 2017 Accepted 2 May 2017 Keywords: Elliptical notch Buckling Hybrid composites Finite element analysis Article history: Received 11 February 2017 Received in revised form 11 April 2017 Accepted 2 May 2017 Keywords: Elliptical notch Buckling Hybrid composites Finite element analysis Article history: Received 11 February 2017 Received in revised form 11 April 2017 Accepted 2 May 2017 Keywords: Elliptical notch Buckling Hybrid composites Finite element analysis © 2017 The Authors. Published by IASE. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). composites have proven to be very efficient in the fabrication of parts of primary structures, due to their performance, quality of lightness and form flexibility (Kweon et al., 2006). The design of this type of structures needs tools to model their sophisticated mechanical behavior, taking into account the specificities of these materials. The numerical methods and in particular the finite element methods are essential for sizing these complex composite structures. Currently, the analysis of the behavior of laminated plates always remains an open research problem, due to their complex behavior (Komur et al., 2010). International Journal of Advanced and Applied Sciences, 4(6) 2017, Pages: 43-49 International Journal of Advanced and Applied Sciences, 4(6) 2017, Pages: 43-49 Contents lists available at Science-Gate 2. Finite element model plates having a crack emanating from a circular notch. They indicated that the critical load of buckling reaches its maximum values when the fibers are oriented in an interval varying from 50° to 90°. Ouinas and Achour (2013) have studied the effect of the presence of an elliptical notch on the buckling of laminated boron/ epoxy plates arranged anti-symmetrically [(θ/ -θ)]. In this study, a thin square plate, 100mm x 100mm x 1.162mm, made of hybrid carbon/epoxy/ aluminum composite material is analyzed. The lower edge of the plate is fixed and the displacement on the upper edge is assumed to be free along the y-axis. The plate is subjected to a uniaxial compression in the vertical direction under the load per unit length 𝜎= 1𝑁/𝑚𝑚. The plate is numerically modeled using the finite element code ABAQUS 6.11. The finite element mesh involved a total of 33328 S8R quadrilateral and 397 STRI65 elements with a refined mesh at the vicinity of the notch as shown in Fig. 1 and Fig. 2. In this study the buckling of composite plates made of hybrid carbon/epoxy/ aluminum material with and without elliptical notches is analyzed using finite element code ABAQUS 6.11. The effects of the notch orientation with respect to x-axis, the orientation of plies and the thickness of the ply on the buckling load have been examined. Moreover, the effect of the thickness of the layer of aluminum has also been considered. Fig. 1: Geometric model of the plate in the presence of an elliptical notch Elliptical notch Fig. 1: Geometric model of the plate in the presence of an elliptical notch Fig. 2: Meshing of the plate in the presence of an elliptical notch Fig. 2: Meshing of the plate in the presence of an elliptical notch The plate is composed of eight plies, six of carbon/epoxy and two of aluminum alloy. The thickness of the carbon/epoxy layer is 0.127 mm and the fibers are symmetrically crossed in an orderly alternate manner according to an angle and   respectively (Fig. 3). The layers of the aluminum are outside of the plate and each has a thickness of 0.2mm. As shown in Fig. 3, several mesh sizes are used in the numerical calculation to examine the accuracy and the convergence of a test case chosen where the ratio of the elliptical notch b/a=3, and the orientation of the layers is [-45/45]3. 1. Introduction The use of materials having specific mechanical properties permits the sizing of lighter structures but equally even more efficient than the conventional ones. The composite materials in form of laminated plates or shells have found a growing use in many industrial applications. Moreover, composite materials reinforced with carbon fiber are now used more and more in many areas such as aeronautics, astronautics, pressurized vessels, pipes, ships and sports equipment due to their high resistance to fatigue and corrosion, therefore, a satisfactory durability. In addition, the minimization of the structural mass in the aeronautical industry leads directly to reducing the aircraft fuel consumption which constitutes an important technological challenge. Several studies of stability of laminated plates have been concentrated on the rectangular plates (Rhodes et al., 1984; Nemeth, 1988; Mroz, 2011; Reddy and Harish, 2014). It is known that the resistance to buckling of rectangular plates depends on the boundary conditions and the orientations of the folds (Hu and Lin, 1995). The thin walled composite structures which are widely used become unstable when they are subjected to mechanical or thermal loads leading to buckling. Accordingly, their behaviors in buckling are significant factors in safe and reliable design (Baba, 2007). Baba and Baltaci (2007) have performed a buckling analysis of a laminated composite rectangular plate having a circular hole at the center. Hamani et al. (2012) have studied the effect of fiber orientation on the critical load of buckling of symmetrical laminated composite The assessment of appropriate levels of security is required by the process of modern design. Particular attention is paid to the behavior to buckling which must be understood and predicted in order to determine effective models and the safe loading conditions. In industry, the laminated 43 Achour et al/ International Journal of Advanced and Applied Sciences, 4(6) 2017, Pages: 43-49 3. 1. Effect of the ply orientations in the presence of two different aluminum layers The buckling parameters calculated by the two types of finite elements shell elements S8R and S8R5 are compared as illustrated in Fig. 4. It is worth noting that the stability of the buckling parameter is marked when the size of the edge element was less than 2 mm, regardless of the type of element used S8R or S8R5. More precision is attained when the size of the element is taken equal to 0.4mm. The mechanical properties of the hybrid composite plate are given in Table 1. Fig. 5 shows the variation of the buckling critical load versus the angle of orientation of the plies where the presence of the aluminum layers is highlight. Two configurations have been analyzed; a carbon/epoxy composite plate and a hybrid composite one, having an outside aluminum layer 0.2 mm thick. Six composite layers are oriented at 𝜃 and −𝜃 without a geometric defect. The evolution of the critical load in first mode of buckling of the composite laminated plate as a function of the angle of the plies is clearly highlighted. It can be noticed that the buckling critical load remains quasi-constant in the range 0° to 30°. Beyond this declination, the load is increased to an asymptotic maximum value corresponding to the angle 𝜃= 900 The buckling critical load in the hybrid plate having an aluminum thickness layer tAl = 0.2mm is found to be more important than of the carbon/epoxy one. The amplification of N has been of the order of 59% when the fibers are at 𝜃= 900. For the hybrid plate having tAl =0.127mm, the amplification factor has reached 27.66 % which indicates that the buckling loads are sensitive when the ply angle is higher than 30°. Table 1: Mechanical properties of the hybrid composite plate Properties Aluminum 2024T3 Carbone/époxy 𝐸1(𝑀𝑃𝑎) 72.4 145 𝐸2(𝑀𝑃𝑎) - 10 𝑣12 0.33 0.25 𝑣23 - 0.25 𝐺12(𝑀𝑃𝑎) 27 7 𝐺13(𝑀𝑃𝑎) - 7 𝐺23(𝑀𝑃𝑎) - 3.7 Table 1: Mechanical properties of the hybrid composite In real applications, notches in composite plates could be of different forms according to the design needs. The shape of the notch has been assumed primarily to be an elliptical hole at the centre of the plate. Fig. 3. Results and discussion Fig. 3: Fiber orientation in the laminated plate 2. Finite element model The classical model theory of laminates is implemented using the shell elements S4R, S8R of the software Abaqus with the composite lay-up option. The linear elements and quadratic with reduced integration S4R and S8R are generally well suited for the calculation of semi-thick or thin shells. Fig. 2: Meshing of the plate in the presence of an elliptical notch 44 Achour et al/ International Journal of Advanced and Applied Sciences, 4(6) 2017, Pages: 43-49 between the major axis of the notch which is the x- axis by 0°, 15, 30, 45, 60, 75, and 90° as shown in Fig. 1. The dimensions of the main diameters of the ellipse are represented by a and b respectively. The ratio of the two geometric parameters of the elliptical notch is kept constant (a/b=1/3). The effect of the circular notch is also analyzed as a special case when a=b. In addition, particular attention has been paid to the number of alternate cross over plies of the laminated plate as well as the ply orientations. Fig. 3: Fiber orientation in the laminated plate 3. 1. Effect of the ply orientations in the presence of two different aluminum layers 5: Effect of the aluminum layer on the buckling critical load 0 10 20 30 40 50 60 70 80 90 0 10 20 30 40 50 Composite plate Hybrid plate (tAl=0,127mm) Hybrid plate (tAl=0,2mm) (kN) Cr N )  Angle ( Fig. 4: Effect of the element size on the convergence of NCR However, the effect of different positions for the elliptical notch on the buckling load is considered. These positions are represented by an angle Fig. 5: Effect of the aluminum layer on the buckling critical load The design of a laminated composite is based on the optimization, therefore the best fiber orientation of each layer should be found. Fig. 6 illustrates 10 buckling modes of a hybrid composite plate when the plies are oriented at 90°. It is clear that the 0 10 20 30 40 50 60 70 80 90 0 10 20 30 40 50 Composite plate Hybrid plate (tAl=0,127mm) Hybrid plate (tAl=0,2mm) (kN) Cr N )  Angle ( 0,4 0,8 1,2 1,6 2,0 2,4 2,8 3,2 3,6 4,0 4,4 4,8 5,2 33,502 33,509 33,516 33,523 33,530 33,537 33,544 Quadratic element S8R S8R5 [(45/-45)3], a/b=1/3 (kN) Cr N Size of element (mm) 0 10 20 30 40 50 60 70 80 90 0 10 20 30 40 50 Composite plate Hybrid plate (tAl=0,127mm) Hybrid plate (tAl=0,2mm) (kN) Cr N )  Angle ( Fig. 4: Effect of the element size on the convergence of NCR 0,4 0,8 1,2 1,6 2,0 2,4 2,8 3,2 3,6 4,0 4,4 4,8 5,2 33,502 33,509 33,516 33,523 33,530 33,537 33,544 Quadratic element S8R S8R5 [(45/-45)3], a/b=1/3 (kN) Cr N Size of element (mm) )  Angle ( Fig. 5: Effect of the aluminum layer on the buckling critical load Fig. 4: Effect of the element size on the convergence of NCR The design of a laminated composite is based on the optimization, therefore the best fiber orientation of each layer should be found. Fig. 6 illustrates 10 buckling modes of a hybrid composite plate when the plies are oriented at 90°. It is clear that the However, the effect of different positions for the elliptical notch on the buckling load is considered. These positions are represented by an angle 45 Achour et al/ International Journal of Advanced and Applied Sciences, 4(6) 2017, Pages: 43-49 buckling critical load increases with the buckling mode for the same ply orientations. sensitive. 3. 1. Effect of the ply orientations in the presence of two different aluminum layers The lowest values of the buckling critical load are obtained when the fibers are oriented perpendicular to the applied load 𝜃= 00 while the maximum values are obtained when the fibers are parallel to the applied load 𝜃= 900. The differences between their trends are quasi-constant. It is very clear that the buckling load of the plate is strongly affected by the stacking order of the composite laminates. Therefore, for the laminated plate to resist better to the buckling load, the greatest ply orientations should be oriented with regards to the x-axis. In addition, the effect of the orientation of the elliptical notch has been considered as well. It is to be noted, that in this case, the increase of the angle of declination of the notch with regards to the x axis lead to an increase in the difference between the buckling loads, and sensitive when the ply orientation is greater than 450 (𝜃> 450). 3.2. Influence of the ply orientation for different declinations on the buckling load 7: Effect of fiber orientation on the buckling load variation (kN) Cr N )  Angle ( 0 10 20 30 40 50 60 70 80 90 2,50 3,00 3,50 4,00 4,50 5,00 a/b=3, tA1=0,2mm Declination of the elliptical notch        0 10 20 30 40 50 60 70 80 90 2,0 3,0 4,0 5,0 6,0 7,0 8,0 9,0 a/b=3, tA1=0,2 mm Declination of the elliptical notch        )  Angle ( N* (kN) Cr N Fig. 7: Effect of fiber orientation on the buckling load variation Fig. 8: Effect of fibre orientation on the buckling load variation For a better illustration of this effect, the variation of a dimensionless reduction factor of the buckling load is shown in Fig. 8 as a function of the declination angle (𝜃). The reduction factor is defined by (Eq. 1): 3.2. Influence of the ply orientation for different declinations on the buckling load 8: Effect of fibre orientation on the buckling load variation 0 10 20 30 40 50 60 70 80 90 2,0 3,0 4,0 5,0 6,0 7,0 8,0 9,0 a/b=3, tA1=0,2 mm Declination of the elliptical notch        )  Angle ( N* (kN) Cr N )  Angle ( 0 10 20 30 40 50 60 70 80 90 2,50 3,00 3,50 4,00 4,50 5,00 a/b=3, tA1=0,2mm Declination of the elliptical notch        Achour et al/ International Journal of Advanced and Applied Sciences, 4(6) 2017, Pages: 43-49 f pp g (g) Mode 7: Ncr = 393,02 N/mm (h) Mode 8: Ncr = 422,22 N/mm (i) Mode 9: Ncr = 520,93 N/mm (j) Mode 10: Ncr = 584,75 N/mm Fig. 6: Different buckling modes of a hybrid composite notchless panel (case 𝜃= 900) (i) Mode 9: Ncr = 520,93 N/mm (i) Mode 9: Ncr = 520,93 N/mm (j) Mode 10: Ncr = 584,75 N/mm ( ) / (j) / Fig. 6: Different buckling modes of a hybrid composite notchless panel (case 𝜃= 900) ( ) / (j) / Fig. 6: Different buckling modes of a hybrid composite notchless panel (case 𝜃= 900) Fig. 7: Effect of fiber orientation on the buckling load variation (kN) Cr N )  Angle ( 0 10 20 30 40 50 60 70 80 90 2,50 3,00 3,50 4,00 4,50 5,00 a/b=3, tA1=0,2mm Declination of the elliptical notch        Fig. 7: Effect of fiber orientation on the buckling load variation Fig. 8: Effect of fibre orientation on the buckling load variation 0 10 20 30 40 50 60 70 80 90 2,0 3,0 4,0 5,0 6,0 7,0 8,0 9,0 a/b=3, tA1=0,2 mm Declination of the elliptical notch        )  Angle ( N* (kN) Cr N )  Angle ( 0 10 20 30 40 50 60 70 80 90 2,50 3,00 3,50 4,00 4,50 5,00 a/b=3, tA1=0,2mm Declination of the elliptical notch        Fig. 3.2. Influence of the ply orientation for different declinations on the buckling load Fig. 7 illustrates the influence of the angle of fiber orientation on the variation of the buckling load of a hybrid composite plate containing an elliptical notch whose major axes ratio is a/b=1/3. Thus, the variation of the declination angle of the elliptical notch with regard to the x-axis is highlighted. It is clear that the critical buckling load increases according to three stages as a function of the fiber angle of declination of the composite material. In the first stage, where 00 ≤𝜃≤300, the critical load has been found almost stable. However, in the second stage where 300 ≤𝜃≤700, the critical buckling is found to increase quadratic ally. Finally, in the last stage, where 𝜃≥700, the increase is found to be less e, where 𝜃≥700, the increase is found to be less (a) Mode 1: Ncr = 100 N/mm (b) Mode 2: Ncr = 128.28N/mm (c) Mode 3: Ncr = 203,08 N/mm (d) Mode 4: Ncr = 232,44 N/mm (e) Mode 5: Ncr= 176,6 N/mm (f) Mode 6: Ncr = 336,681 N/mm (a) Mode 1: Ncr = 100 N/mm (b) Mode 2: Ncr = 128.28N/mm (c) Mode 3: Ncr = 203,08 N/mm (d) Mode 4: Ncr = 232,44 N/mm (e) Mode 5: Ncr= 176,6 N/mm (f) Mode 6: Ncr = 336,681 N/mm (b) Mode 2: Ncr = 128.28N/mm (f) Mode 6: Ncr = 336,681 N/mm (e) Mode 5: Ncr= 176,6 N/mm (f) Mode 6: Ncr = 336,681 N/mm 46 Achour et al/ International Journal of Advanced and Applied Sciences, 4(6) 2017, Pages: 43-49 (g) Mode 7: Ncr = 393,02 N/mm (h) Mode 8: Ncr = 422,22 N/mm (i) Mode 9: Ncr = 520,93 N/mm (j) Mode 10: Ncr = 584,75 N/mm Fig. 6: Different buckling modes of a hybrid composite notchless panel (case 𝜃= 900) Fig. 7: Effect of fiber orientation on the buckling load variation For a better illustration of this effect, the Fig. 3.3. Influence of the aluminum layer thickness To examine the thickness effect of the aluminum layer and the position of the elliptical notch on the evolution of the critical load of the hybrid plate, three cases are considered. For the first case, the notch major axis is taken parallel to the x-axis; the second case, the notch minor axis is perpendicular to the y-direction and the third case, the notch major axis has a 45° declination with respect to the x-axis. The orientations of the composite plies were [Al/(0)6/Al], [Al/(45/-45)3/Al] and [Al/(90)6/Al]. The results obtained are represented in Fig. 9. 𝑁∗= 1 − 𝑁𝑤𝑛 𝑁𝑤𝑡𝑛 (1) 𝑁∗= 1 − 𝑁𝑤𝑛 𝑁𝑤𝑡𝑛 (1) where, 𝑁𝑤𝑛 and 𝑁𝑤𝑡𝑛 are the buckling loads for the hybrid plate with and without the elliptical notch respectively. From Fig. 8, the critical stress was found to be reduced by 2.8% when the elliptical notch was oriented at 90° and then moved to 8.5% for an orientation of 0°. The maximum reduction was obtained for an orientation of the fibers at 45°and this regardless of the orientation of the elliptical notch with respect to the applied load. It is clear that the critical buckling load increases exponentially with the increase of the thickness of the aluminum layer and this regardless of the orientation of the plies and the orientation of the 47 Achour et al/ International Journal of Advanced and Applied Sciences, 4(6) 2017, Pages: 43-49 elliptical notch. The increase of the aluminum layer from 0.127 mm to 0.8 mm increased the buckling critical load by 94.62%, 93.36% and 89.44% respectively for the [Al/ (0)6 /Al], [Al/ (45/-45)3 /Al] and [Al/ (90)6/ Al] plies when the notch orientation 𝜑= 00 Regarding the two other orientations, the increase is of the order of 94,624 %, 93,395% and 89.4% for 𝜑= 450 and 94,645 %, 93,435% and 90.026% for 𝜑= 900. It is worth noting that the effect of the notch declination on the critical load when the aluminum layer became important is not significant. Fig. 10 shows the thickness effect of the aluminum layer on the critical load amplification for the three previous cases. To illustrate this, the variation of a dimensionless reduction factor 𝑁∗ of the buckling load as a function of the thickness of the aluminum layer is drawn. 3.3. Influence of the aluminum layer thickness 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 4,20 4,30 4,40 4,50 4,60 4,70 4,80 4,90 5,00 5,10 5,20 5,30 5,40 5,50 l/(6Al l/(3Al l/(6Al Declination of the notch =45° Al Thickness t 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 6,60 6,70 6,80 6,90 7,00 7,10 7,20 7,30 7,40 7,50 7,60 7,70 7,80 7,90 8,00 l/(6Al l/(3Al l/(6Al Declination of the notch =0° Al Thickness t N* N* 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 6,60 6,70 6,80 6,90 7,00 7,10 7,20 7,30 7,40 7,50 7,60 7,70 7,80 7,90 8,00 l/(6Al l/(3Al l/(6Al Declination of the notch =0° Al Thickness t N* 48 when the aluminum layer became important is not significant. Fig. 9: Effect of the aluminum layer thickness on the critical load Fig. 10: Effect of the aluminum layer thickness on the buckling load amplification For the orientations of the plies at 0° and 45°, the reduction factor increased quadratically with the increase of the thickness of the aluminum layer up to 0.8mm. It reached respectively 7.4% and 7.6% in the presence of a notch oriented at 00. An inverse 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 2,60 2,80 3,00 3,20 3,40 3,60 3,80 4,00 4,20 l/(6Al l/(3Al l/(6Al Declination of the notch =90° Thickness tAl 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 4,20 4,30 4,40 4,50 4,60 4,70 4,80 4,90 5,00 5,10 5,20 5,30 5,40 5,50 l/(6Al l/(3Al l/(6Al Declination of the notch =45° Al Thickness t 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 6,60 6,70 6,80 6,90 7,00 7,10 7,20 7,30 7,40 7,50 7,60 7,70 l/(6Al l/(3Al l/(6Al Declination of the notch =0° Al Thickness t NCr (kN) Al Thickness t 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,00 4,38 8,76 13,15 17,53 21,91 26,29 30,67 l/(6Al l/(3Al l/(6Al Declination of the notch =90° (kN) Cr N Al Thickness t 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,00 4,38 8,76 13,15 17,53 21,91 26,29 30,67 l/(6Al l/(3Al l/(6Al Declination of the notch =45° 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,00 4,38 8,76 13,15 17,53 21,91 26,29 30,67 l/(3Al l/(3Al l/(93Al Declination of the notch =0° Al Thickness t (kN) Cr N N* N* N* 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,00 4,38 8,76 13,15 17,53 21,91 26,29 30,67 l/(3Al l/(3Al l/(93Al Declination of the notch =0° Al Thickness t (kN) Cr N Fig. 4. Conclusion Hamani N, Ouinas D, Benderdouche N, and Sahnoun M (2012). Buckling analyses of the antisymetrical composite laminate plate with a crack from circular notch. Advanced Materials Research, 365: 56-61. In this study the buckling response of a hybrid composite square plate in the presence of an elliptical notch is investigated. The effects of, the orientation angle of the elliptical notch, the notch geometry, the orientation of the plies of the composite material and the thickness of the aluminum layer on the variation of the buckling load are examined leading to the following findings: Hu HT and Lin BH (1995). Buckling optimization of symmetrically laminated rectangular plates with various geometry and end conditions. Composite Science and Technology, 55(3): 277– 285. Komur MA, Sen F, Atas A, and Arslan N (2010). Buckling analysis of laminated composite plates with an elliptical/circular cutout using FEM. Advances in Engineering Software, 41(2): 161–164.  The critical buckling load amplification in the notchless hybrid plate is of the order of 59% and 27.66% respectively for the thickness of the aluminum layer tAl= 0.2mm and 0.127mm, when the fibers are oriented at 𝜃= 900. Kweon JH, Jung JW, Kim TH, Choi JH, and Kim DH (2006). Failure of carbon composite-toaluminum joints with combined mechanical fastening and adhesive bonding. Composite Structures, 75(1): 192–198.  The buckling load increases exponentially with regard to the increase of the orientation angle of the composite material fibers. This increase is much faster and more important for angles 𝜃≥ 450. Mroz A (2011). Stability analysis of a plane, rectangular, boron- epoxy laminated plate basing on strength properties determined by different methods. Mechanics and Mechanical Engineering, 15(2): 161-181. Nemeth MP (1988). Buckling behavior of compression-loaded symmetrically laminated angle-ply plates with holes. The American Institute of Aeronautics and Astronautics Journal, 26(3): 330–336.  For an aluminum layer of 0.2mm in the hybrid plate, the critical stress is reduced by 2.8% and 8.5% when the angles of declination of the elliptical notch are respectively 900 and 00. The maximum reduction is obtained for an orientation of the fibers at 450 and this regardless of the orientation of the elliptical notch with respect to the applied load. Ouinas D and Achour B (2013). Buckling analysis of laminated composite plates [(θ/− θ)] containing an elliptical notch. Composites Part B: Engineering, 55: 575-579. Reddy PR and Harish T (2014). References Baba B O (2007). Buckling behavior of laminated composite plates. Journal of Reinforced Plastic Composites, 26(16): 1637–1655. Baba BO and Baltaci A (2007). Buckling characteristics of symmetrically and anti-symmetrically laminated composite plates with central cutout. Applied Composite Materials, 14(4): 265-276. 3.3. Influence of the aluminum layer thickness 10: Effect of the aluminum layer thickness on the buckling load amplification 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 2,60 2,80 3,00 3,20 3,40 3,60 3,80 4,00 4,20 l/(6Al l/(3Al l/(6Al Declination of the notch =90° Thickness tAl 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 4,20 4,30 4,40 4,50 4,60 4,70 4,80 4,90 5,00 5,10 5,20 5,30 5,40 5,50 l/(6Al l/(3Al l/(6Al Declination of the notch =45° Al Thickness t 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 Al Thickness t N* N* Al Thickness t 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 4,20 4,30 4,40 4,50 4,60 4,70 4,80 4,90 5,00 5,10 5,20 5,30 5,40 5,50 l/(6Al l/(3Al l/(6Al Declination of the notch =45° Al Thickness t N* Al Thickness t (kN) Cr N Al Thickness t 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,00 4,38 8,76 13,15 17,53 21,91 26,29 30,67 l/(6Al l/(3Al l/(6Al Declination of the notch =45° Al Thickness t 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 2,60 2,80 3,00 3,20 3,40 3,60 3,80 4,00 4,20 l/(6Al l/(3Al l/(6Al Declination of the notch =90° Thickness tAl N* Al Thickness t NCr (kN) Al Thickness t 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,00 4,38 8,76 13,15 17,53 21,91 26,29 30,67 l/(6Al l/(3Al l/(6Al Declination of the notch =90° Fig. 10: Effect of the aluminum layer thickness on the buckling load amplification For the orientations of the plies at 0° and 45°, the reduction factor increased quadratically with the increase of the thickness of the aluminum layer up to 0.8mm. It reached respectively 7.4% and 7.6% in the presence of a notch oriented at 00. An inverse Fig. 9: Effect of the aluminum layer thickness on the critical load 48 Achour et al/ International Journal of Advanced and Applied Sciences, 4(6) 2017, Pages: 43-49 Acknowledgment behavior has been observed when the plies are at 900; the factor 𝑁∗ increased exponentially with the decrease of the aluminum layer. The maximum value was attained for the smallest thickness which was of the order of about 8 %. When the elliptical notch was declined at 450, the factor 𝑁∗ decrease from 5.44% and 5.07% for tAl=0.127mm down to 5.22 % and 5.03% for tAl=0.8mm for both orientations 𝜃= 450 and 𝜃= 900. The research reported herein was funded by the Deanship of Scientific Research at the University of Hail, Saudi Arabia, under the contract (0150089). The authors would like to express their deepest gratitude to the Deanship of Scientific Research and to the College of Engineering at the University of Hail for providing necessary support to conducting this research. The lowest reductions are obtained for 𝜃= 900. For this last case, the maximum reduction is 4.92% for tAl=0.8mm. Comparing the last case (𝜑= 900) and the first case (𝜑= 00), the reduction factor 𝑁∗ is found to be roughly halved for both the extreme thicknesses (tAl = 0.127mm and tAl = 0.8mm) and for the different ply orientations 𝜑= 00, 𝜑= 450 and 𝜑= 900. 4. Conclusion Buckling analysis of orthotropic laminated composite plate with rectangular Cut-Outs by using FEA. Journal of Emerging Technologies in Computational and Applied Sciences, 10(1): 75-81.  The elliptical notch declined at 0°, reduces the maximum load four times more than when declined at 900. Rhodes MD, Mikulas MM, and McGowan PE (1984). Effects of orthotropy and width on the compression strength of graphite–epoxy panels with holes. AIAA Journal, 22(9): 1283– 1292. 49
https://openalex.org/W2167663354	https://repository.ubn.ru.nl/bitstream/handle/2066/94055/3/94055.pdf	English	null	Search for Higgs bosons of the minimal supersymmetric standard model in <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" altimg="si1.gif" overflow="scroll"><mml:mi>p</mml:mi><mml:mover accent="true"><mml:mi>p</mml:mi><mml:mo>¯</mml:mo></mml:mover></mml:math> collisions at <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" altimg="si2.gif" overflow="scroll"><mml:msqrt><mml:mi>s</mml:mi></mml:msqrt><mml:mo>=</mml:mo><mml:mn>1.96</mml:mn><mml:mtext> </mml:mtext><mml:mtext>TeV</mml:…	Physics letters. B	2,012	cc-by	10,469	Search for Higgs bosons of the minimal supersymmetric standard model in pp collisions at s=1.96TeV 2012, Article / Letter to editor (Physics Letters B, 710, 4-5, (2012), pp. 569-577) Doi link to publisher: https://doi.org/10.1016/j.physletb.2012.03.021 Doi link to publisher: https://doi.org/10.1016/j.physletb.2012.03.021 Version of the following full text: Publisher’s version Downloaded from: http://hdl.handle.net/2066/94055 Download date: 2024-10-24 Contents lists available at SciVerse ScienceDirect Contents lists available at SciVerse ScienceDirect Note: To cite this publication please use the final published version (if applicable). Physics Letters B 710 (2012) 569–577 Contents lists available at SciVerse ScienceDirect 0370-2693 © 2012 Elsevier B.V. http://dx.doi.org/10.1016/j.physletb.2012.03.021 Open access under CC BY license. D0 Collaboration Negret g, 0370 2693 © 2012 Elsevier B V Open access under CC BY license V.M. Abazov ah, B. Abbott bt, B.S. Acharya ab, M. Adams av, T. Adams at, G.D. Alexeev ah, G. Alkhazov al, A. Alton bh,1, G. Alverson bg, M. Aoki au, A. Askew at, B. Åsman an, S. Atkins be, O. Atramentov bl, K. Augsten i, C. Avila g, J. BackusMayes ca, F. Badaud l, L. Bagby au, B. Baldin au, D.V. Bandurin at, S. Banerjee ab, E. Barberis bg, P. Baringer bc, J. Barreto c, J.F. Bartlett au, U. Bassler q, V. Bazterra av, A. Bean bc, M. Begalli c, C. Belanger-Champagne an, L. Bellantoni au, S.B. Beri z, G. Bernardi p, R. Bernhard u, I. Bertram ao, M. Besançon q, R. Beuselinck ap, V.A. Bezzubov ak, P.C. Bhat au, S. Bhatia bj, V. Bhatnagar z, G. Blazey aw, S. Blessing at, K. Bloom bk, A. Boehnlein au, D. Boline bq, E.E. Boos aj, G. Borissov ao, T. Bose bf, A. Brandt bw, O. Brandt v, R. Brock bi, G. Brooijmans bo, A. Bross au, D. Brown p, J. Brown p, X.B. Bu au, M. Buehler au, V. Buescher w, V. Bunichev aj, S. Burdin ao,2, T.H. Burnett ca, C.P. Buszello an, B. Calpas n, E. Camacho-Pérez ae, M.A. Carrasco-Lizarraga bc, B.C.K. Casey au, H. Castilla-Valdez ae, S. Chakrabarti bq, D. Chakraborty aw, K.M. Chan ba, A. Chandra by, E. Chapon q, G. Chen bc, S. Chevalier-Théry q, D.K. Cho bv, S.W. Cho ad, S. Choi ad, B. Choudhary aa, S. Cihangir au, D. Claes bk, J. Clutter bc, M. Cooke au, W.E. Cooper au, M. Corcoran by, F. Couderc q, M.-C. Cousinou n, A. Croc q, D. Cutts bv, A. Das ar, G. Davies ap, S.J. de Jong ag, E. De La Cruz-Burelo ae, F. Déliot q, R. Demina bp, D. Denisov au, S.P. Denisov ak, S. Desai au, C. Deterre q, K. DeVaughan bk, H.T. Diehl au, M. Diesburg au, P.F. Ding aq, A. Dominguez bk, T. Dorland ca, A. Dubey aa, L.V. Dudko aj, D. Duggan bl, A. Duperrin n, S. Dutt z, A. Dyshkant aw, M. Eads bk, D. Edmunds bi, J. Ellison as, V.D. Elvira au, Y. Enari p, H. Evans ay, A. Evdokimov br, V.N. Evdokimov ak, G. Facini bg, T. Ferbel bp, F. Fiedler w, F. Filthaut ag, W. Fisher bi, H.E. Fisk au, M. Fortner aw, H. D0 Collaboration Fox ao, S. Fuess au, A. Garcia-Bellido bp, G.A. García-Guerra ae,3, V. Gavrilov ai, P. Gay l, W. Geng n,bi, D. Gerbaudo bm, C.E. Gerber av, Y. Gershtein bl, G. Ginther au,bp, G. Golovanov ah, A. Goussiou ca, P.D. Grannis bq, S. Greder r, H. Greenlee au, Z.D. Greenwood be, E.M. Gregores d, G. Grenier s, Ph. Gris l, J.-F. Grivaz o, A. Grohsjean q,9, S. Grünendahl au, M.W. Grünewald ac, T. Guillemin o, G. Gutierrez au, P. Gutierrez bt, A. Haas bo,4, S. Hagopian at, J. Haley bg, L. Han f, K. Harder aq, A. Harel bp, J.M. Hauptman bb, J. Hays ap, T. Head aq, T. Hebbeker t, D. Hedin aw, H. Hegab bu, A.P. Heinson as, U. Heintz bv, C. Hensel v, I. Heredia-De La Cruz ae, K. Herner bh, G. Hesketh aq,5, M.D. Hildreth ba, R. Hirosky bz, T. Hoang at, J.D. Hobbs bq, B. Hoeneisen k, M. Hohlfeld w, Z. Hubacek i,q, V. Hynek i, I. Iashvili bn, Y. Ilchenko bx, R. Illingworth au, A.S. Ito au, S. Jabeen bv, M. Jaffré o, D. Jamin n, A. Jayasinghe bt, R. Jesik ap, K. Johns ar, M. Johnson au, A. Jonckheere au, P. Jonsson ap, J. Joshi z, A.W. Jung au, A. Juste am, K. Kaadze bd, E. Kajfasz n, D. Karmanov aj, P.A. Kasper au, I. Katsanos bk, R. Kehoe bx, S. Kermiche n, N. Khalatyan au, A. Khanov bu, A. Kharchilava bn, Y.N. Kharzheev ah, J.M. Kohli z, A.V. Kozelov ak, J. Kraus bi, S. Kulikov ak, A. Kumar bn, A. Kupco j, T. Kurˇca s, V.A. Kuzmin aj, S. Lammers ay, G. Landsberg bv, P. Lebrun s, H.S. Lee ad, S.W. Lee bb, W.M. Lee au, J. Lellouch p, H. Li m, L. Li as, Q.Z. Li au, S.M. Lietti e, J.K. Lim ad, D. Lincoln au, J. Linnemann bi, V.V. Lipaev ak, R. Lipton au, Y. Liu f, A. Lobodenko al, M. Lokajicek j, R. Lopes de Sa bq, H.J. Lubatti ca, R. Luna-Garcia ae,6, A.L. Lyon au, A.K.A. Maciel b, D. Mackin by, R. Madar q, R. Magaña-Villalba ae, S. Malik bk, V.L. Malyshev ah, Y. Maravin bd, J. Martínez-Ortega ae, R. McCarthy bq, C.L. McGivern bc, M.M. Meijer ag, A. Melnitchouk bj, D. Menezes aw, P.G. Mercadante d, M. Merkin aj, A. Meyer t, J. Meyer v, F. Miconi r, N.K. Mondal ab, G.S. Muanza n, M. Mulhearn bz, E. D0 Collaboration V.M. Abazov ah, B. Abbott bt, B.S. Acharya ab, M. Adams av, T. Adams at, G.D. Alexeev ah, G. Alkhazov al, A. Alton bh,1, G. Alverson bg, M. Aoki au, A. Askew at, B. Åsman an, S. Atkins be, O. Atramentov bl, K. Augsten i, C. Avila g, J. BackusMayes ca, F. Badaud l, L. Bagby au, B. Baldin au, D.V. Bandurin at, S. Banerjee ab, E. Barberis bg, P. Baringer bc, J. Barreto c, J.F. Bartlett au, U. Bassler q, V. Bazterra av, A. Bean bc, M. Begalli c, C. Belanger-Champagne an, L. Bellantoni au, S.B. Beri z, G. Bernardi p, R. Bernhard u, I. Bertram ao, M. Besançon q, R. Beuselinck ap, V.A. Bezzubov ak, P.C. Bhat au, S. Bhatia bj, V. Bhatnagar z, G. Blazey aw, S. Blessing at, K. Bloom bk, A. Boehnlein au, D. Boline bq, E.E. Boos aj, G. Borissov ao, T. Bose bf, A. Brandt bw, O. Brandt v, R. Brock bi, G. Brooijmans bo, A. Bross au, D. Brown p, J. Brown p, X.B. Bu au, M. Buehler au, V. Buescher w, V. Bunichev aj, S. Burdin ao,2, T.H. Burnett ca, C.P. Buszello an, B. Calpas n, E. Camacho-Pérez ae, M.A. Carrasco-Lizarraga bc, B.C.K. Casey au, H. Castilla-Valdez ae, S. Chakrabarti bq, D. Chakraborty aw, K.M. Chan ba, A. Chandra by, E. Chapon q, G. Chen bc, S. Chevalier-Théry q, D.K. Cho bv, S.W. Cho ad, S. Choi ad, B. Choudhary aa, S. Cihangir au, D. Claes bk, J. Clutter bc, M. Cooke au, W.E. Cooper au, M. Corcoran by, F. Couderc q, M.-C. Cousinou n, A. Croc q, D. Cutts bv, A. Das ar, G. Davies ap, S.J. de Jong ag, E. De La Cruz-Burelo ae, F. Déliot q, R. Demina bp, D. Denisov au, S.P. Denisov ak, S. Desai au, C. Deterre q, K. DeVaughan bk, H.T. Diehl au, M. Diesburg au, P.F. Ding aq, A. Dominguez bk, T. Dorland ca, A. Dubey aa, L.V. Dudko aj, D. Duggan bl, A. Duperrin n, S. Dutt z, A. Dyshkant aw, M. Eads bk, D. Edmunds bi, J. Ellison as, V.D. Elvira au, Y. Enari p, H. Evans ay, A. Evdokimov br, V.N. Evdokimov ak, G. Facini bg, T. Ferbel bp, F. Fiedler w, F. Filthaut ag, W. Fisher bi, H.E. Fisk au, M. Fortner aw, H. Fox ao, S. Fuess au, A. Garcia-Bellido bp, G.A. García-Guerra ae,3, V. Gavrilov ai, P. D0 Collaboration Gay l, W. Geng n,bi, D. Gerbaudo bm, C.E. Gerber av, Y. Gershtein bl, G. Ginther au,bp, G. Golovanov ah, A. Goussiou ca, P.D. Grannis bq, S. Greder r, H. Greenlee au, Z.D. Greenwood be, E.M. Gregores d, G. Grenier s, Ph. Gris l, J.-F. Grivaz o, A. Grohsjean q,9, S. Grünendahl au, M.W. Grünewald ac, T. Guillemin o, G. Gutierrez au, P. Gutierrez bt, A. Haas bo,4, S. Hagopian at, J. Haley bg, L. Han f, K. Harder aq, A. Harel bp, J.M. Hauptman bb, J. Hays ap, T. Head aq, T. Hebbeker t, D. Hedin aw, H. Hegab bu, A.P. Heinson as, U. Heintz bv, C. Hensel v, I. Heredia-De La Cruz ae, K. Herner bh, G. Hesketh aq,5, M.D. Hildreth ba, R. Hirosky bz, T. Hoang at, J.D. Hobbs bq, B. Hoeneisen k, M. Hohlfeld w, Z. Hubacek i,q, V. Hynek i, I. Iashvili bn, Y. Ilchenko bx, R. Illingworth au, A.S. Ito au, S. Jabeen bv, M. Jaffré o, D. Jamin n, A. Jayasinghe bt, R. Jesik ap, K. Johns ar, M. Johnson au, A. Jonckheere au, P. Jonsson ap, J. Joshi z, A.W. Jung au, A. Juste am, K. Kaadze bd, E. Kajfasz n, D. Karmanov aj, P.A. Kasper au, I. Katsanos bk, R. Kehoe bx, S. Kermiche n, N. Khalatyan au, A. Khanov bu, A. Kharchilava bn, Y.N. Kharzheev ah, J.M. Kohli z, A.V. Kozelov ak, J. Kraus bi, S. Kulikov ak, A. Kumar bn, A. Kupco j, T. Kurˇca s, V.A. Kuzmin aj, S. Lammers ay, G. Landsberg bv, P. Lebrun s, H.S. Lee ad, S.W. Lee bb, W.M. Lee au, J. Lellouch p, H. Li m, L. Li as, Q.Z. Li au, S.M. Lietti e, J.K. Lim ad, D. Lincoln au, J. Linnemann bi, V.V. Lipaev ak, R. Lipton au, Y. Liu f, A. Lobodenko al, M. Lokajicek j, R. Lopes de Sa bq, H.J. Lubatti ca, R. Luna-Garcia ae,6, A.L. Lyon au, A.K.A. Maciel b, D. Mackin by, R. Madar q, R. Magaña-Villalba ae, S. Malik bk, V.L. Malyshev ah, Y. Maravin bd, J. Martínez-Ortega ae, R. McCarthy bq, C.L. McGivern bc, M.M. Meijer ag, A. Melnitchouk bj, D. Menezes aw, P.G. Mercadante d, M. Merkin aj, A. Meyer t, J. Meyer v, F. Miconi r, N.K. Mondal ab, G.S. Muanza n, M. Mulhearn bz, E. Nagy n, M. Naimuddin aa, M. Narain bv, R. Nayyar aa, H.A. Neal bh, J.P. D0 Collaboration Sirotenko au, P. Skubic bt, P. Slattery bp, D. Smirnov ba, K J S ith bn G R S bk J S bs S S d br S Söld R b ld aq L S h i t K. Soustruznik h, J. Stark m, V. Stolin ai, D.A. Stoyanova ak, M. Strauss bt, D. Strom av, L. Stutte au, L. Suter aq P Svoisky bt M Takahashi aq A Tanasijczuk a M Titov q V V Tokmenin ah Y T Tsai bp y , , j , , , , n-Grimm bq, D. Tsybychev bq, B. Tuchming q, C. Tully bm, L. Uvarov al, S. Uvarov al, f k y j K. Tschann-Grimm bq, D. Tsybychev bq, B. Tuchming q, C. Tully bm, L. Uvarov al, S. Uvarov al, S. Uzunyan aw, ay af av ar ak s y y g y W.M. van Leeuwen af, N. Varelas av, E.W. Varnes ar, I.A. Vasilyev ak, P. Verdier s, h i L.S. Vertogradov ah, M. Verzocchi au, M. Vesterinen aq, D. Vilanova q, P. Vokac i, H.D. Wahl at, M.H.L.S. Wang au, J. Warchol ba, G. Watts ca, M. Wayne ba, M. Weber au,8, J. Weichert w, L. Welty-Rieger ax, A. White bw, D. Wicke y, M.R.J. Williams ao, G.W. Wilson bc, M. Wobisch be, D.R. Wood bg, T.R. Wyatt aq, Y. Xie au, R. Yamada au, W.-C. Yang aq, T. Yasuda au, Y.A. Yatsunenko ah, W. Ye bq, Z. Ye au, H. Yin au, K. Yip br, S.W. Youn au, T. Zhao ca, B. Zhou bh, J. Zhu bh, M. Zielinski bp, D. Zieminska ay, L. D0 Collaboration Nagy n, M. Naimuddin aa, M. Narain bv, R. Nayyar aa, H.A. Neal bh, J.P. Negret g, 0370 2693 © 2012 Elsevier B V Open access under CC BY license Z.D. Greenwood be, E.M. Gregores d, G. Grenier s, Ph. Gris l, J.-F. Grivaz o, A. Grohsjean q,9, S. Grünendahl au, M.W. Grünewald ac, T. Guillemin o, G. Gutierrez au, P. Gutierrez bt, A. Haas bo,4, S. Hagopian at, J. Haley bg, L. Han f, K. Harder aq, A. Harel bp, J.M. Hauptman bb, J. Hays ap, T. Head aq, T. Hebbeker t, D. Hedin aw, D0 Collaboration / Physics Letters B 710 (2012) 569–577 570 P. Neustroev al, S.F. Novaes e, T. Nunnemann x, G. Obrant al,10, J. Orduna by, N. Osman n, J. Osta ba, G.J. Otero y Garzón a, M. Padilla as, A. Pal bw, N. Parashar az, V. Parihar bv, S.K. Park ad, R. Partridge bv,4, N. Parua ay, A. Patwa br, B. Penning au, M. Perﬁlov aj, Y. Peters aq, K. Petridis aq, G. Petrillo bp, P. Pétroff o, R. Piegaia a, M.-A. Pleier br, P.L.M. Podesta-Lerma ae,7, V.M. Podstavkov au, P. Polozov ai, A.V. Popov ak, M. Prewitt by, D. Price ay, N. Prokopenko ak, J. Qian bh, A. Quadt v, B. Quinn bj, M.S. Rangel b, K. Ranjan aa, P.N. Ratoff ao, I. Razumov ak, P. Renkel bx, M. Rijssenbeek bq, I. Ripp-Baudot r, F. Rizatdinova bu, b i P. Neustroev al, S.F. Novaes e, T. Nunnemann x, G. Obrant al,10, J. Orduna by, N. Osman n, J. Osta ba, G.J. Otero y Garzón a, M. Padilla as, A. Pal bw, N. Parashar az, V. Parihar bv, S.K. Park ad, R. Partridge bv,4, N. Parua ay, A. Patwa br, B. Penning au, M. Perﬁlov aj, Y. Peters aq, K. Petridis aq, G. Petrillo bp, P. Pétroff o, R. Piegaia a, M.-A. Pleier br, P.L.M. Podesta-Lerma ae,7, V.M. Podstavkov au, P. Polozov ai, A.V. Popov ak, M. Prewitt by, D. Price ay, N. Prokopenko ak, J. Qian bh, A. Quadt v, B. Quinn bj, M.S. Rangel b, K. Ranjan aa, P.N. Ratoff ao, I. Razumov ak, P. Renkel bx, M. Rijssenbeek bq, I. Ripp-Baudot r, F. Rizatdinova bu, M. Rominsky au, A. Ross ao, C. Royon q, P. Rubinov au, R. Ruchti ba, G. Safronov ai, G. Sajot m, P. Salcido aw, A. Sánchez-Hernández ae, M.P. Sanders x, B. Sanghi au, A.S. D0 Collaboration Zivkovic bv a Universidad de Buenos Aires, Buenos Aires, Argentina a Universidad de Buenos Aires, Buenos Aires, Argentina b LAFEX, Centro Brasileiro de Pesquisas Físicas, Rio de Janeiro, Brazil c Universidade do Estado do Rio de Janeiro, Rio de Janeiro, Brazil d Universidade Federal do ABC, Santo André, Brazil Instituto de Física Teórica, Universidade Estadual Paulista, São Paulo, Br f University of Science and Technology of China, Hefei, People’s Republic of China g Universidad de los Andes, Bogotá, Colombia h Charles University, Faculty of Mathematics and Physics, Center for Particle Physics, Prague, Czech Republic i h Charles University, Faculty of Mathematics and Physics, Center for P Czech Technical University in Prague, Prague, Czech Republic y g g p j Center for Particle Physics, Institute of Physics, Academy of Sciences of the Czech Republic, Prague, Czech Republic k Center for Particle Physics, Institute of Physics, Academy of Sciences of t k Universidad San Francisco de Quito, Quito, Ecuador l LPC, Université Blaise Pascal, CNRS/IN2P3, Clermont, France m LPSC, Université Joseph Fourier Grenoble 1, CNRS/IN2P3, Institut National Polytechnique de Grenoble, Grenoble, France m LPSC, Université Joseph Fourier Grenoble 1, CNRS/IN2P3, Insti n CPPM, Aix-Marseille Université, CNRS/IN2P3, Marseille, France n CPPM, Aix-Marseille Université, CNRS/IN2P3, Marseille, France o LAL, Université Paris-Sud, CNRS/IN2P3, Orsay, France p LPNHE, Universités Paris VI and VII, CNRS/IN2P3, Paris, France r IPHC, Université de Strasbourg, CNRS/IN2P3, Strasbourg, France s IPNL, Université Lyon 1, CNRS/IN2P3, Villeurbanne, and Université de Lyon, Lyon, France III. Physikalisches Institut A, RWTH Aachen University, Aachen, German u Physikalisches Institut, Universität Freiburg, Freiburg, Germany v II. Physikalisches Institut, Georg-August-Universität Göttingen, Gö w Institut für Physik, Universität Mainz, Mainz, Germany x Ludwig-Maximilians-Universität München, München, Germany y Fachbereich Physik, Bergische Universität Wuppertal, Wuppertal z Panjab University, Chandigarh, India aa Delhi University, Delhi, India ab Tata Institute of Fundamental Research, Mumbai, India ac University College Dublin, Dublin, Ireland y g ad Korea Detector Laboratory, Korea University, Seoul, Republic of Korea af Nikhef, Science Park, Amsterdam, The Netherlands af Nikhef, Science Park, Amsterdam, The Netherlands f ag Radboud University Nijmegen, Nijmegen, and Nikhef, Science Park, Amsterdam, The Netherlands h ag Radboud University Nijmegen, Nijmegen, and Nikh ah Joint Institute for Nuclear Research, Dubna, Russia ai Institute for Theoretical and Experimental Physics, Moscow, Russia aj Moscow State University, Moscow, Russia aj Moscow State University, Moscow, Russia ak Institute for High Energy Physics, Protvino, Russia al Petersburg Nuclear Physics Institute, St. D0 Collaboration Santos e, G. Savage au, L. Sawyer be, T. Scanlon ap, R.D. Schamberger bq, Y. Scheglov al, H. Schellman ax, T. Schliephake y, S. Schlobohm ca, C. Schwanenberger aq, R. Schwienhorst bi, J. Sekaric bc, H. Severini bt, E. Shabalina v, V. Shary q, A.A. Shchukin ak, R.K. Shivpuri aa, V. Simak i, V. Sirotenko au, P. Skubic bt, P. Slattery bp, D. Smirnov ba, K.J. Smith bn, G.R. Snow bk, J. Snow bs, S. Snyder br, S. Söldner-Rembold aq, L. Sonnenschein t, K. Soustruznik h, J. Stark m, V. Stolin ai, D.A. Stoyanova ak, M. Strauss bt, D. Strom av, L. Stutte au, L. Suter aq, P. Svoisky bt, M. Takahashi aq, A. Tanasijczuk a, M. Titov q, V.V. Tokmenin ah, Y.-T. Tsai bp, K. Tschann-Grimm bq, D. Tsybychev bq, B. Tuchming q, C. Tully bm, L. Uvarov al, S. Uvarov al, S. Uzunyan aw, R. Van Kooten ay, W.M. van Leeuwen af, N. Varelas av, E.W. Varnes ar, I.A. Vasilyev ak, P. Verdier s, L.S. Vertogradov ah, M. Verzocchi au, M. Vesterinen aq, D. Vilanova q, P. Vokac i, H.D. Wahl at, M.H.L.S. Wang au, J. Warchol ba, G. Watts ca, M. Wayne ba, M. Weber au,8, J. Weichert w, L. Welty-Rieger ax, A. White bw, D. Wicke y, M.R.J. Williams ao, G.W. Wilson bc, M. Wobisch be, D.R. Wood bg, T.R. Wyatt aq, Y. Xie au, R. Yamada au, W.-C. Yang aq, T. Yasuda au, Y.A. Yatsunenko ah, W. Ye bq, Z. Ye au, H. Yin au, K. Yip br, S.W. Youn au, T. Zhao ca, B. Zhou bh, J. Zhu bh, M. Zielinski bp, D. Zieminska ay, L. Zivkovic bv , , , j , pp , , M. Rominsky au, A. Ross ao, C. Royon q, P. Rubinov au, R. Ruchti ba, G. Safronov ai, G. Sajot m, P. Salcido aw, A Sánche Hernánde ae M P Sanders x B Sanghi au A S Santos e G Sa age au L Sa er be , , g , , g , y , T. Scanlon ap, R.D. Schamberger bq, Y. Scheglov al, H. Schellman ax, T. Schliephake y, S. Schlobohm ca, C S h b aq R S h i h bi J S k i bc H S i i bt E Sh b li v V Sh q g J y A.A. Shchukin ak, R.K. Shivpuri aa, V. Simak i, V. D0 Collaboration Petersburg, R am Institució Catalana de Recerca i Estudis Avançats (ICREA) and Institut de Física d’Altes Energies (IFAE), Barcelona, Spain am Institució Catalana de Recerca i Estudis Avançats (ICREA) and Institut de Física d’Altes Energies (IFAE), Barcelona, Spain an Stockholm University, Stockholm, and Uppsala University, Upps ao Lancaster University, Lancaster LA1 4YB, United Kingdom ao Lancaster University, Lancaster LA1 4YB, United Kingdom ap Imperial College London, London SW7 2AZ, United Kingdom aq The University of Manchester, Manchester M13 9PL, United Kingdom ar University of Arizona, Tucson, AR 85721, USA ar University of Arizona, Tucson, AR 85721, USA as University of California Riverside, Riverside, CA 92521, USA as University of California Riverside, Riverside, CA 92521, USA at Florida State University, Tallahassee, FL 32306, USA at Florida State University, Tallahassee, FL 32306, USA 571 D0 Collaboration / Physics Letters B 710 (2012) 569–577 au Fermi National Accelerator Laboratory, Batavia, IL 60510, USA av University of Illinois at Chicago, Chicago, IL 60607, USA aw Northern Illinois University, DeKalb, IL 60115, USA ax Northwestern University, Evanston, IL 60208, USA ay Indiana University, Bloomington, IN 47405, USA az Purdue University Calumet, Hammond, IN 46323, USA ba University of Notre Dame, Notre Dame, IN 46556, USA bb Iowa State University, Ames, IA 50011, USA bc University of Kansas, Lawrence, KS 66045, USA bd Kansas State University, Manhattan, KS 66506, USA be Louisiana Tech University, Ruston, LA 71272, USA bf Boston University, Boston, MA 02215, USA bg Northeastern University, Boston, MA 02115, USA bh University of Michigan, Ann Arbor, MI 48109, USA bi Michigan State University, East Lansing, MI 48824, USA bj University of Mississippi, University, MS 38677, USA bk University of Nebraska, Lincoln, NE 68588, USA bl Rutgers University, Piscataway, NJ 08855, USA bm Princeton University, Princeton, NJ 08544, USA bn State University of New York, Buffalo, NY 14260, USA bo Columbia University, New York, NY 10027, USA bp University of Rochester, Rochester, NY 14627, USA bq State University of New York, Stony Brook, NY 11794, USA br Brookhaven National Laboratory, Upton, NY 11973, USA bs Langston University, Langston, OK 73050, USA bt University of Oklahoma, Norman, OK 73019, USA bu Oklahoma State University, Stillwater, OK 74078, USA bv Brown University, Providence, RI 02912, USA bw University of Texas, Arlington, TX 76019, USA bx Southern Methodist University, Dallas, TX 75275, USA by Rice University, Houston, TX 77005, USA bz University of Virginia, Charlottesville, VA 22901, USA ca University of Washington, Seattle, WA 98195, USA au Fermi National Accelerator Laboratory, Batavia, IL 60510, USA av University of Illinois at Chicago, Chicago, IL 60607, USA aw Northern Illinois University, DeKalb, IL 60115, USA ax Northwestern University, Evanston, IL 60208, USA ay Indiana University, Bloomington, IN 47405, USA az Purdue University Calumet, Hammond, IN 46323, USA ba University of Notre Dame, Notre Dame, IN 46556, USA bb Iowa State University, Ames, IA 50011, USA bc University of Kansas, Lawrence, KS 66045, USA bd Kansas State University, Manhattan, KS 66506, USA be Louisiana Tech University, Ruston, LA 71272, USA bf Boston University, Boston, MA 02215, USA bg Northeastern University, Boston, MA 02115, USA bh University of Michigan, Ann Arbor, MI 48109, USA bi Michigan State University, East Lansing, MI 48824, USA bj University of Mississippi, University, MS 38677, USA bk University of Nebraska, Lincoln, NE 68588, USA bl Rutgers University, Piscataway, NJ 08855, USA bm Princeton University, Princeton, NJ 08544, USA bn State University of New York, Buffalo, NY 14260, USA bo Columbia University, New York, NY 10027, USA bp University of Rochester, Rochester, NY 14627, USA bq State University of New York, Stony Brook, NY 11794, USA br Brookhaven National Laboratory, Upton, NY 11973, USA bs Langston University, Langston, OK 73050, USA bt University of Oklahoma, Norman, OK 73019, USA bu Oklahoma State University, Stillwater, OK 74078, USA bv Brown University, Providence, RI 02912, USA bw University of Texas, Arlington, TX 76019, USA bx Southern Methodist University, Dallas, TX 75275, USA by Rice University, Houston, TX 77005, USA bz University of Virginia, Charlottesville, VA 22901, USA ca University of Washington, Seattle, WA 98195, USA a b s t r a c t Article history: Received 22 December 2011 Received in revised form 5 March 2012 Accepted 8 March 2012 Available online 13 March 2012 Editor: H. Weerts We report results from searches for neutral Higgs bosons produced in p ¯p collisions recorded by the D0 experiment at the Fermilab Tevatron Collider. We study the production of inclusive neutral Higgs boson in the ττ ﬁnal state and in association with a b quark in the bττ and bbb ﬁnal states. These results are combined to improve the sensitivity to the production of neutral Higgs bosons in the context of the minimal supersymmetric standard model (MSSM). The data are found to be consistent with expectation from background processes. Upper limits on MSSM Higgs boson production are set for Higgs boson masses ranging from 90 to 300 GeV. We exclude tanβ > 20–30 for Higgs boson masses below 180 GeV. These are the most stringent constraints on MSSM Higgs boson production in p ¯p collisions. © 2012 Elsevier B.V. Open access under CC BY license. 1 Visitor from Augustana College, Sioux Falls, SD, USA. 2 Visitor from The University of Liverpool, Liverpool, UK. 3 Visitor from UPIITA-IPN, Mexico City, Mexico. 4 Visitor from SLAC, Menlo Park, CA, USA. 5 Visitor from University College London, London, UK. 6 Visitor from Centro de Investigacion en Computacion – IPN, Mexico City, Mexico. 7 Visitor from ECFM, Universidad Autonoma de Sinaloa, Culiacán, Mexico. 8 Visitor from Universität Bern, Bern, Switzerland. 9 Visitor from DESY, Hamburg, Germany. 10 Deceased. 1. Introduction pseudoscalar Higgs boson. Radiative corrections introduce addi- tional dependencies on other model parameters. Although tanβ is a free parameter in the MSSM, some indications suggest it should be large (tanβ ≳20). A value of tanβ ≈35 [2] would naturally ex- plain the top to bottom quark mass ratio. The observed density of dark matter also points towards high tanβ values [3]. In the minimal supersymmetric standard model (MSSM) [1], the SU(2) symmetry is broken via two Higgs doublets; the ﬁrst dou- blet couples to down-type fermions only while the second couples to up-type fermions. This leads to ﬁve physical Higgs bosons: two neutral CP-even bosons, h and H, one neutral CP-odd boson A, and two charged bosons H±. The neutral Higgs bosons are col- lectively denoted as φ. At leading order the mass spectrum and the couplings of the Higgs bosons are determined by only two pa- rameters, conventionally chosen to be tanβ, the ratio of the two Higgs doublet vacuum expectation values, and M A, the mass of the p g β [ ] At large tanβ, one of the CP-even Higgs bosons (h or H) is approximately degenerate in mass with the A boson. In addi- tion, they have similar couplings to fermions, which are enhanced (suppressed) by tanβ compared to the standard model (SM) for down-type (up-type) fermions. This enhancement has several con- sequences. First, the main decay modes become φ →b¯b and φ →τ +τ −with respective branching ratios B(φ →b¯b) ≈90% and B(φ →τ +τ −) ≈10%. Secondly, the main production processes at a hadron collider involve b quarks originating from the sea. Inclusive Higgs boson production is dominated by gluon fusion (ggφ) and b¯b annihilation (bbφ), as shown in Fig. 1. The latter process may produce a b quark in the acceptance of the detector in addition to the Higgs boson. This associated production gb →φb (bgbφ) is shown in Fig. 1(c). In this case, the detection of the associated b quark is a powerful experimental handle for reducing backgrounds. MSSM Higgs boson masses below 93 GeV have been excluded by experiments at the CERN e+e−collider (LEP) [4]. The CDF and 1 Visitor from Augustana College, Sioux Falls, SD, USA. 2 Visitor from The University of Liverpool, Liverpool, UK. 3 Visitor from UPIITA-IPN, Mexico City, Mexico. 4 Visitor from SLAC, Menlo Park, CA, USA. 5 Visitor from University College London, London, UK. D0 Collaboration Petersburg, Russia al Petersburg Nuclear Physics Institute, St. Table 1 Hence, we re-analyse here the inclusive φ →ττ production: we require that there are no b jets, we extend the dataset to 7.3 fb−1 of integrated lumi- nosity, and we increase the trigger acceptance and reﬁne the treat- ment of systematic uncertainties. The di-tau channels are restricted to ﬁnal states where one τ lepton (τμ) decays via τ →μνμντ and the other (τh) decays hadronically. Jets are reconstructed from energy deposits in the calorime- ter [18] using the midpoint cone algorithm [16] with a radius of 0.5. All jets are required to have at least two reconstructed tracks originating from the p ¯p interaction vertex matched within R(track, jet-axis) = (η)2 + (ϕ)2 < 0.5 (where ϕ is the az- imuthal angle). To identify jets originating from b quark decay, a neural network b-tagging algorithm (NNb) [19] has been devel- oped. It uses lifetime-based information involving the track impact parameters and secondary vertices as inputs. The presence of neutrinos is inferred from the missing trans- verse energy, /E T , which is reconstructed as the negative of the vector sum of the transverse energy of calorimeter cells with \|η\| < 3.2, corrected for the energy scales of all reconstructed ob- jects and for muons. 2. Detector and event reconstruction The data analysed in the different studies presented here have been recorded by the D0 detector [12]. It has a central-tracking system, consisting of a silicon microstrip tracker and a central ﬁ- bre tracker, both located within a 2 T superconducting solenoidal magnet, with designs optimised for tracking and vertexing at pseu- dorapidities [13] \|η\| < 3 and \|η\| < 2.5, respectively. A liquid-argon and uranium calorimeter has a central section covering pseudora- pidities \|η\| up to ≈1.1, and two end calorimeters that extend cov- erage to \|η\| ≈4.2, with all three housed in separate cryostats [14]. An outer muon system, at \|η\| < 2, consists of a layer of tracking detectors and scintillation trigger counters in front of 1.8 T toroids, followed by two similar layers after the toroids. 1. Introduction 6 Visitor from Centro de Investigacion en Computacion – IPN, Mexico City, Mexico. 7 Visitor from ECFM, Universidad Autonoma de Sinaloa, Culiacán, Mexico. 8 Visitor from Universität Bern, Bern, Switzerland. 9 Visitor from DESY, Hamburg, Germany. 10 Deceased. MSSM Higgs boson masses below 93 GeV have been excluded by experiments at the CERN e+e−collider (LEP) [4]. The CDF and D0 Collaboration / Physics Letters B 710 (2012) 569–577 572 Fig. 1. Main Higgs boson production mechanisms in the MSSM in the 5-ﬂavor scheme where c and b quarks are included in parton density functions. The gluon fusion (a) and b¯b annihilation (b) processes dominate the inclusive production, while (c) is the dominant process for associated bφ production. Fig. 1. Main Higgs boson production mechanisms in the MSSM in the 5-ﬂavor scheme where c and b quarks are included in parton density functions. The gluon fusion (a) nd b¯b annihilation (b) processes dominate the inclusive production, while (c) is the dominant process for associated bφ production. Fig. 1. Main Higgs boson production mechanisms in the MSSM in the 5-ﬂavor scheme where c and b quarks are included in par and b¯b annihilation (b) processes dominate the inclusive production, while (c) is the dominant process for associated bφ producti hanisms in the MSSM in the 5-ﬂavor scheme where c and b quarks are included in parton density functions. The gluon fusion (a) te the inclusive production, while (c) is the dominant process for associated bφ production. Muons are reconstructed from track segments in the muon sys- tem. They are matched to tracks in the inner tracking system. The timing of associated hits in the scintillators must be consistent with the beam crossing to veto cosmic muons. Table 1 Table 1 Searches combined in this Letter. Final state L (fb−1) Reference φ →τμτh (b-jet veto) 7.3 bφ →bτμτh 7.3 [7] bφ →b¯bb 5.2 [8] Searches combined in this Letter. Hadronic tau decays are characterised by narrow jets that are reconstructed using a jet cone algorithm with a radius of 0.3 [16] in the calorimeter and by low track multiplicity [17]. We split the τh candidates into three different categories that approximately correspond to one-prong τ decays with no π 0 meson (τh type 1), one-prong decay with π 0 mesons (τh type 2), and multi-prong decay (τh type 3). In addition, a neural-network-based τh identi- ﬁcation (NNτ ) has been trained to discriminate light parton jets (u, d, s quarks or gluon) from hadronic τ decays [17]. We select τh candidates requiring NNτ > 0.9 (0.95 for τh type 3). This condi- tion has an eﬃciency of approximately 65% while rejecting ∼99% of quark/gluon jets. D0 Collaborations have searched for MSSM neutral Higgs bosons decaying to tau pairs both inclusively [5,6] and in association with a b quark [7]. They have also searched for bφ →bbb production [8,9], which is challenging due to the high rate of multijet (MJ) production. Since these results have comparable sensitivities, com- bining them further enhances the potential reach. Recently, similar searches were performed at the LHC [10,11]. In this Letter, we present a combination of three searches performed by the D0 Col- laboration in the φ →ττ , bφ →bττ , and bφ →bbb ﬁnal states. D0 Collaborations have searched for MSSM neutral Higgs bosons decaying to tau pairs both inclusively [5,6] and in association with a b quark [7]. They have also searched for bφ →bbb production [8,9], which is challenging due to the high rate of multijet (MJ) production. Since these results have comparable sensitivities, com- bining them further enhances the potential reach. Recently, similar searches were performed at the LHC [10,11]. In this Letter, we present a combination of three searches performed by the D0 Col- laboration in the φ →ττ , bφ →bττ , and bφ →bbb ﬁnal states. Since the inclusive and bgbφ production signal samples in the di- tau ﬁnal states are not mutually exclusive, the D0 result presented in [6] cannot be directly combined with [7]. 4. Analysis strategy In this section, we describe the search strategy as well as the selection of the ﬁnal signal samples. Further details of the bττ and bbb analyses can be found in Refs. [7] and [8], respectively. 4.1.3. MJ background estimation J g In both di-tau channels, the MJ background is estimated from data control samples applying two different methods. The ﬁrst is based on the small correlation between the electric charge of muon and τh in MJ events. For each analysis, we select a data sample with identical criteria as the signal sample but with the two leptons having the same electric charge (SS). We subtract the residual contribution from other SM backgrounds from this MJ- dominated SS sample. We measure the ratio of the number of OS to SS events to be 1.09 ± 0.01 and 1.07 ± 0.01, respectively, in the ττ and bττ channels. We then multiply the SS sample yields by this ratio. This method is used in the inclusive ττ chan- nel but it suffers from large statistical uncertainties of the bττ SS sample. Therefore, we develop an alternate method that uses a MJ-enriched control sample with identical requirements as applied to the signal samples but reversing the muon isolation criteria. In a MJ-dominated SS sample, obtained without any requirement on the number of jets (Njets), the ratio of the probabilities for a muon of a MJ-event to appear isolated or not isolated, Riso/iso ≡ P(μiso\|MJ)/P(μiso\|MJ), is measured as function of ητh , pτh T , and leading-jet pT (if Njets > 0). The ratio Riso/iso is then applied to the distributions of the non-isolated-muon sample, predicting the MJ background in the two signal samples. This method is used in the bττ study. In each analysis, the alternate method is used to determine the systematic uncertainty on the MJ-background nor- malisation. 4.1.1. Inclusive ττ selection 4.1.1. Inclusive ττ selection For the inclusive ττ selection, we tighten the requirements on the τh transverse momentum to suppress the MJ background: pτh T > 12.5 GeV (15 GeV for τh type 3) and ptrk T > 12.5/7/15 GeV respectively for τh type 1/2/3. We further reduce the W + jets background by requiring MT (μ,/E T ) < 40 GeV. We deﬁne Mhat, which represents the minimum centre-of-mass energy consistent with the decay of a di-tau resonance, by g g The alpgen samples are processed through pythia for show- ering and hadronisation. tauola [23] is used to decay τ leptons and evtgen [24] to model b hadron decays. All samples are fur- ther processed through a detailed geant [25]-based simulation of the D0 detector. The output is then combined with data events recorded during random beam crossings to model the effects of detector noise and pile-up energy from multiple interactions and different beam crossings. Finally, the same reconstruction algo- rithms as for data are applied to the simulated events. Data control samples are used to correct the simulation for object identiﬁ- cation eﬃciencies, energy scales and resolutions, trigger eﬃcien- cies, and the longitudinal p ¯p vertex distribution. Signal, t¯t pair, and diboson yields are normalised to the product of their accep- tance and detector eﬃciency (both determined from the simula- tion), their corresponding theoretical cross section and the lumi- nosity. Mhat ≡ Eμτh −pμτh z + /E T 2 − ⃗pτh T + ⃗pτμ T + ⃗/E T 2, where Eμτh is the energy of the μτh system and pμτh z is its momentum component along the beam axis. We require Mhat > 40 GeV to suppress the MJ background. Finally, to prevent any overlap with the bττ sample, we select only events for which no jet has NNb > 0.25. 4.1. Di-tau ﬁnal states The ττ and bττ searches follow a similar strategy. We ﬁrst deﬁne a common selection by retaining events with one recon- structed p ¯p interaction vertex with at least three tracks, exactly one isolated muon and exactly one reconstructed τh. We require the muon to have a transverse momentum pτμ T > 15 GeV, \|ημ\| < 1.6, and to be isolated in the calorimeter and in the central track- ing system, i.e., R(μ, jet) > 0.5 relative to any reconstructed jet. The τh candidate must have a transverse momentum, as measured in the calorimeter with appropriate energy corrections, pτh T > 10 GeV, \|ητh\| < 2.0, R(τh,μ) > 0.5 relative to any muon, and τh tracks must not be shared with any reconstructed muons in the event. The sum of the transverse momenta, ptrk T , of all tracks associated with the τh candidate must satisfy ptrk T > 7/5/10 GeV, respectively, for τh types 1/2/3. We require the distance along the beam axis between the τh and the muon, at their point of closest approach to the p ¯p interaction vertex, z(τh,μ) < 2 cm. In addi- tion, the τh and the muon must have an opposite electric charge (OS) and a transverse mass MT (μ,/E T ) < 60 GeV (100 GeV for τh type 2) where MT (μ,/E T ) = 2 · pτμ T · /E T · [1 −cosϕ(μ,/E T )]. The distributions of Mhat for the ττ study and two different D f discriminants for the bττ analysis are presented in Fig. 2. The observed data, expected signal and background yields are given in Table 2 for the two di-tau event selections. 3. Signal and background Monte Carlo simulation Signal samples are generated with the LO event generator pythia [20]. The inclusive production is simulated with the SM ggφ process. We checked that the kinematic differences between bbφ and ggφ do not have any impact on our ﬁnal result. The associated production with a b-quark is generated with the SM gb →φb process. The contributions to the bφ cross section and event kinematics from next-to-leading order (NLO) diagrams are taken into account by using mcfm [21] to calculate correction fac- tors for the pythia generator as a function of the leading b quark pT and η in the range pb T > 12 GeV and \|ηb\| < 5. The integrated luminosities (L) [15] associated with each search are summarised in Table 1. Di-tau events were recorded using a mixture of single high-pT muon, jet, tau, muon plus jet, and muon plus tau triggers. The eﬃciency of this inclusive trigger condition is measured in a Z →τμτh data sample with respect to single muon triggers. We also verify this measurement in a sample of Z(→τμτh) + jets events. Depending on the kinematics and on the decay topology of the τh, the trigger eﬃciency ranges from 80% to 95%. For the bbb analysis, we employ triggers selecting events with at least three jets. In addition, 95% of the bbb data sam- ple was recorded with b-tagging requirements at the trigger level. The trigger eﬃciency for mφ = 150 GeV is approximately 60% for events passing the analysis requirements. In the ﬁnal states with a tau pair, the dominant backgrounds are due to Z →ττ(+ jets), diboson (WW, WZ and ZZ), W + jets, t¯t pair and MJ production, the latter being estimated from data. Dibo- son events are simulated with pythia while the Z + jets, W + jets, and t¯t samples are generated using alpgen [22]. In the bbb chan- nel, the dominant background is due to MJ production. We simu- late MJ background events from the b¯b j, b¯b jj, c¯c j, c¯c jj, b¯bc¯c, and b¯bb¯b processes, where j denotes a light parton, with the alpgen event generator. The small contribution from t¯t production to the background is also simulated with alpgen. The contribution from D0 Collaboration / Physics Letters B 710 (2012) 569–577 573 other processes, such as Z + b¯b and single top quark production, is negligible. 4.1.2. bττ selection In the bbb ﬁnal state, the relative contribution of the different MJ backgrounds is determined from data; its overall normalisa- tion is constrained by a ﬁt done in the ﬁnal limit-setting pro- cedure which exploits the dijet-mass shape differences between signal and background. In the di-tau channels, a dedicated treat- ment of the dominant Z →ττ background has been developed to reduce its systematic uncertainties. The simulation of the Z boson kinematics is corrected by comparing a large sample of Z →μμ events in data and in the simulation. We measure correction fac- tors in each jet multiplicity bin as a function of the Φ∗quantity introduced in Ref. [26], leading jet η, and leading b-tagged jet NNb. This affects both the normalisation and the kinematic dis- tributions. For the W + jets background, the muon predominantly arises from the W boson decay while the τh candidate is a mis- reconstructed jet. The W + jets simulation is normalised to data, for each jet multiplicity bin, using a W (→μν) + jets data control sample. The complementary sample with at least one b-tagged jet with NNb > 0.25 constitutes the bττ sample. This b-tagged sample suf- fers from large Z + jets, t¯t and MJ backgrounds. We build separate multivariate discriminants, DMJ and Dt¯t, to discriminate against the MJ and t¯t processes. We require DMJ > 0.1 and Dt¯t > 0.1, then we combine NNb, DMJ, and Dt¯t, to form a set of ﬁnal discriminat- ing variables D f (one for each τh type and mφ) to be used in the limit-setting procedure. Further details can be found in Ref. [7]. Table 2 Table 3 Observed data yield and expected signal yields in the bbb channel. The signal yields are given for the scenario described in Table 2. Njets 3 4 Data 15 214 10 417 Signal mφ = 100 GeV 335 166 Signal mφ = 190 GeV 70 36 background model in a control sample selected using an inverted likelihood criterion Dbbb < 0.12. Table 2 Expected background yield, observed data yield, and expected signal yields for the di-tau selections with their total systematic uncertainties. The signal yields are given for the mmax h scenario (μ = +200 GeV and tanβ = 40). Observed data yield and expected signal yields in the bbb channel. The signal yields are given for the scenario described in Table 2. Njets 3 4 Data 15 214 10 417 Signal mφ = 100 GeV 335 166 Signal mφ = 190 GeV 70 36 ττ bττ Z(+jets) 11 547 ± 634 218 ± 17 t¯t 25 ± 4 183 ± 32 MJ 1343 ± 236 36 ± 6 Other 560 ± 25 40 ± 2 Total background 13 474 ± 684 476 ± 40 Data 13 344 488 Signal mφ = 100 GeV 1165 81 Signal mφ = 190 GeV 70 12 background model in a control sample selected using an inverted likelihood criterion Dbbb < 0.12. 4.3. Systematic uncertainties 4.2. bbb ﬁnal state In the bbb analysis, at least three jets, each satisfying pT > 15 GeV, \|η\| < 2.5 and NNb > 0.775, are required. The two lead- ing jets must have pT > 25 GeV. To improve the signal sensitivity, the events are separated into two channels, containing exactly 3 or D0 Collaboration / Physics Letters B 710 (2012) 569–577 574 Fig. 2. Distribution of Mhat in the inclusive ττ sample on (a) linear and (b) logarithmic scale. (c) D f in the bττ sample trained for mφ = 100 GeV, and (d) for mφ = 190 GeV adding the ﬁnal requirements on DMJ and Dt¯t. All τh types are combined. The predicted signal is shown in the case of the mmax h scenario (μ = +200 GeV and tanβ = 40) for mφ = 100 GeV in (a) and (c), and mφ = 190 GeV in (b) and (d). Fig. 2. Distribution of Mhat in the inclusive ττ sample on (a) linear and (b) logarithmic scale. (c) D f in the bττ sample trained for mφ = 100 GeV, and (d) for mφ = 190 GeV adding the ﬁnal requirements on DMJ and Dt¯t. All τh types are combined. The predicted signal is shown in the case of the mmax h scenario (μ = +200 GeV and tanβ = 40) for mφ = 100 GeV in (a) and (c), and mφ = 190 GeV in (b) and (d). 4.3. Systematic uncertainties Further sources of uncertainty affecting the shape of the ﬁnal discriminant are considered: the jet energy scale (10%) and the modelling of the b-tagging eﬃciency (∼4%) mostly affect the bττ signal modelling but are negligible in the ττ channel, while the τh D0 Collaboration / Physics Letters B 710 (2012) 569–577 575 Fig. 3. Distributions of the dijet invariant mass, taken from Ref. [8], in the signal region deﬁned by Dbbb > 0.65 (a) and in a control region deﬁned by Dbbb < 0.12 (b) for the dominant bbb 3-jet channel is shown. The line shows the background model, the solid histogram the component coming from bbb, the points with error bars show the data. The background is normalised to the data yield for illustration purposes. The difference between data and the background model is shown at the bottom of each panel. Fig. 4. Comparison of the expected limits in the (tan channels separately, and their combination for the mm h and (b) μ > 0. the b-quark eﬃciency, luminosity, and jet en assumed to be 100% correlated. The theore the signal are other sources of correlated s among all channels. They are dominated by tion uncertainties, renormalisation and facto sign an uncertainty of 15% on the theoretica correlated across all processes. 5. Results We combine the ττ , bττ and bbb channe frequentist approach [27]. The test statistic of proﬁled likelihoods [28]: p(data\|H ) Fig. 3. Distributions of the dijet invariant mass, taken from Ref. [8], in the signal region deﬁned by Dbbb > 0.65 (a) and in a control region deﬁned by Dbbb < 0.12 (b) for the dominant bbb 3-jet channel is shown. The line shows the background model, the solid histogram the component coming from bbb, the points with error bars show the data. The background is normalised to the data yield for illustration purposes. The difference between data and the background model is shown at the bottom of each panel. F c a t a t a t s c 5 f o Fig. 4. Comparison of the expected limits in the (tanβ, M A) plane for the three channels separately, and their combination for the mmax h scenario with (a) μ < 0 and (b) μ > 0. Fig. 4. 4.3. Systematic uncertainties Comparison of the expected limits in the (tanβ, M A) plane for the three channels separately, and their combination for the mmax h scenario with (a) μ < 0 and (b) μ > 0. the b-quark eﬃciency, luminosity, and jet energy scale, which are assumed to be 100% correlated. The theoretical uncertainties on the signal are other sources of correlated systematic uncertainty among all channels. They are dominated by parton density func- tion uncertainties, renormalisation and factorisation scales. We as- sign an uncertainty of 15% on the theoretical cross sections that is correlated across all processes. Fig. 3. Distributions of the dijet invariant mass, taken from Ref. [8], in the signal region deﬁned by Dbbb > 0.65 (a) and in a control region deﬁned by Dbbb < 0.12 (b) for the dominant bbb 3-jet channel is shown. The line shows the background model, the solid histogram the component coming from bbb, the points with error bars show the data. The background is normalised to the data yield for illustration purposes. The difference between data and the background model is shown at the bottom of each panel. 4.3. Systematic uncertainties Depending on the source, we consider the effect of systematic uncertainties on the normalisation and/or on the shape of the dif- ferential distributions of the ﬁnal discriminants. 4 jets. The data and signal yields are given in Table 3. In addition, a likelihood discriminant, Dbbb, based on six kinematic variables is employed. Two separate likelihoods, one for the mass region 90 ⩽M A < 140 GeV and the other for 140 ⩽M A < 300 GeV, are used. The dominant heavy ﬂavor multijet backgrounds are esti- mated using a data driven technique. The background in the triple b-tagged sample is estimated by applying a 2D-transformation in Mb¯b and Dbbb, derived from the ratio of the number of MC events in the triple and double b-tagged samples, to the double b-tagged data sample. The method signiﬁcantly reduces the sensitivity of the background model to the underlying kinematics of the simu- lated events and the modelling of the geometric acceptance of the detector. The appropriate composition of the simulated samples is determined by comparing the sum of the transverse momenta of the jets in each event in simulation and data for various b- tagging criteria. The invariant mass distribution of the jet pairing with the highest Dbbb value is used as the ﬁnal discriminant. The distribution for the dominant 3-jet channel is shown in Fig. 3(a). In Fig. 3(b), good agreement is observed between the data and In the di-tau channels, the Z(+ jets) background uncertainties are estimated using Z/γ ∗→μ+μ−data control samples, resulting in normalisation uncertainties of 3.2% (5%) for Z(+b-tagged jets) boson production, an inclusive trigger eﬃciency uncertainty of 3% (common to all simulated backgrounds) and a shape-dependent uncertainty of ∼1% from the modelling of the Z boson kinemat- ics. The MJ-background uncertainty ranges from 10% to 40% on the bττ channel yields while it is found to be shape dependent in the ττ channel (up to 100% at high Mhat). For the remaining backgrounds and for signal, we consider uncertainties affecting the normalisation: luminosity (6.1%), muon reconstruction eﬃciency (2.9%), τh reconstruction eﬃciency (4–10%), single muon trigger eﬃciency (1.3%), t¯t (11%) and diboson (7%) production cross sec- tions. 5. Results Nexp ττ+X L = Aggφ × σ model ggφ + Abbφ × σ model bbφ −σ model bgbφ + Abgbφ × σ model bgbφ , + Abgbφ × σ model bgbφ , where the acceptances A are computed using the simulation and include the experimental eﬃciency. The two ﬁrst terms of this equation refers to Higgs boson production without any b quark within the acceptance, while the third term is used for bgbφ pro- duction. There is no difference in the experimental acceptance for the ggφ and bbφ processes with no outgoing b quark within the acceptance. Therefore, we set Abbφ ≡Aggφ. The Higgs boson width, calculated with feynhiggs, is also taken into account [8]. 5. Results We combine the ττ , bττ and bbb channels using the modiﬁed frequentist approach [27]. The test statistic is a negative log-ratio of proﬁled likelihoods [28]: LLR = −2 ln p(data\|H1) p(data\|H0), energy scale (∼10%) only impacts signiﬁcantly the ττ search for both Z boson background and signal Mhat distribution. With the exception of the τh reconstruction eﬃciency, τh energy scale and MJ estimation, which are evaluated for each τh type, these un- certainties are assumed to be 100% correlated across both di-tau channels. where H1 is the test (background + signal) hypothesis, H0 is the null (background only) hypothesis and p are the proﬁle likelihoods based on Poisson probabilities for obtaining the observed number of events under each hypothesis. We deﬁne CLs by CLs ≡CLs+b/CLb, where CLs+b and CLb are the conﬁdence levels for the test and null hypothesis respectively. We exclude signal yields with CLs < 0.05. In the bbb channel, for the dominant MJ background, only sys- tematic variations in the shape of the Mb¯b distribution are con- sidered, as only the shape, and not the normalisation, is used to distinguish signal from background [8]. The dominant sources arise from the measurement of the rate at which light partons fake a heavy ﬂavor jet and the b-tagging eﬃciency. For the signal model, the b-tagging eﬃciency (11–18%), the luminosity (6.1%) and the jet energy scale (2–10%) dominate the experimental uncertainties. The LLR quantity is computed from the Mhat distribution for the ττ channel, the D f distributions for the bττ channel and the Mb¯b distribution for the bbb channel. The NNLO SM cross sections σggφ and σbbφ are taken from [29–36] and [37], respectively, while the NLO SM cross section σbgbφ is taken from mcfm. The model- dependent MSSM to SM cross section ratios are computed with feynhiggs [38]. To avoid double counting between the bbφ and bgbφ processes, we obtain the expected signal yield Nexp ττ+X in the di-tau channels by Most of the experimental uncertainties are uncorrelated be- tween the di-tau and the bbb analyses with the exceptions of D0 Collaboration / Physics Letters B 710 (2012) 569–577 576 Fig. 5. Constraints in the (tanβ, M A) plane from the di-tau combination in the mmax h scenario. These limits very weakly depend on the other MSSM parameters. 5. Results ate M A, the ττ and bττ channels have similar sensitivities, while at high M A, the bbb sensitivity becomes appreciable especially in μ < 0 scenarios. While the sensitivity in the ττ + X channels are barely sensitive to other MSSM parameters than M A and tanβ, the bbb signal yields is much more model dependent. Therefore we also provide a combination of the ττ and bττ searches only. We do not observe any signiﬁcant excess in data above the expected background ﬂuctuations and we proceed to set limits. The limit from the ττ + X combination is shown in Fig. 5 and the full com- bination limits in different MSSM scenarios are shown in Fig. 6. ate M A, the ττ and bττ channels have similar sensitivities, while at high M A, the bbb sensitivity becomes appreciable especially in μ < 0 scenarios. While the sensitivity in the ττ + X channels are barely sensitive to other MSSM parameters than M A and tanβ, the bbb signal yields is much more model dependent. Therefore we also provide a combination of the ττ and bττ searches only. We do not observe any signiﬁcant excess in data above the expected background ﬂuctuations and we proceed to set limits. The limit from the ττ + X combination is shown in Fig. 5 and the full com- bination limits in different MSSM scenarios are shown in Fig. 6. In summary, we present MSSM Higgs boson searches in three ﬁnal states: ττ , bττ and bbb. These different searches are com- bined to set limits in the (tanβ, M A) plane in four different MSSM scenarios. Furthermore, we combine the ττ and bττ channels to obtain MSSM-scenario-independent limits. We exclude a substan- tial region of the MSSM parameter space, especially for M A < 180 GeV where we exclude tanβ > 20–30. These are the tightest constraints from the Tevatron on the production of neutral Higgs bosons in the MSSM and are comparable to the published LHC lim- its [10,11], especially at low M A. Fig. 5. Constraints in the (tanβ, M A) plane from the di-tau combination in the mmax h scenario. These limits very weakly depend on the other MSSM parameters. References [20] T. Sjöstrand, et al., J. High Energy Phys. 0605 (2006) 026. [21] J. Campbell, R.K. Ellis, F. Maltoni, S. Willenbrock, Phys. Rev. D 67 (2003) 095002. [21] J. Campbell, R.K. Ellis, F. Maltoni, S. Willenbrock, Phys. Rev. [22] M.L. Mangano, et al., J. High Energy Phys. 0307 (2003) 001. [1] H.P. Nilles, Phys. Rep. 110 (1984) 1; [1] H.P. Nilles, Phys. Rep. 110 (1984) 1; H.E. Haber, G.L. Kane, Phys. Rep. 11 [23] Z. Was, Nucl. Phys. Proc. Suppl. 98 (2001) 96, Version 2.5.04. H.E. Haber, G.L. Kane, Phys. Rep. 117 (1985) 75. [24] D.J. Lange, Nucl. Instrum. Methods Phys. Res. A 462 (2001) 152, Version 9.39. [2] B. Ananthanarayan, G. 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Acknowledgements We thank the staffs at Fermilab and collaborating institutions, and acknowledge support from the DOE and NSF (USA); CEA and CNRS/IN2P3 (France); FASI, Rosatom and RFBR (Russia); CNPq, FAPERJ, FAPESP and FUNDUNESP (Brazil); DAE and DST (India); Colciencias (Colombia); CONACyT (Mexico); KRF and KOSEF (Ko- rea); CONICET and UBACyT (Argentina); FOM (The Netherlands); STFC and the Royal Society (United Kingdom); MSMT and GACR (Czech Republic); CRC Program and NSERC (Canada); BMBF and DFG (Germany); SFI (Ireland); The Swedish Research Council (Swe- den); and CAS and CNSF (China). We test two MSSM benchmark scenarios [39], no-mixing and mmax h , and we vary the sign of the Higgsino mass parameter, μ. The expected sensitivities for two mmax h scenarios are shown in Fig. 4 for the three different searches and for their combination. At low M A, the bττ channel dominates the sensitivity. For intermedi- Fig. 6. Constraints in the (tanβ, M A) plane for different MSSM scenarios from the combined Higgs bosons searches. Fig. 6. Constraints in the (tanβ, M A) plane for different MSSM scenarios from the combined Higgs bosons searches. D0 Collaboration / Physics Letters B 710 (2012) 569–577 577 References Rev. Lett. 88 (2002) 201801. [10] S. Chatrchyan, et al., CMS Collaboration, Phys. Rev. Lett. 106 (2011) 231801. [30] C. Anastasiou, K. Melnikov, Nucl. Phys. B 646 (2002) 220 1] G. Aad, et al., ATLAS Collaboration, Phys. Lett. B 705 (2011) 174 [31] V. Ravindran, J. Smith, W.L. van Neerven, Nucl. Phys. B 665 (2003) 325. [31] V. Ravindran, J. Smith, W.L. van Neerven, Nucl. Phys. B 6 [12] V.M. Abazov, et al., D0 Collaboration, Nucl. Instrum. Methods Phys. Res. A 565 (2006) 463; M. Abolins, et al., Nucl. Instrum. Methods Phys. Res. A 584 (2008) 75; R. Angstadt, et al., Nucl. Instrum. Methods Phys. Res. A 622 (2010) 298. [12] V.M. Abazov, et al., D0 Collaboration, Nucl. Instrum. Methods Phys. Res. 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https://openalex.org/W4392172177	https://pubs.rsc.org/en/content/articlepdf/2024/ta/d4ta00427b	English	null	Copper Shape-Templated N-Doped Carbons: Exercising Selective Surface Area Control for Lithium-Ion Batteries & Beyond	Journal of materials chemistry. A	2,024	cc-by	8,214	aDepartment of Chemistry, The University of Texas at Austin, Austin, Texas 78712-1224, USA. E-mail: mullins@che.utexas.edu bMcKetta Department of Chemical Engineering, The University of Texas at Austin, Austin, Texas 78712-1589, USA cTexas Materials Institute, The University of Texas at Austin, Austin, Texas 78712- 1591, USA † Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4ta00427b Open Access Article. Published on 26 February 2024. Downloaded on 10/24/2024 This article is licensed under a Creative Commons Attribution 3.0 U Doped-carbons are attractive alternatives to traditional graphite anodes in lithium-ion batteries because they maintain the low cost and operating voltage window of carbon while providing higher capacities. Key challenges lie in creating useful doped-carbons with controllable properties and understanding which properties are most beneﬁcial for these materials. Here, N-doped carbons are synthesized via a metal-templating platform, which provides selective control of both the chemical composition and surface area, allowing the two properties to be decoupled from one another. Copper metal particles in dendritic, spheroidal, and ﬂake shapes are utilized as metal templates with graphitic carbon nitride precursor to create N-doped carbons. The resulting carbons are chemically similar, deﬁned by XPS, pXRD and Raman spectroscopy. However, the surface area and pore distributions are distinctly diﬀerent, as quantiﬁed by gas adsorption analysis and observed via SEM, which results in capacity diﬀerences of 100 mA h g−1 when applied as active materials in lithium-ion batteries. Received 18th January 2024 Accepted 26th February 2024 Received 18th January 2024 Accepted 26th February 2024 DOI: 10.1039/d4ta00427b rsc.li/materials-a have increased specic capacities. N can be doped into sp2 hybridized carbon structures in one of four typical bonding motifs – pyrrolic, pyridinic, graphitic, and oxidized.11 Pyrrolic and pyridinic N groups are sp2 hybridized, substituted into ve and six member carbon rings, respectfully, while graphitic and oxidized N groups are sp3 hybridized, substituted into six member carbon rings.9 Pyridinic and pyrrolic N functionalities are generally preferred for LIB anodes as the lone pair provides an additional reversible lithiation site, boosting capacity, while graphitic N functionalities are less preferred due to their tendency to bind irreversibly with lithium.12 The concentration of N dopant is also critical, as excessive dopant levels can decrease the electrical conductivity of the material.13 Therefore, it is important that both the type and quantity of N dopants are balanced. 1. J. Mater. Chem. A, 2024, 12, 7923–7931 \| 7923 Journal of Materials Chemistry A Open Access Article. Published on 26 February 2024. Downloaded on 10/24/2024 5:37:26 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. View Article Online View Journal \| View Issue Copper shape-templated N-doped carbons: exercising selective surface area control for lithium-ion batteries & beyond† Samantha N. Lauro, a James N. Burrow, b Benjamin G. Broekhuis, b Philippe E. Papa ab and C. Buddie Mullins abc Copper shape-templated N-doped carbons: exercising selective surface area control for lithium-ion batteries & beyond† Copper shape-templated N-doped carbons: exercising selective surface area control for lithium-ion batteries & beyond† Cite this: J. Mater. Chem. A, 2024, 12, 7923 Samantha N. Lauro, a James N. Burrow, b Benjamin G. Broekhuis, b Philippe E. Papa ab and C. Buddie Mullins abc Samantha N. Lauro, a James N. Burrow, b Benjamin G. Broekhuis, b Philippe E. Papa ab and C. Buddie Mullins abc 1. Introduction Lithium-ion batteries (LIBs) have become a pivotal technology in enabling the global transition to renewable energy sources.1 Technological advancements demanding batteries with perfor- mance tailored towards each application have led to extensive research into novel chemistries for both electrolytes2–4 and active materials,5–7 with one such component being the anode. The traditional anode active material in LIBs is graphite, in which lithium ions are stored between the carbon sheets by intercalation, resulting in a specic capacity of ∼372 mA h g−1.8 Graphite is low in cost and abundant compared to other active materials, but higher energy and power densities are needed for future technologies. Doped-carbons are an alternative anode material to graphite, with synthetic platforms that can employ virtually any organic or bio-derived precursor, leading to a variety of materials that can be produced with diﬀerent dopants, surface areas, pore distributions, and crystallinities.9,10 There any many synthetic methods to produce N-doped carbons, usually consisting of the carbonization of a nitrogen- containing precursor.14 The precursor usually falls into one of three categories-bio-derived precursors, such as an organic waste product from the food industry, lab-synthesized nitrogen containing polymers, or graphitic carbon nitride. Bio-derived precursors are readily available, such as food and animal waste,15,16 and is an attractive platform as a solution for reducing waste products. However, these materials oen have low purity, since many dopants besides nitrogen are present, making chemical analysis of the source of LIB performance benets challenging to isolate. Polymeric compounds on the other hand, such as polyacrylonitrile and polyaniline, can be synthetically controlled to include only desired dopants, highly Nitrogen (N)-doped carbons have been of specic interest because N-doped carbons are simple to synthesize, and can J. Mater. Chem. A, 2024, 12, 7923–7931 \| 7923 This journal is © The Royal Society of Chemistry 2024 View Article Online Paper w Article Online Journal of Materials Chemistry A Paper puried, and produce unique nanostructures, but the synthetic process may involve toxic chemicals and be time intensive, and the resulting N-dopant levels aer carbonization are moderate (usually less than 20 atomic%).17,18 In contrast, graphitic carbon nitride (g-C3N4) can be produced from inexpensive precursors such as urea and melamine,19 consists of only carbon and nitrogen, and can result in N-doped carbons with nitrogen contents upwards of 30 atomic%, making it an ideal material to use for fundamental studies on N-doped carbon anodes. Open Access Article. Published on 26 February 2024. Downloaded on 10/24/2024 5:37:26 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. All materials were used as received without further purication. Urea (synthetic grade), copper spheroidal powder (99%), copper dendritic powder (99.7%), nitric acid (70%, ACS reagent grade), and dimethyl carbonate (DMC, >99%) were purchased from Millipore Sigma. Copper ake powder (99.9%) was acquired from Beantown Chemical, lithium foil (750 mm thick, 99.9%) from ThermoFisher Scientic, and lithium bis(uorosulfonyl) imide salt (LiFSI, 99%) from Oakwood Chemical. 1-Methyl-2- pyrrolidone (NMP, 99%), ethylene carbonate (EC, >99.0%), and uoroethylene carbonate (FEC, >98.0%) were acquired from Tokyo Chemical Industry. Copper foil, polyvinylidene uoride (PVDF, >99.5%) and conductive carbon black (SuperP) were obtained from MTI Corporation. One synthetic technique, metal-templating, has shown promise in controlling the nal properties of N-doped carbons.22–24 While metals such as zinc and copper have been readily used as nanostructured hosts for LIB anodes,25–28 in the context of carbon synthesis, metal-templating refers to the process in which a carbon precursor is pyrolyzed in the presence of a metal, usually as a mixture of powders. The metal is removed aer the carbonization, oen by acid-washing, to yield resulting carbon materials with unique properties. Fahlman et al. mixed magnesium particles with graphitic carbon nitride (g-C3N4) before pyrolysis to create N-doped carbon anodes with highly preferred pyrrolic N functionalities.20 Mao et al. created hollow N-doped carbon spheres by using a zinc template, and altered the N concentration by performing the pyrolysis over a range of temperatures (650–850 °C).29 Previous work from our group investigated the use of iron and copper metal templates, and revealed that the choice of metal allowed for control over the type of N dopants present.24 Iron templates resulted in N- doped carbons with preferred graphitic N functionalities, while copper promoted pyridinic and pyrrolic functionalities, with the latter being more preferential for anodes of LIBs. Copper is a unique metal-template option, because it remains a solid at the temperatures of carbon pyrolysis (unlike zinc and magnesium, with melting points at ∼420 °C and 650 °C, respectively), and does not form a carbide intermediate like other templates (such as zinc and iron). Copper is low in cost, abundant, and can be produced in diﬀerent nanostructured- shapes at the commercial level and in research labs, providing an array of particle size and shape options to choose from.30–32 As a metal template, changing the contact surface area between the carbon and copper resulted in diﬀerent physical morphol- ogies of the N-doped carbons. 2.2 Carbon synthetic methods Urea derived graphitic carbon nitride (g-C3N4) was synthesized by a thermal condensation method.37 Briey, 75 g urea was placed in an alumina crucible covered with aluminum foil. The crucible was heated in a box furnace under air atmosphere using a ramp rate of 2.3 °C min−1 and held at 550 °C for four hours. The crucible cooled naturally to room temperature and the carbon nitride was collected. To create nitrogen doped carbons, 1.2 g of g-C3N4 was mixed with 1.2 g of copper metal particles with a mortar and pestle. Three diﬀerent samples were prepared, where the copper particles were either dendritic, spheroidal, or ake shaped. These shapes were according to the manufacturer label. The mixture was then transferred to a covered alumina crucible and carbonized under owing argon (120 cc min−1) in a quartz tube furnace. The temperature was ramped at a rate of 5 °C min−1 and held at 40 °C for one hour to purge residual air from the tube, then held at 100 °C for one hour to remove residual water, and carbonized at a nal temperature of 750 °C for two hours before cooling naturally back to room temperature. The resulting carbons were washed with concentrated nitric acid (HNO3) three times to remove the copper and residual salt by-products and then washed with excess deionized water until pH neutral. The N-doped carbons were dried in a box furnace at 120 °C overnight and collected. The N-doped carbons are named by the shape of copper template used (Dendritic, Spheroidal, Flake) and therefore correspond to dendritic-shape copper, spheroidal-shape copper, and ake-shaped copper templated carbons, respectively. Importantly, the tunability of this synthetic platform enables fundamental studies on the role of diﬀerent properties of N- doped carbons for a given application. A majority of previous studies have shown that the choice of metal template dictates the surface chemical bonding motifs of these N-doped carbons, and has focused on changing the chemical identity of the metal (i.e., magnesium, zinc, iron, copper).20,22–24 In contrast, the Open Access Article. Published on 26 February 2024. Downloaded on 10/24/2024 5:37:26 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Physical morphology encom- passes particle shape, size, texture, surface area, and porosity, which in an LIB anode inuences ionic transport through both the electrode lm and through the carbon particles, solid electrolyte interphase growth (SEI), and the contribution of capacitive and pseudo-capacitive surface storage mechanisms to overall observed specic capacity and rate capability.33–36 1. Introduction Carbon nitride can be converted to an N-doped carbon through pyrolysis, sometimes in the presence of metals, metal-salts, and salt mixtures.20,21 present study investigates and demonstrates that altering the physical shape of the metal template controls the resulting physical morphology (and porosity) of the carbons, without signicantly impacting surface chemistry. 7924 \| J. Mater. Chem. A, 2024, 12, 7923–7931 2.3 Material characterization Crystallite size was determined by obtaining powder X-ray Diﬀraction (pXRD) patterns by using a Rigaku MiniFlex 600II (Cu Ka radiation, l = 1.5418 Å) in continuous scan mode from 7924 \| J. Mater. Chem. A, 2024, 12, 7923–7931 This journal is © The Royal Society of Chemistry 2024 View Article Online View Article Online Paper Journal of Materials Chemistry A Journal of Materials Chemistry A 2q = 10° to 80° with a step width of 0.02 at 2.5° min−1 with a single crystal zero background silicon sample holder. The crystallite sizes and d-spacings were determined by using a Lorentz t for peaks at approximately 24° and 44° to account for diﬀraction of 002 and 101 planes, respectively. The degree of graphitization of each sample was determined by Raman spectroscopy. Spectra were obtained with a Renishaw inVia Raman Microscope equipped with a 100 mW green laser which was employed to excite the samples at 514 nm with a spectral shicentered at 1500 cm−1. The ID/IG ratio was determined by tting the spectra with four Voigt peaks representing the D, D, D**, and G bands at approximately 1230, 1366, 1521, and 1595 cm−1, respectively, and using the ratio between the D and G band intensity. X-ray photoelectron spectroscopy (XPS) spectra were acquired on a Kratos Axis Ultra X-ray photoelectron spectrometer. CasaXPS soware was used to t survey scans and the high-resolution spectra of each elemental region (C 1s, N 1s, O 1s), and all spectra were shied to account for the adventi- tious carbon peak at 284.7 eV.21 Scanning electron micrographs (SEM) were collected on a FEI Quanta 650 ESEM at a scanning voltage of 30.0 kV. Textural properties of the resulting carbons were studied by acquiring isotherms of N2 at 77 K and CO2 at 273 K using a Quantachrome Autosorb iQ-MP (Anton Paar). Before gas adsorption analysis, around 100 mg of powdered sample was loaded into glass sample tubes and degassed under dynamic vacuum at 200 °C overnight. With ASiQ WinTM so- ware, surface areas were quantied with multi-point BET anal- ysis with optimized point selection to account for the Rouquerol criteria and the microporous nature of these materials. These multi-point BET analyses are provided in the ESI (Fig. S7†). 3. Results & discussion Fig. 1a outlines the synthetic procedure. Commercially available copper particles of three diﬀerent morphologies – dendritic, spheroidal, and ake shaped (as labelled by the manufacturers) – were used as templates. N-doped carbons were produced by carbonizing a mixture of the copper templates with urea-derived g-C3N4 at 750 °C for two hours. The resulting product was washed with concentrated nitric acid (HNO3) to remove the metal templates and any salt by-products. Scanning electron microscopy (SEM) revealed the morphology of the resulting carbon materials. The g-C3N4 precursor exhibited a highly textured, amorphous particle morphology as seen in Fig. 1b, which was not retained in the nal N-doped carbons (Fig. 1f–h). The copper metal templates have distinct morphological diﬀerences: the dendritic copper has a branched morphology and asymmetrical shape (Fig. 1c); the spheroidal copper is round, with a smooth surface (Fig. 1d); and the ake copper appears to be an agglomeration of secondary string-like particles (Fig. 1e). The resulting N-doped carbons had morphologies reminiscent of their respective templates and visually distinctive from each other. The dendritic templated carbon (Fig. 1f) exhibited a textured surface with grooves and wrinkles, with approximately 30-micron sized particles, and asymmetrical particles shapes. The spheroidal templated carbons (Fig. 1g) were smaller in size (∼5–10 mm) and had a smoother observed texture, while the ake templated carbons (Fig. 1h) were irregular in shape and size (around 10 mm). Additional SEM images of the carbon materials are shown in Fig. S1,† and population images of the copper templates and carbons are shown in Fig. S2.† The carbon secondary particle size appears to be related to the size of the copper templates used, with large dendritic templates resulting in larger sized N- doped carbon particles, and smaller spheroidal templates resulting in smaller carbon particle sizes. The metal template remains in a solid phase during synthesis (as opposed to salt- melts), so the surface area of the copper templates available 2.3 Material characterization Pore size distributions were derived from the adsorption branch of the isotherm with quenched-solid density functional theory (QSDFT) assuming a convolution of spherical, cylindrical, and slit-shaped pores for N2 adsorption, while non-local density functional theory (NLDFT) was employed for analysis of ultra- microporosity from CO2 isotherms at 273 K on carbon. The total pore size distribution was acquired by stitching together results from CO2 (<1.5 nm) and N2 (1.5–50 nm). Microporosity was dened as the cumulative pore volume at a pore diameter of 2 nm. Total pore volume (up to 200 nm) was quantied using total quantity of adsorbed N2 at P/P0 = 0.99. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. with 150 mL of electrolyte. The slurry-cast materials were cut into 13 mm diameter electrodes and tested against a 750 mm thick lithium metal counter electrode using a 1.2 M lithium bis(- uorosulfonyl)imide (LiFSI) in 3 : 7 (v/v) ethylene carbonate (EC) : dimethyl carbonate (DMC) with 5% v/v uoroethylene carbonate (FEC) electrolyte system. Long-term and variable rate cycling tests were performed using a multi-channel battery test system (Neware BTS4000- 5V10 mA) using a 10 mV–3 V potential window. Long-term cycling consisted of three conditioning cycles at a rate of 0.02 A g−1 and 200 subsequent cycles at a rate of 0.2 A g−1. Variable rate cycling consisted of three cycles at 0.02 A g−1 fol- lowed by ve cycles of each faster rate (0.05 A g−1, 0.1 A g−1, 0.2 A g−1, 0.5 A g−1, 1.0 A g−1, and 2.0 A g−1) and recovered at a rate of 0.2 A g−1. Cyclic voltammograms were obtained over the potential window of 10 mV–3 V vs. Li/Li+ at a scan rate of 0.1 mV s−1 on a multi-channel battery test system (Arbin BT 2043). All reported electrochemical results are from duplicate cells (Fig. S7 and S8†). This journal is © The Royal Society of Chemistry 2024 J. Mater. Chem. A, 2024, 12, 7923–7931 \| 7925 2.4 Electrochemical measurements Electrode lms were fabricated by mixing the active carbon materials with a conductive carbon black additive (SuperP) and polyvinylidene uoride (PVDF) binder in an 80 : 10 : 10 mass ratio in 1-methyl-2-pyrrolidone (NMP) solvent. The slurries were coated onto 8 mm thick copper foil using a doctor blade set at 150 mm, and lms were transferred to an oven to dry for 3 hours at 120 °C before pulling vacuum for 12 hours to remove residual solvent. Resulting electrodes had a mass loading between 1.5– 1.8 mg cm−2 and lm thicknesses ∼80 mm. Materials were tested electrochemically in stainless steel 2032 coin cells assembled in an argon atmosphere glovebox using three 500 mm thick stainless steel spacers, one circular spring, and one polypropylene separator (Celgard 2400) ooded J. Mater. Chem. A, 2024, 12, 7923–7931 \| 7925 This journal is © The Royal Society of Chemistry 2024 View Article Online Journal of Materials Chemistry A Journal of Materials Chemistry A Fig. 1 Schematic illustration of N-doped carbon synthesis procedure for using diﬀerent shaped copper metal templates (a). SEM images of g- C3N4 precursor (b), dendritic copper particle (c), spheroidal copper particle (d), ﬂake copper particle (e),and the N-doped carbons produced using dendritic (f), spheroidal (g), and ﬂake (h) shaped copper templates. cle is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 1 Schematic illustration of N-doped carbon synthesis procedure for using diﬀerent shaped copper metal templates (a). SEM images of g- C3N4 precursor (b), dendritic copper particle (c), spheroidal copper particle (d), ﬂake copper particle (e),and the N-doped carbons produced using dendritic (f), spheroidal (g), and ﬂake (h) shaped copper templates. Fig. 1 Schematic illustration of N-doped carbon synthesis procedure for using diﬀerent shaped copper metal templates (a). SEM images of g- C3N4 precursor (b), dendritic copper particle (c), spheroidal copper particle (d), ﬂake copper particle (e),and the N-doped carbons produced using dendritic (f), spheroidal (g), and ﬂake (h) shaped copper templates. to the g-C3N4 is likely responsible for particle shape and morphology of the nal N-doped carbons. Going forward, the carbon nomenclature will be indicated by their copper template shape (i.e. dendritic, sphere, and ake carbon). and G bands peak intensities (example tting shown in Fig. S3 and values in Table S2†) were found to be nearly identical for the three carbons (ID/IG values of 1.20, 1.19, and 1.19 for ake, spheroidal, and dendritic carbons, respectively). 2.4 Electrochemical measurements ( ) To better understand the nanostructure of the resulting carbon particles, powdered X-ray diﬀraction (XRD) was per- formed (Fig. 2a). All of the carbon samples produced a broad diﬀraction pattern with a large peak centered at approximately 2q = 27.5°, representing the 002 reection. The broadening of this peak as compared to the sharper peak from the g-C3N4 precursor represents a turbostratic carbon structure, which is indicative of crystallites with short-range but a lack of long range order.38 Scherrer analysis gives an estimation of the crystallite parameters, shown in Table S1.† The d-spacing in the 002 plane is ∼3.4 Å for all of the carbons. However, there is a slight diﬀerence in the lateral crystallite coherence length (La), with the dendritic carbon having a larger La of 8.71 Å compared to the spheroidal and ake carbon, which had La values of 6.98 and 6.45 Å, respectively. The La represents the in-plane order of the crystallite and is directly related to the defect density.39 Therefore, as a larger La value correlates to longer range order, the dendritic carbon likely exhibited slightly fewer structural defects than the spheroidal and ake carbons. Nonetheless, all three samples show similar crystal structures overall. The graphitic order of the carbons was further compared via Raman spectroscopy (Fig. 2b). The ID/IG ratios calculated from the D Only very small diﬀerences in the surface chemistry of the carbons were observed with X-ray photoelectron spectroscopy (XPS). The total elemental composition summarized in Fig. 2c is in line with previous studies of copper templating,24 comprised of ∼63% carbon, 30% nitrogen, 5% oxygen, and less than 2% of residual copper. The corresponding XPS survey spectra can be found in Fig. S4.† The N bonding motifs were deconvoluted by tting the N 1s region in Fig. S5† with four peaks at binding energies of ∼398.1, 398.7, 399.8, and 400.8 eV to represent the pyridinic, pyrrolic, graphitic, and oxidized N functionalities, respectively.40 The diﬀerence in chemical motifs is near iden- tical, with only 1% or less diﬀerence between the carbons, and preferential formation of pyridinic N from the use of copper (Fig. 2d). As demonstrated above, the carbons resulting from the diﬀerent copper templates were nearly identical in chemical structure and nanocrystalline (dis)order. In contrast, the phys- ical morphology diﬀered signicantly depending on the morphology of the templates employed. This journal is © The Royal Society of Chemistry 2024 2.4 Electrochemical measurements While SEM provided a qualitative view of the carbon particle morphology at the micron-scale, gas sorption isotherms of nitrogen (N2) and carbon dioxide (CO2) were used to generate a more quantitative 7926 \| J. Mater. Chem. A, 2024, 12, 7923–7931 This journal is © The Royal Society of Chemistry 2024 7926 View Article Online Paper Journal of Materials Chemistry A Fig. 2 Characterization of dendritic, spheroidal, and ﬂake templated carbons. (a) XRD patterns of carbons and g-C3N4 precursor. (b) Raman spectra of carbons. (c) Summary of elemental composition of carbons from XPS analysis of survey spectra. (d) Summary of nitrogen bonding motifs in carbons from XPS analysis of the N 1s spectra. Fig. 2 Characterization of dendritic, spheroidal, and ﬂake templated carbons. (a) XRD patterns of carbons and g-C3N4 precursor. (b) Raman spectra of carbons. (c) Summary of elemental composition of carbons from XPS analysis of survey spectra. (d) Summary of nitrogen bonding motifs in carbons from XPS analysis of the N 1s spectra. Fig. 2 Characterization of dendritic, spheroidal, and ﬂake templated carbons. (a) XRD patterns of carbons and g-C3N4 precursor. (b) Raman spectra of carbons. (c) Summary of elemental composition of carbons from XPS analysis of survey spectra. (d) Summary of nitrogen bonding motifs in carbons from XPS analysis of the N 1s spectra. comparison of the carbons' surface areas and pore size distributions,41–44 as summarized in Fig. 3a. The dendritic carbon exhibited the smallest BET surface area,44 measured at 308 m2 g−1, while the spheroidal carbon had the largest at 406 m2 g−1. Meanwhile, the ake carbon had an intermediate surface area of at 380 m2 g−1. This diﬀerence in exposed surface area is primarily attributed to the volume of mesopores. comparison of the carbons' surface areas and pore size distributions,41–44 as summarized in Fig. 3a. The dendritic carbon exhibited the smallest BET surface area,44 measured at 308 m2 g−1, while the spheroidal carbon had the largest at 406 m2 g−1. Meanwhile, the ake carbon had an intermediate surface area of at 380 m2 g−1. This diﬀerence in exposed surface area is primarily attributed to the volume of mesopores. comparison of the carbons' surface areas and pore size distributions,41–44 as summarized in Fig. 3a. The dendritic carbon exhibited the smallest BET surface area,44 measured at 308 m2 g−1, while the spheroidal carbon had the largest at 406 m2 g−1. 2.4 Electrochemical measurements Meanwhile, the ake carbon had an intermediate surface area of at 380 m2 g−1. This diﬀerence in exposed surface area is primarily attributed to the volume of mesopores. Although the cumulative volume of micropores is similar between the templated carbons, each carbon exhibits a unique pore size distribution (Fig. 3b). While all of the carbons dis- played a similar volume of micropores (<2 nm), the volume of meso- (2–50 nm) and macropores (50–200 nm) in the dendritic carbon was less than half of that in the spheroidal and ake carbon, with the spheroidal carbon having the largest volume of Although the cumulative volume of micropores is similar between the templated carbons, each carbon exhibits a unique pore size distribution (Fig. 3b). While all of the carbons dis- played a similar volume of micropores (<2 nm), the volume of meso- (2–50 nm) and macropores (50–200 nm) in the dendritic carbon was less than half of that in the spheroidal and ake carbon, with the spheroidal carbon having the largest volume of Fig. 3 Surface area characterization of dendritic, spheroidal, and ﬂake templated carbons. (a) BET derived surface area and pore volume of carbons. (b) Pore size distributions derived via combination of quenched solid and nonlocal density functional theory (QSDFT and NLDFT) analysis of N2 and CO2 isotherms, respectively. Fig. 3 Surface area characterization of dendritic, spheroidal, and ﬂake templated carbons. (a) BET derived surface area and pore volume of carbons. (b) Pore size distributions derived via combination of quenched solid and nonlocal density functional theory (QSDFT and NLDFT) analysis of N2 and CO2 isotherms, respectively. J. Mater. Chem. A, 2024, 12, 7923–7931 \| 7927 This journal is © The Royal Society of Chemistry 2024 View Article Online Paper ew Article Online Journal of Materials Chemistry A Paper 17 mA h g−1 of capacity compared to 54 and 92 mA h g−1 for spheroidal and ake carbon, respectively. The diﬀerence in performance between the templated carbons can be further understood by examining the diﬀerential capacity curves (dQ/ dV, mA h g−1 V−1) for insight into the lithium storage processes. The normalized diﬀerential capacities at 0.05 A g−1 for the dendritic, spheroidal, and ake copper are overlayed in Fig. 4b (derived from the charge discharge proles in Fig. 4c–e). 2.4 Electrochemical measurements The extraction of lithium from the N-doped carbons can be attrib- uted to a mixture of three processes: faradaic storage in the carbon layers (<∼0.8 V), (pseudo)capacitive storage at defects and in pores (∼0.8–2.2 V), and reversible binding of lithium- ions to nitrogen sites (>∼2.2 V).24,52–54 Although there is signif- icant overlap in the specic potentials at which these lithium storage mechanisms occur, our choice of representative potential ranges is in accordance with previous literature on copper templated N-doped carbons.24 As clearly visible in Fig. 4b, the dendritic carbon exhibited proportionally less capacity contribution from (pseudo)capacitance in pores and defects than did the ake or spheroidal carbons. This result explains the reduced specic capacity of the dendritic carbon at faster rates, where capacitive mechanisms of lithium-ion storage become dominant, and can be directly related to the smaller BET surface area and porosity of the dendritic carbon.55 The signicance of this diﬀerence is further exemplied in the long-term cycling at a rate of 0.2 A g−1, shown in Fig. 5. Over the course of 200 cycles, the dendritic, spheroidal, and ake carbons demonstrated average capacities of 416, 483, and 541 mA h g−1, respectively. Despite identical chemical compo- sition, more than 100 mA h g−1 of capacity diﬀerence between the dendritic and ake carbon material seemingly resulted from the diﬀerences in physical morphology and nanostructure alone. However, while the spheroidal carbon exhibited the largest measurable BET surface area of the three templated carbons, the ake carbon achieved the largest capacities at faster cycling rates. This diﬀerence in performance between the ake and spheroidal carbon is likely due to the diﬀerence in the pore-size distribution, especially in the ultra-micropore (<0.7 nm) region, as shown in Fig. 3b. The ake carbon exhibits three populations of ultra-micropores at approximately 0.35, 0.48, and 0.6 nm in size, while the spheroidal and dendritic carbons only exhibit a single population of approximately 0.52 nm. The full mechanistic role of ultra-micropores in lithium storage is currently a topic of heavy debate in the literature. Kang et al. has reported that ultra-micropores <0.4 nm increase carbon rate performance by a suggested size-sieving mechanism, where the pores are accessible to lithium-ions but inaccessible to elec- trolyte molecules, reducing decomposition and irreversible capacity loss.56 Lee et al. This journal is © The Royal Society of Chemistry 2024 2.4 Electrochemical measurements utilizes a rst principles approach to demonstrate that lithium-ions can occupy the pore cavity, surfaces, and walls in micro and ultra-micropores.57 Ultra- microporosity is suggested to provide active sites for lithium- ions with thin walls which shorten diﬀusion time and these types of pores. This diﬀerence in the volume of pores with larger characteristic length scales (meso/microporosity) is inuenced by the amount of contact area with the metal template. However, increased exposed surface area accom- panies increases in porosity, and a larger electrochemically active surface area requires the formation of more solid elec- trolyte interphase (SEI) for passivation. Since the SEI consumes the Li inventory and is associated with irreversible capacity losses during initial cycling, increased porosity does not always correlate with improved electrochemical performance of carbon anodes in LIBs. In this way, templating the nanostructure, while keeping surface chemistry and crystalline/defect structure consistent, can enable improved performance by exerting a ner control on porosity and surface area, which inuence SEI formation and rate performance. Open Access Article. Published on 26 February 2024. Downloaded on 10/24/2024 5:37:26 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. A, 2024, 12, 7923–7931 This journal is © The Royal Society of Chemistry 2024 7928 View Article Online Journal of Materials Chemistry A Journal of Materials Chemistry A Fig. 4 Variable rate cycling of the dendritic, spheroidal, and ﬂake copper shape templated carbons at current densities of 0.05, 0.1, 0.2, 0.5, 1.0, and 2.0 A g−1 with recovery at 0.2 A g−1. (a) Normalized diﬀerential capacity curves (dQ/dV vs. V) of lithium extraction at 0.05 A g−1 cycling rate for dendritic, spheroidal, and ﬂake carbons. (b) Charge–discharge proﬁles from variable rate cycling of dendritic (c), spheroidal (d), and ﬂake (e) templated carbons. Paper Journal of Materials Chemistry A Fig. 4 Variable rate cycling of the dendritic, spheroidal, and ﬂake copper shape templated carbons at current densities of 0.05, 0.1, 0.2, 0.5, 1.0, and 2.0 A g−1 with recovery at 0.2 A g−1. (a) Normalized diﬀerential capacity curves (dQ/dV vs. V) of lithium extraction at 0.05 A g−1 cycling rate for dendritic, spheroidal, and ﬂake carbons. (b) Charge–discharge proﬁles from variable rate cycling of dendritic (c), spheroidal (d), and ﬂake (e) templated carbons. packs, which can alter the size, length, and continuity of pores in the lm, and subsequently change the ionic diﬀusion pathway through the electrolyte, though is diﬃcult to quantify and visualize in amorphous carbon systems due to the other carbonaceous components used in the electrode lm.34,49,60 Fig. 5 Long-term cycling of dendritic, spheroidal, and ﬂake carbons at a cycling rate of 0.2 A g−1 over 200 cycles. The surface area of carbon anodes is also well-known to inuence the rst-cycle irreversible capacity loss due to the formation of the solid electrolyte interphase (SEI).61 Aer cycling, post-mortem analysis was conducted on all carbon electrodes, via SEM and energy dispersive X-ray (EDX) analysis shown in Fig. S10.† SEI is observed to have grown for all carbons, though no lm fracture or cracking is observed indi- cating minimal volumetric expansion and structural stability. EDX reveals the presence of carbon, nitrogen, oxygen, sulfur and uorine in the SEI, an expected result from the breakage of the carbonate solvents and LiFSI salt used in the electrolyte. Cyclic voltammetry (CV) (Fig. Open Access Article. Published on 26 February 2024. Downloaded on 10/24/2024 5:37:26 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. As mentioned previously, pyridinic and pyrrolic nitrogen defects have been shown to provide reversible lithium-ion storage sites due to the availability of lone pairs of electrons, while graphitic defects have oen resulted in irreversible capacity loss.45 Pender et al. demonstrated that carbons derived from Cu-templated g-C3N4 exhibited pyridinic and pyrrolic defects and obtained high capacities for Li storage.24 The chemical composition of the anode largely inuences the mechanisms of lithium-storage, especially for faradaic processes; however, physical morphology also inuences the nal LIB performance. For example, for capacitive and/or pseudocapacitive storage mechanisms, large (electrochemically-active) surface area is critical.46 Moreover, highly porous structures oen result in increased capacity at fast rates of charge/discharge due to a mixture of increased contribution from capacitive storage processes (which occur over smaller time scales than faradaic storage processes)47 and more electrolyte penetration, increasing the ease of mass transport.48 Additionally, particle size and shape also inuence how the anode material is packed into electrode lms, which can alter the porosity and electronic conductivity of the lm, further altering electrochemical performance.49,50 As a result, even with identical chemical makeup, careful tuning of the physical particle morphology is imperative for the development of high quality battery anodes. The electrochemical performance at variable cycling rates for each of the templated carbons is shown in Fig. 4a. Lithium half- cells of the dendritic, spheroidal, and ake carbons were cycled for ve consecutive cycles at current densities of 0.05, 0.12, 0.2, 0.5, 1.0, and 2.0 A g−1 to evaluate the diﬀerence in rate capa- bility between the materials. All reported cycling is shown as an average between duplicate cells. Individual cell cycling is shown in Fig. S7† and capacities reported with standard deviation in Table S5.† At the slower cycling rate of 0.05 A g−1, the dendritic carbon delivers the largest capacity of 640 mA h g−1, compared to the ake and spheroidal carbons which delivered 610 and 544 mA h g−1, respectively, which are near double the capacity of traditional graphite at the same cycling rate, ∼330 mA h g−1.51 In contrast, at rates of 0.2 A g−1 and above, the dendritic carbon achieved the smallest specic capacity of the three carbons. For example, at a fast charge/discharge rate of 2.0 A g−1 (∼3.7 mA cm−2), the dendritic carbon exhibited only 7928 \| J. Mater. Chem. This journal is © The Royal Society of Chemistry 2024 Open Access Article. Published on 26 February 2024. Downloaded on 10/24/2024 5:37:26 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. measured surface area) did show the smallest absolute capacity loss compared to the spheroidal and ake carbons. Both the peak corresponding to SEI formation and the peaks related to faradaic reactions occurred at the same potentials for all carbon samples (1.2 and 0.1 V vs. Li/Li+, respectively), indicative of their similar chemical composition. The CV curves also have a non- negligible box shape, further indicating (pseudo)capacitive contributions towards the overall Li-ion storage capacities.47 Overall, our results suggest that the ake carbon achieves the largest capacities in the present study for both the long-term and variable rate cycling, and maintains higher and competi- tive capacities to graphite under the same cycling rate condi- tions.51 However, additional considerations, such as initial capacity loss, capacity fade, and lithiation potential/overall cell voltage, are important factors to consider when optimizing anode active materials for LIBs for industrial scalability. The long-term cycling in Fig. 5, for example, has both capacity growth and decay present in the prole. The ake, spheroidal, and dendritic carbons start with initial capacities of 473, 442, and 405 mA h g−1, respectfully, and increase over the rst 125 cycles to values of 578, 503, and 450 mA h g−1, representing a capacity increase of 18, 12, and 10%, respectfully. This phenomena is commonly observed for N-doped carbon anodes in LIBs, and is ascribed to increased electrolyte penetration with the activation of defects and pores.24,62 Aer the 125th cycle, capacity fade starts to occur, with nal capacities at the 200th cycle reaching values of 535, 458, and 402 mA h g−1 for the ake, spheroidal, and dendritic carbons, respectfully, representing a capacity fade of approximately 7.4, 9, and 10.6% from the highest values reached at the 125th cycle. This ux in capacity is informative from a research perspective, but problematic for real-world batteries, which need to provide consistent, predictable performance per application needs. The ake carbon has the highest percent of capacity increase at the 125th cycle and smallest amount of capacity fade at the 200th cycle, while the dendritic carbon demonstrates the opposite trend, suggesting that purposeful structuring of carbon materials may aid in alleviating capacity fade in N-doped carbon anodes. Additional scalability concerns are the coulombic eﬃciency for these materials, tabulated in Table S6,† at approximately 99% for all the carbons, which is high for research materials, but falls shy of the 99.9% required for commercial applications. Open Access Article. Published on 26 February 2024. Downloaded on 10/24/2024 5:37:26 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. S11†) performed at a scan rate of 0.1 mV s−1 and analysis of the peak area decrease between the rst and second CV cycles reveal that 38, 43, and 42% of the charge passed in the initial cycle resulted in irreversible capacity loss for the dendritic, spheroidal, and ake carbons. Although only a maximum of a 5% diﬀerence in rst cycle irreversibility was observed between samples despite signicant morpholog- ical diﬀerences, the dendritic carbon (with the smallest Fig. 5 Long-term cycling of dendritic, spheroidal, and ﬂake carbons at a cycling rate of 0.2 A g−1 over 200 cycles. morphological diﬀerences, which cannot be captured by BET analysis alone, may also play a role in inuencing the lithium- accessible surface area, which is dened by the ability of lithium to reach electrochemically active surfaces sites, span- ning the nano and micro-scale of the active material particle surface area to the macro organization of pore channels within the electrode composite lm itself. The shape and size of the active material particles can change how the electrode lm This journal is © The Royal Society of Chemistry 2024 J. Mater. Chem. A, 2024, 12, 7923–7931 \| 7929 View Article Online Paper Journal of Materials Chemistry A Paper all carbons through XPS, pXRD, and Raman spectroscopy, while the physical morphology diﬀered as qualitatively observed via SEM of diﬀering secondary particle shapes. Surface area and porosity diﬀerences quantied through gas physisorption analysis revealed that surface area diﬀerences up to 100 m2 per gram could be obtained with diﬀering micropore network distributions, despite the use of the same chemical precursors. These morphological diﬀerences inuence the electrochemical evaluation of doped carbon materials, resulting in specic capacity diﬀerences of up to ∼100 mA h g−1 for chemically identical materials. The dendritic, spheroidal, and ake copper shapes are commercially available templates that are easily obtained at scale. 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https://openalex.org/W4297450261	https://zenodo.org/records/7121003/files/BD11.pdf	English	null	AN ANALYSIS OF 'STRESS' IN ENGLISH LANGUAGE:A STUDY OF THE RULES AND APPROACHES.	Zenodo (CERN European Organization for Nuclear Research)	2,013	cc-by	3,480	KEYWORDS: Syllable, word & sentence stress, contrastive stress, stressed & syllable timed language, accent. KEYWORDS: Syllable, word & sentence stress, contrastive stress, stressed & syllable timed language, accent. Bipin Bihari Dash College Of Engineering And Technology, Bhubaneswar A Constituent College Of B.P.U.T, Odisha Electronic Electronic International Interdisciplinary Research Journal (EIIRJ) Bi-monthly Reviewed Journal Jan/Feb 2013 ISSN 2277-8721 Electronic Electronic International Interdisciplinary Research Journal (EIIRJ) Bi-monthly Reviewed Journal Jan/Feb 2013 ISSN 2277-8721 Electronic Electronic International Interdisciplinary Research Journal (EIIRJ) Bi-monthly Reviewed Journal Jan/Feb 2013 ISSN 2277-8721 ISSN 2277-8721 Electronic Electronic International Interdisciplinary Research Journal (EIIRJ) -monthly Reviewed Journal Jan/Feb 2013 AN ANALYSIS OF ‘STRESS’ IN ENGLISH LANGUAGE: ABSTRACT: In linguistics stress is the relative emphasis that may be given to certain syllables in a word, or to certain words in a phrase or sentence. English is what is known as a stressed language; stressed languages that are with differing levels of emphasis for the different words and syllables in the sentence. Native speakers of English use stress naturally. Word phrase or sentence is so natural for them that they don‟t even know they use it. Non-native speakers, who speak English to native speakers without using word stress, encounter two problems. They find it difficult to understand native speakers, especially those speaking fast and the speakers of English will get insight in learning the rules of stress. SYLLABLE According to Das et al, a syllable is a cluster of sounds which forms either a complete word having a meaning (e.g. red), or a part of a word (e.g. the syllable „ab‟ which forms the first of the word „absent‟). Normally, each syllable contains only one vowel (a, e, I, o, or u) or vowel sound. ISSN 2277-8721 language. It is also believed that other skills cannot be taught effectively until speech is placed on the top priority. In this context, Saleem views: “the sensitivity of speaking to form the nucleus of all linguistic activity”(1983, p.168.). Stress, also known as accent is a phenon that has been termed a key to the pronunciation of an English word. This is also a linguistic feature that creates rhythm in English language. Gilani (1996, p.3) writes: “the study of stress/ accent as a paralinguistic feature is so vital that no one can learn the pronunciation of an English word without learning its accent”. Sethi and Dhamija (1989, p.132) points out: “the word accent is an essential part of the word shape”. INTRODUCTION There are four skills of language, listening, speaking, reading, & writing (LSRW). Listening & reading are receiving skills whereas reading & writing are Productive skills. In the language learning process speaking comes after listening. It is proved that effective language learning starts from speech training good speech in English involves command over the system of sounds, stress, and intonation to the V o l - I I I s s u e s - I Page 121 Electronic Electronic International Interdisciplinary Research Journal (EIIRJ) Bi-monthly Reviewed Journal Jan/Feb 2013 ISSN 2277-8721 Electronic Electronic International Interdisciplinary Research Journal (EIIRJ) -monthly Reviewed Journal Jan/Feb 2013 Electronic Electronic International Interdisciplinary Research Journal (EIIRJ) Bi-monthly Reviewed Journal Jan/Feb 2013 ISSN 2277-8721 Electronic Electronic International Interdisciplinary Research Journal (EIIRJ) Bi-monthly Reviewed Journal Jan/Feb 2013 ISSN 2277-8721 ISSN 2277-8721 ISSN 2277-8721 STRESS In English some syllables are spoken with a greater degree of force than the others are. Syllables that are pronounced more forcibly than the others are said to be stressed. Syllables that are pronounced without much force are called unstressed. Roach (1993, p. 85-86) writes: all stressed syllables have one characteristic in common, and that is called prominence; stressed syllables are recognized as stressed because they are more prominent than unstressed syllables. How important is teaching to place stress on the right syllables in English, it would be worth quoting to Clifford et. al. (1985, p.19) who says that stress: is the key to the pronunciation of an English word and the location of the accent always be learned with the word. Examples of primary stress: 'Accident, com'mittee, under „stand, 'calculate, enter'tain LEVELS OF STRESS There are two kinds of stress primary stress & secondary stress. According to Bansal & Harrison “The syllable on which there is a pitch change is said to have the primary or tonic accent. Any other prominent syllable is said to have secondary accent. Primary accent is marked with a vertical bar above and in front of the syllable to which it refers secondary accent with a bar below and in front of the syllable. Examples of secondary stress: , Anthropology, organization, edu, cate, calcu, late, over, simplification SYLLABLE TIMED AND STRESS TIMED LANGUAGES: Language is the vital means of communication. Verities of people and communities are living in the world having different languages with their different written and spoken forms. So far as speaking is concerned these languages may be broadly divided into two categories:  Stress timed  Syllable timed English language is called a stress timed language because the time taken to speak a sentence depends on the number of stressed syllables and not on the total number of syllables. Languages such as Hindi, odia, Bengali etc. on the other hand, are called syllable timed languages, because the time taken to go from one syllable to the next, whether stressed or unstressed, is always the same. The time taken to speak a sentence depends on the total number of syllables and not on the number of stressed syllables, as in English. V o l - I I I s s u e s - I Page 122 w w w . a a r h a t . c o m I S S N 2 2 7 7 - 8 7 2 1 I S S N 2 2 7 7 - 8 7 2 1 I S S N 2 2 7 7 - 8 7 2 1 Electronic Electronic International Interdisciplinary Research Journal (EIIRJ) Bi-monthly Reviewed Journal Jan/Feb 2013 ISSN 2277-8721 STRESS Electronic Electronic International Interdisciplinary Research Journal (EIIRJ) Bi-monthly Reviewed Journal Jan/Feb 2013 ISSN Electronic Electronic International Interdisciplinary Research Journal (EIIRJ) Bi-monthly Reviewed Journal Jan/Feb 2013 ISSN 2277-8721 Electronic Electronic International Interdisciplinary Research Journal (EIIRJ) Bi-monthly Reviewed Journal Jan/Feb 2013 Electronic Electronic International Interdisciplinary Research Journal (EIIRJ) Bi-monthly Reviewed Journal Jan/Feb 2013 ISSN 2277-8721 LEXICAL & PROSODIC STRESS: The stress placed on syllables within words is called word stress or lexical stress. The stressed placed on words within sentences is called sentences or prosodic stress. Languages in which the position of the stress can usually be predicted by a simple rule are said to have fixed stress. For example, in Czech, Finnish, & Hungarian the stress almost always in first syllable of a word. French words are sometimes said to be stressed on the final syllable. Languages in which the position V o l - I I I s s u e s - I Page 123 w w w . a a r h a t . c o m I S S N 2 2 7 7 - 8 7 2 1 Electronic Electronic International Interdisciplinary Research Journal (EIIRJ) Bi-monthly Reviewed Journal Jan/Feb 2013 ISSN 2277-8721 of stress in a word is less predictable are said to have variable stress. This applies to English, Russian, Italian, & Spanish. Electronic Electronic International Interdisciplinary Research Journal (EIIRJ) -monthly Reviewed Journal Jan/Feb 2013 Electronic Electronic International Interdisciplinary Research Journal (EIIRJ) Bi-monthly Reviewed Journal Jan/Feb 2013 ISSN ISSN 2277-8721 of stress in a word is less predictable are said to have variable stress. This applies to English, Russian, Italian, & Spanish. of stress in a word is less predictable are said to have variable stress. This applies to English, Russian, Italian, & Spanish. Category Category Concept of stress with structure and content words: English words can be divided into two categories: content words and structure words. Content words are usually stressed, while structure words are usually unstressed. In certain cases they may receive stress. Here is a list of the structure/ function words: E.g. who are you speaking 'to?  when two structure words are contrasted E.g. I go 'to and 'from the station every day. STRESS VARIATION There are several pairs of words in English which have the same spelling but belong different grammatical classes. Such words can be used either as nouns or as verbs. However, there is a shift in stress in these words. When they are used as nouns, the stress is on the first syllable and when used as verbs, they are stressed on the second syllable. Examples; Nouns verbs 'con-duct con-'duct 'ob-ject ob-'ject 'pre-sent pre-'sent 'pro-cess pro-'cess 'ex-port ex-' port Examples Examples  determiners a, an, the, some, much, more  pronouns he, they, anybody, it, one  conjunctions and, as , but, then, that, when, or  prepositions at, on, in, since, for, to  auxiliaries is, am, are, have, has, had, do does did  modals shall, may, can would must  Particles no, not nor, as, down ,up a, an, the, some, much, more he, they, anybody, it, one and, as , but, then, that, when, or at, on, in, since, for, to is, am, are, have, has, had, do does did shall, may, can would must no, not nor, as, down ,up Structure words are pronounced in their strong forms in the following cases: Structure words are pronounced in their strong forms in the following cases:  auxiliary verbs are generally stressed when placed before sentences to form questions e.g.; 'are you coming to the party?  When they occur in final position of a sentence. E.g. could you please tell me where the hospital 'is?  when they are emphasized  when they are emphasized E.g. I am not sure he will recover. But he 'is recovering.  pronouns are stressed when they contrast as in: E.g. my bike is superior to 'yours. V o l - I I I s s u e s - I Page 124 Electronic Electronic International Interdisciplinary Research Journal (EIIRJ) Bi-monthly Reviewed Journal Jan/Feb 2013 ISSN 2277-8721  reflexive pronouns are always stressed when they are used as subject intensifier: Electronic Electronic International Interdisciplinary Research Journal (EIIRJ) Bi-monthly Reviewed Journal Jan/Feb 2013 ISSN 2277-8721 Electronic Electronic International Interdisciplinary Research Journal (EIIRJ) -monthly Reviewed Journal Jan/Feb 2013 ISSN 2277-8721  reflexive pronouns are always stressed when they are used as subject intensifier:  reflexive pronouns are always stressed when they are used as subject intensifier: E.g. he did the work him „self. E.g. he did the work him „self.  prepositions are stressed when: They occur at the end of a sentence E.g. who are you speaking 'to? E.g. who are you speaking 'to? STRESS SHIFT It should not be assumed that words with the same stem will keep the primary stress on the same syllable. Indeed, stress shift in derivatives is quite normal, e.g., V o l - I I I s s u e s - I Page 125 w w w . a a r h a t . c o m I S S N 2 2 7 7 - 8 7 2 1 Electronic Electronic International Interdisciplinary Research Journal (EIIRJ) Bi-monthly Reviewed Journal Jan/Feb 2013 ISSN 2277-8721 Electronic Electronic International Interdisciplinary Research Journal (EIIRJ) -monthly Reviewed Journal Jan/Feb 2013 Electronic Electronic International Interdisciplinary Research Journal (EIIRJ) Bi-monthly Reviewed Journal Jan/Feb 2013 ISSN 2277-8721 ISSN 2277-8721 a' cademy, aca 'demic, a cade' mician indi' vidual, individu' ality, individual' listic 'politics, po' litical, poli' tician ' photograph, pho 'tography, photo' graphic 'diplomat, 'di plomacy, diplo' matic 'certify, cer 'tificate certify' cation a' cademy, aca 'demic, a cade' mician CONTRASTIVE STRESS; Stress is not always pre-determined, but can be moved from its normal position in a sentence. take, for example, the sentence „I did not take the test yesterday‟. Normally, stresses would fall on the content words only, giving us the pattern: I did not 'take the 'test 'yesterday‟. WHY IS WORD STRESS IMPORTANT? Word stress is not used in all languages. Some languages, Japanese or French for example, pronounce each syllable with equal emphasis. other languages, English for example, use word stress.english speakers use word stress to communicate rapidly and accurately, even in difficult conditions. if for example, you do not hear a word clearly, you can still understand the word because of the position of the stress. WHAT IS WORD STRESS? In English, we do not say each syllable with the same force or strength. In one word, we accentuate ONE syllable. We say one syllable very loudly(big, strong) and all other syllables very quietly. in compound verbs, the stress is on the second part E.g. over „flow Under „stand I did not 'take the 'test 'yesterday‟. But if the stresses are placed on other syllables, which do not normally take stress special meanings can be created. For instance:  'I did not take the test yesterday. (Somebody else did.)  I' did not 'take the test yesterday.( I did not take it)  I did not' take the test yesterday.(I did something else with it)  I did not take 'the test yesterday. (I took a different way)  I did not take the 'test yesterday.( I took something else)  I did not take the test 'yesterday.( I took it some other day) COMPOUND WORDS: In compound words, that is, words consisting of combinations of two words, the primary accent is generally on one element. Examples: Examples:  primary accent on the first element e.g. 'Anything V o l - I I I s s u e s - I Page 126 Electronic Electronic International Interdisciplinary Research Journal (EIIRJ) Bi-monthly Reviewed Journal Jan/Feb 2013 ISSN 2277-8721 Electronic Electronic International Interdisciplinary Research Journal (EIIRJ) Bi-monthly Reviewed Journal Jan/Feb 2013 ISSN 2277-8721 Electronic Electronic International Interdisciplinary Research Journal (EIIRJ) -monthly Reviewed Journal Jan/Feb 2013 ISSN 2277-8721 RULES OF WORD STRESS IN ENGLISH There are two very simple rules about word stress: There are two very simple rules about word stress:  One word has only one stress.  We can only stress vowels, not consonants. V o l - I I I s s u e s - I Page 127 w w . a a r h a t . c o m I S S N 2 2 7 7 - 8 7 2 1 I S S N 2 2 7 7 - 8 7 2 1 Electronic Electronic International Interdisciplinary Research Journal (EIIRJ) Bi-monthly Reviewed Journal Jan/Feb 2013 ISSN 2277-8721 ANALYSIS OF STRESS RULES ON THE WORD Here are a few rules for accentual patterns in English words. (1) Words with weak prefixes are accented on the root, and not the prefix. E.g. a „broad, ad „vice, a „mount, at „tend, be'tween, com'pose, de'velop, re'duce (1) Words with weak prefixes are accented on the root, and not the prefix. E.g. a „broad, ad „vice, a „mount, at „tend, be'tween, com'pose, de'velop, re'duce (2) The inflectional suffixes –es, -ing, -ed, and the following derivational suffixes do not affect the accent: -age, -dom, -en, -er, -ess, -ful, -less, -let, -ly, -ment, -ness, -or, - some, -ward.e.g., (2) The inflectional suffixes –es, -ing, -ed, and the following derivational suffixes do not affect the accent: -age, -dom, -en, -er, -ess, -ful, -less, -let, -ly, -ment, -ness, -or, - some, -ward.e.g., match ' matches begin be „ginning want ' wanted break ' breakage free 'freedom bright 'brighten board ' boarder god ' goddess care ' careful class ' classify aim 'aimless book „ booklet bad ' badly appoint ap'pointment bitter ' bitterness conquer ' conqueror match ' matches begin be „ginning want ' wanted break ' breakage free 'freedom bright 'brighten board ' boarder god ' goddess care ' careful class ' classify aim 'aimless book „ booklet bad ' badly appoint ap'pointment bitter ' bitterness conquer ' conqueror V o l - I I I s s u e s - I Page 128 Electronic Electronic International Interdisciplinary Research Journal (EIIRJ) Bi-monthly Reviewed Journal Jan/Feb 2013 ISSN 2277-8721 fear „ fearsome back „ backward Electronic Electronic International Interdisciplinary Research Journal (EIIRJ) Bi-monthly Reviewed Journal Jan/Feb 2013 ISSN 2277-8721 fear „ fearsome Electronic Electronic International Interdisciplinary Research Journal (EIIRJ) Bi-monthly Reviewed Journal Jan/Feb 2013 ISSN 2277-8721 fear „ fearsome back „ backward ISSN 2277-8721 (3) words ending in –ion have the primary accent on the last syllable but one. e.g. qualifi'cation, culti'vation, imagi'nation, exami'nation (3) words ending in –ion have the primary accent on the last syllable but one. e.g. qualifi'cation, culti'vation, imagi'nation, exami'nation (4) Words ending in –ic, -ical, -ically have the primary accent on the syllable preceding the suffix, e.g., apolo'getic, e'lectrical, patri'otic, sympa'thetically. (4) Words ending in –ic, -ical, -ically have the primary accent on the syllable preceding the suffix, e.g., apolo'getic, e'lectrical, patri'otic, sympa'thetically. (4) Words ending in –ic, -ical, -ically have the primary accent on the syllable preceding the suffix, e.g., apolo'getic, e'lectrical, patri'otic, sympa'thetically. CONCLUSION: The important thing from the learners point of view is that putting stress on the right syllables when one speak English is extremely vital. It is clear that there are no strict rules for determining the stress in English. Here we have presented the valuable and fundamental things about stress. In order to need the clarity about the expression the use of stress is very essential. it is expected that my effort would help the learners knowing and learning about the nature of stress in English language. This analysis would encourage and support the students to explore the subject in a greater depth. ANALYSIS OF STRESS RULES ON THE WORD (4) Words ending in –ic, -ical, -ically have the primary accent on the syllable preceding the suffix, e.g., apolo'getic, e'lectrical, patri'otic, sympa'thetically. (5) Words ending in –ity, are accented on the syllable preceding the suffix, that is, on the third syllable from the end- the ante-penultimate syllable, (5) Words ending in –ity, are accented on the syllable preceding the suffix, that is, on the third syllable from the end- the ante-penultimate syllable, e.g., e'quality, possi'bility, mo'rality, origi'nality e.g., e'quality, possi'bility, mo'rality, origi'nality (6) Words ending in –ial, -ially have the primary accent on the syllable preceding the suffix, e.g., cere'monial, es'sential, confi'dentially, (6) Words ending in –ial, -ially have the primary accent on the syllable preceding the suffix, e.g., cere'monial, es'sential, confi'dentially, (7) In words of more than two syllables ending in –ate, the primary stress is placed two syllables before the suffix, that is, on the third syllable from the end, e.g.,' fortunate, 'separate, „educate (7) In words of more than two syllables ending in –ate, the primary stress is placed two syllables before the suffix, that is, on the third syllable from the end, e.g.,' fortunate, 'separate, „educate (8) Words ending in-ian are stressed on the syllable preceding the suffix, e.g., li'brarian, mu'sician, poli'tician (9) Words ending in-ious are accented on the syllable preceding the suffix. (9) Words ending in-ious are accented on the syllable preceding the suffix. e.g., in‟dustrious, lu'xurious, vic'torious (10) The following suffixes take the primary accent on their first syllable. -aire million'aire Electronic Electronic International Interdisciplinary Research Journal (EIIRJ) Bi-monthly Reviewed Journal Jan/Feb 2013 ISSN 2277-8721 Electronic Electronic International Interdisciplinary Research Journal (EIIRJ) Bi-monthly Reviewed Journal Jan/Feb 2013 ISSN 2277-8721 -escence effer'vescence -esque gro'tesque -ique phy'sique -it is neu'ritis CONCLUSION: Electronic Electronic International Interdisciplinary Research Journal (EIIRJ) Bi-monthly Reviewed Journal Jan/Feb 2013 ISSN 2277-8721 -escence effer'vescence -esque gro'tesque -ique phy'sique -it is neu'ritis CONCLUSION: Electronic Electronic International Interdisciplinary Research Journal (EIIRJ) Bi-monthly Reviewed Journal Jan/Feb 2013 ISSN 2277-8721 Electronic Electronic International Interdisciplinary Research Journal (EIIRJ) -monthly Reviewed Journal Jan/Feb 2013 ISSN 2277-8721 REFERENCES Bansal, R.K. & Harrison, J.B. (2002).Spoken English a manual of speech and phonetics. New Delhi: Orient Longman Bansal, R.K. & Harrison, J.B. (2002).Spoken English a manual of speech and phonetics. New Delhi: Orient Longman Bansal, R.K. & Harrison, J.B. (2002).Spoken English a manual of speech and phonetics. New Delhi: Orient Longman Das, et.al.An Introduction to Professional English and Soft skills (2009).New Delhi: CUP. Das, et.al.An Introduction to Professional English and Soft skills (2009).New Delhi: CUP. Gilani, Syed (1996). Study of stress: A paralinguistic Feature of English Speech. Gilani, Syed (1996). Study of stress: A paralinguistic Feature of English Speech. O‟ CONNER, J. D. (2006). Better English Pronunciation, CUP. O‟ CONNER, J. D. (2006). Better English Pronunciation, CUP. Roach, Peter (1993). English Phonetics and Phonology.CUP. Saleemi Anjum.P & Samina Amin (1988). The application of the principles of intonation, Islamabad: AIOU Press. V o l - I I I s s u e s - I Page 130 w w w . a a r h a t . c o m I S S N 2 2 7 7 - 8 7 2 1 I S S N 2 2 7 7 - 8 7 2 1 I S S N 2 2 7 7 - 8 7 2 1
https://openalex.org/W2110277235	https://www.scielo.br/j/sa/a/xxmDcyPgnWGFxt5L4p3HKcq/?lang=en&format=pdf	English	null	Grain quality of upland rice cultivars in response to cropping systems in the Brazilian tropical savanna	Scientia Agrícola	2,008	cc-by	5,662	468 468 Carlos Alexandre Costa Crusciol1; Orivaldo Arf2; Rogério Peres Soratto1; Gustavo Pavan Mateus3 1UNESP/FCA - Depto. de Produção Vegetal, Fazenda Experimental Lageado, C.P. 237 - 18603-970 - Botucatu, SP - Brasil. 2UNESP/FE - Depto. de Fitotecnia, Tecnologia de Alimentos e Sócio-Economia, C.P. 31 - 15385-000 - Ilha Solteira, SP - Brasil. 3APTA - Pólo Regional de Desenvolvimento Tecnológico dos Agronegócios do Extremo Oeste, C.P. 67 - 16900-000 - Andradina, SP - Brasil. Corresponding author <crusciol@fca.unesp.br> 1UNESP/FCA - Depto. de Produção Vegetal, Fazenda Experimental Lageado, C.P. 237 - 18603-970 - Botucatu SP - Brasil. 2 2UNESP/FE - Depto. de Fitotecnia, Tecnologia de Alimentos e Sócio-Economia, C.P. 31 - 15385-000 - Ilh Solteira, SP - Brasil. 3 Solteira, SP - Brasil. 3APTA - Pólo Regional de Desenvolvimento Tecnológico dos Agronegócios do Extremo Oeste, C.P. 67 - 16900-000 - Andradina, SP - Brasil. *C di th < i l@f b > 3APTA - Pólo Regional de Desenvolvimento Tecnológico dos Agronegócios do Extremo Oeste, C.P. 67 - 16900-000 - Andradina, SP - Brasil. ABSTRACT: The industrial quality of grain is an important parameter to determine the commercial value of rice and can be influenced by water availability and type of cultivar. The present study aimed to evaluate the milling yield as well as the protein and nutrient contents of polished grains originated from two upland rice cultivars grown under rainfed and sprinkler-irrigated conditions. A randomized block design was used in a split-plot arrangement with eight replicates. Plots consisted of two cropping systems (rainfed and sprinkler-irrigated) with subplots consisting of two cultivars recommended for upland ecosystems (IAC 201 and Carajás). The sprinkler irrigation system increased upland rice milling (8.0%) and head rice yield (23.7%), for years with hot and dry weather spells occurring during the reproductive and maturation stages. Under conditions of lower water availability, the Carajás cultivar showed a milled yield value 5.1% higher than that of cultivar IAC 201. Lower water availability provided increases in protein, N, P, Ca, Mg, Fe, and Zn contents but reductions in S and Cu in the polished grains. Cultivars IAC 201 and Carajás had similar nutrient contents in the polished grains. Key words: Oryza sativa, sprinkler irrigation, milling yield, head rice yield QUALIDADE DE GRÃOS DE CULTIVARES DE ARROZ DE TERRAS ALTAS EM FUNÇÃO DO SISTEMA DE CULTIVO NA REGIÃO DO CERRADO BRASILEIRO RESUMO: A qualidade industrial do grão beneficiado é parâmetro importante para determinar o valor de comercialização do arroz e pode estar relacionada com a disponibilidade hídrica e o cultivar utilizado. O trabalho teve como objetivo avaliar o rendimento de engenho e teores de nutrientes e proteínas nos grãos brunidos provenientes de dois cultivares de arroz de terras altas conduzidos sob condições de sequeiro e irrigados por aspersão. O delineamento experimental utilizado foi o de blocos ao acaso, em esquema de parcela subdividida, com oito repetições. As parcelas foram constituídas por dois sistemas de cultivo (sequeiro e irrigado por aspersão) e as subparcelas por dois cultivares indicados para o ecossistema de terras altas (IAC 201 e Carajás). A irrigação por aspersão aumentou o rendimento de engenho (8,0%) e de grãos inteiros (23,7%) do arroz de terras altas, em anos com ocorrência de veranicos nas fases reprodutiva e de maturação. Sob condições de baixa disponibilidade hídrica o cultivar Carajás apresenta rendimento de benefício 5,1% maior do que o cultivar IAC 201. A baixa disponibilidade hídrica promove acréscimo nos teores de proteína, N, P, Ca, Mg, Fe e Zn nos grãos brunidos, porém reduz os teores de S e Cu. Os teores de nutrientes nos grãos brunidos dos cultivares IAC 201 e Carajás são similares. RESUMO: A qualidade industrial do grão beneficiado é parâmetro importante para determinar o valor de comercialização do arroz e pode estar relacionada com a disponibilidade hídrica e o cultivar utilizado. Palavras-chave: Oryza sativa, irrigação por aspersão, rendimento de benefício, rendimento de grãos inteiros INTRODUCTION der the dryland system in general presents lower grain quality when compared to grains originating from low- land systems. A major reason for this, aside from In addition to low yield, upland rice grown un- Sci. Agric. (Piracicaba, Braz.), v.65, n.5, p.468-473, September/October 2008 Grain quality of upland rice cultivars 469 cording to the methodology proposed by Raij & Quaggio (1983). The mean annual rain precipitation is 1,370 mm, the mean annual temperature is 23.5ºC and the relative air humidity ranges from 70 to 80%. causes due to genetic variability, is the frequent peri- ods of water deficiency the plant is exposed to during the cycle, in highly productive regions (Crusciol et al., 1999b). The presence of broken grains in a rice lot is an undesirable characteristic, since it decreases qual- ity and economic value, and also leads to a reduction of the milling yield since small grains may be thrown away along with the hulls (Crusciol et al., 1999a). Grain breakage, verified during the milling process, is caused by cracks or weaker regions in the grains, and occurs especially during the hulling process and when the grains are polished (Crusciol et al., 1999b). Variety traits, inadequate crop management as well as weather conditions (temperature, humidity, and precipitation) may influence milling yield and broken-grain ratio, de- creasing the product’s physical quality (Chen & Kunze, 1983; Srinivas & Bhashyam, 1985). A randomized complete block design, in a split-plot scheme and eight replications was used. Plots consisted of two cultivation systems (rainfed and sprinkler-irrigated) with subplots consisting of two cultivars recommended for upland ecosystems (IAC 201 and Carajás). The distance between plots was 8 m. Each subplot consisted of six rows of plants, six meter length, spaced 0.40 m between rows. Only the four central rows were usable areas, whereas 0.50 m of the edge of each row of plants and the two outer rows were considered border. IAC 201 cultivar in the state of Mato Grosso do Sul presents the following primary characteristics: medium size, precocious cycle, long and slender grain type, translucent and high-yield whole grains in mill- ing with an insignificant chalk and outstanding culi- nary qualities. Carajás cultivar shows size and cycle similar to IAC 201 cultivar, although it shows better plant architecture, higher finishing and water defi- ciency tolerance and long grain type (Bazoni et al., 1995). INTRODUCTION Besides representing a means of minimizing crop loss risk and improving productivity in upland ecosystems, sprinkler irrigation may also allow higher quality grains to be obtained, since the grain filling pro- cess is continuous, leading to higher head rice and threshed-grain yields, consequently reducing broken- grain yield (Oliveira, 1994), especially in years with the occurrence of hot and dry weather spells (Rodrigues, 1998; Arf et al., 2002). In addition, rice nutritional quality is also strongly influenced by the environment. During the experiment, the minimum and maxi- mum air temperatures were daily determined at the weather station, 500 m distant from the experimental location. The rainfall was determined with a Ville de Paris raingange installed at the center of the experi- mental area. Weather elements are shown in Figure 1. In order to establish treatments under sprinkler irriga- tion, the available water capacity (AWC), in mm was determined by the following equation: AWC=[(FC- WPP)/100].d.h, where FC is the field capacity (%); WPP is the wilt permanent point (%); d is the soil den- sity (1,250 kg m -3); h is the rooting effective depth (0.20 m). In this way, the employed AWC of the soil was established as 14.80 mm. In the context of sprinkler-irrigated crop sys- tems, information is still scarce regarding upland-rice grain-nutrient content and industrial quality. Therefore, the present work aimed to evaluate milling yield and protein and nutrient contents in polished grains of cul- tivars IAC 201 and Carajás under rainfed and sprin- kler-irrigated conditions. MATERIAL AND METHODS This work was performed under field and labo- ratory conditions, where grains from experiments car- ried out in field conditions were used, in the cropping seasons of 1994/95 and 1995/96 in Selvíria State of Mato Grosso do Sul, Brazil (51º22’ W, 20º22’ S, at altitude 335 m). The chemical characteristics of the clayey-textured Typic Haplustox at the experiment site (Table 1) were established before the experiment, ac- In sprinkler-irrigated treatments, the water sup- ply was conveyed by a fixed conventional sprinkler system, with outflow of 3.3 mm hour-1. The irriga- tions were performed every time the crop maximum evapotranspiration (ETm) reached 8.25 mm, in other words, 45% of the AWC. The ETm was estimated from the equation: ETm = Kc.ETo; where ETm = Sci. Agric. (Piracicaba, Braz.), v.65, n.5, p.468-473, September/October 2008 Table 1 - Soil chemical characteristics for depth from 0 to 0.20 m. Selvíria, Mato Grosso do Sul state, Brazil. 1Determined in a 0.01 mol-1 Cacl2 solution. 2 Extracted by resin. r a e Y . M . O H p 1 P2 K a C g M l A + H C E C V g k g 1 - m d g m 3 - l o m m ------------------------ c m d 3 - ------------------------ % 5 9 / 4 9 9 1 6 2 4 . 5 4 2 3 . 1 4 2 5 1 9 2 3 . 9 6 8 5 6 9 / 5 9 9 1 3 2 1 . 5 6 2 9 . 1 8 2 8 8 2 9 . 5 6 8 5 Table 1 - Soil chemical characteristics for depth from 0 to 0.20 m. Selvíria, Mato Grosso do Sul stat hemical characteristics for depth from 0 to 0.20 m. Selvíria, Mato Grosso do Sul state, Brazil. ci. Agric. (Piracicaba, Braz.), v.65, n.5, p.468-473, September/October 2008 Sci. Agric. (Piracicaba, Braz.), v.65, n.5, p.468-473, September/October 2008 Crusciol et al. 470 crop’s maximum evapotranspiration (mm day -1), ETo = reference evapotranspiration (mm day-1) and Kc = crop coefficient. flowering; 1.3 for the period 11 to three days before flowering; 1.0 for the period from three days to five days after flowering; and 0.7 for the period between five and 12 days after flowering. The amount and dis- tribution of the water received by the rice crop are shown in Table 2. MATERIAL AND METHODS 1995/96 When approximately 90% of panicles had grains of typical mature coloration, manual harvest was carried out. Panicles were left to dry in the sun for one or two days and later submitted to mechanical threshing in a research plot thresher. Figure 1 - Rain ( ), sprinkler-irrigation ( ), maximum ( ) and minimum temperatures ( ) obtained for the experimental area during the period from November to March in the 1994/95 and 1995/96 cropping seasons at Selvíria, Mato Grosso do Sul state, Brazil. Rainf.: treatment without irrigation, receiving only natural precipitation; Irrig.: sprinkler-irrigated system; S: sowing; E: emergence; PI: panicle initiation; F: flowering and M: maturation. From each subplot, one sample of 100 g of hulled rice grains was collected, and a (SUZUKI) proof mill model MT was used for 1 minute; then the pol- ished grains were weighed and the found values were considered the milling yield percentage. Later, the pol- ished grains were placed in a “trieur” 2 and the grains separated for 30 seconds. The grains that remained in Sci. Agric. (Piracicaba, Braz.), v.65, n.5, p.468-473, September/October 2008 m ets y s g ni p p o r C s e g ats p o r C lat o T e vitate g e V e vitc u d o r p e R n oita r u ta M 5 9 / 4 9 9 1 d ef nia R ) 7 4 ( 7 . 3 3 3 ) 1 ( ) 0 3 ( 9 . 3 4 3 ) 0 2 ( 4 . 5 3 1 ) 7 9 ( 0 . 3 1 8 d eta girri-r el k nir p S ) 9 3 ( 3 . 3 9 2 ) 0 3 ( 6 . 2 2 3 ) 4 2 ( 6 . 3 3 2 ) 3 9 ( 5 . 9 4 8 6 9 / 5 9 9 1 d ef nia R ) 6 4 ( 2 . 5 4 2 ) 0 3 ( 4 . 8 2 2 ) 2 2 ( 2 . 4 2 1 ) 8 9 ( 8 . 8 9 5 d eta girri-r el k nir p S ) 0 4 ( 4 . 1 5 2 ) 0 3 ( 3 . 7 7 2 ) 2 2 ( 1 . MATERIAL AND METHODS The water evaporation (mm) was daily ob- tained from a Class A pan. The Class A pan coeffi- cient (Kp) was the one proposed by Doorenbos & Pruitt (1976), calculated according to the surround- ing area, wind speed and relative humidity. Different Kc values were employed in the different crop devel- opment periods, according to Reichardt (1987): 0.4 for the period between the emergence and 30 days before flowering; 0.7 for the period from 30 to 19 days be- fore flowering; 1.0 between 19 and 11 days before Soil was tilled and two leveling disks employed, the first one soon after the soil tilling and the second shortly before the sowing. The mineral fertilization, during the two cropping seasons, was made by ap- plying into the sowing furrow using 12 kg ha -1 of N, 90 kg ha -1 of P2O5, 30 kg ha -1 of K2O (formula NPK 04-30-10) and 40 kg ha -1 of FTE BR-12 as source of nutrients (2.17% B, 9.2% Zn, 0.8% Cu, 3.82% Fe, 3.47% Mn and 0.132% Mo). Sowings were completed on 24 November 1994 and 13 November 1995, at the density of 100 viable seeds per square meter. Also 1.5 kg ha -1 of carbofuran 5G (a.i.) was added into the sow- ing furrow to control termites (Synterms molestus, Procorniterms striatus and Cornitermes lespesii) and elasmo caterpillars (Elasmopalpus lignosellus). The emergence of plants occurred on 02 December 1994 and 21 November 1995, respectively. Weed control was accomplished with oxadiazon (1 kg ha -1 a.i.) her- bicide in the pre-emergence, one day after the sowing and 2.4D (670 g ha-1 a.i.) in the post-emergence. Ni- trogen sidedressing was performed at tillering, with 30 kg ha -1 of N urea source. Figure 1 - Rain ( ), sprinkler-irrigation ( ), maximum ( ) and minimum temperatures ( ) obtained for the experimental area during the period from November to March in the 1994/95 and 1995/96 cropping seasons at Selvíria, Mato Grosso do Sul state, Brazil. Rainf.: treatment without irrigation, receiving only natural precipitation; Irrig.: sprinkler-irrigated system; S: sowing; E: emergence; PI: panicle initiation; F: flowering and M: maturation. Rain / Sprinkler-irrigation (mm day ) -1 Temperature (°C) 0 30 60 90 0 15 30 45 Rainf. S E PI F M Irrig. 1994/95 0 30 60 90 November December January February March 0 15 30 45 Rainf. S E PI F M Irrig. MATERIAL AND METHODS 9 5 1 ) 2 9 ( 8 . 7 8 6 Table 2 - Amount and distribution of water received by upland rice crops (mm) and crop stage duration (days) under rainfed and sprinkler-irrigated systems. Selvíria, Mato Grosso do Sul state, Brazil. (1)Values in parentheses are crop stage duration in days. Table 2 - Amount and distribution of water received by upland rice crops (mm) and crop stage duration (days) under rainfed and sprinkler-irrigated systems. Selvíria, Mato Grosso do Sul state, Brazil. Table 2 - Amount and distribution of water received by upland rice crops (mm) and crop stage duration (days) under rainfed and sprinkler-irrigated systems. Selvíria, Mato Grosso do Sul state, Brazil. Sci. Agric. (Piracicaba, Braz.), v.65, n.5, p.468-473, September/October 2008 Sci. Agric. (Piracicaba, Braz.), v.65, n.5, p.468-473, September/October 2008 Grain quality of upland rice cultivars 471 the “trieur” were weighed to determine percentages of head rice yield and broken grains. moted root growth, allowing better plant nutrition and grain filling, increasing milling yield, and allowing a con- tinuous supply of water during periods when no pre- cipitation was available (dry and hot weather spells, lo- cally known as “veranicos”). Increased milling yields for all studied cultivars under sprinkler irrigation were also observed by Rodrigues (1998) and Arf et al. (2002). In 1995/96, the polished grains were dried in an oven at 105ºC, after which grains milling were ground (to pass a 40-mesh stainless steel screen) and subjected to nutrient content determinations. Nitrogen was analyzed by acid sulfuric digestion and later quan- tification by semi-micro-Kjeldahl method (Bataglia et al., 1983). Analyses of P, K, Ca, Mg, S, Fe, Zn, Cu, and Mn contents were performed following to nitric- perchloric acid digestion (Bataglia et al., 1983). Pol- ished-grain protein content was calculated by multi- plying the nitrogen content by the factor 5.95. y g ( ) ( ) Head rice yield indicates the weight of whole grains obtained after industrial processing. This is one of the most important parameters in rice commercial value determination (Arf et al., 2002). There was a cropping system effect on this characteristic only in the first year of the experiment (p ≤ 0.05) (Table 3). Sprinkler irrigation provided higher head rice yields in both cultivars. Therefore, adequate soil water availabil- ity enhances water and nutrient absorption as well as photosynthate translocation into the grains, allowing continuous grain filling and adequate development. RESULTS AND DISCUSSION During the first cropping season, milled grain yield was influenced by cropping system and cultivar as well as by the interaction between these factors (p ≤ 0.05) (Table 3). Based on partitioned interaction observations (Table 4), it was verified that under rainfed conditions the Carajás cultivar showed a milled yield value 5.1% higher than that of cultivar IAC 201, corroborating Oliveira (1994) and Crusciol (1998), who verified a contrasting performance of those cul- tivars in terms of response to sprinkler irrigation. The lower tolerance to water deficit shown by the IAC 201 cultivar can be explained by the fact that this cultivar originated from a cross between cultivar IAC 165 (rec- ommended for upland crops) and cultivar Labelle (rec- ommended for deepwater rice crops) (Arf et al., 2002). Sprinkler irrigation provided higher milling yields in both cultivars, both during the 1994/95 cropping season and the following year, indicating that water supplied through sprinkler irrigation may have pro- Table 3 - Total milled rice yield, head rice yield and broken grains obtained from upland rice cultivars grown under rainfed and sprinkler-irrigated systems. Selvíria, Mato Grosso do Sul state, Brazil. Table 3 - Total milled rice yield, head rice yield and broken grains obtained from upland rice cultiv and sprinkler-irrigated systems. Selvíria, Mato Grosso do Sul state, Brazil. S i A i (Pi i b B ) 65 5 468 473 S t b /O t b 2008 stn e m ta e r T e cir d elli m lat o T e cir d a e H s nia r g n e k o r B 5 9 / 4 9 9 1 6 9 / 5 9 9 1 5 9 / 4 9 9 1 6 9 / 5 9 9 1 5 9 / 4 9 9 1 6 9 / 5 9 9 1 ------------------------------------------------- % ------------------------------------------------- m ets y s g ni p p o r C d ef nia R b 9 . 3 6 ) 1 ( b 9 . 7 6 b 8 . 8 3 1 . 5 4 1 . 5 2 8 . 2 2 d eta girri-r el k nir p S a 0 . 9 6 a 0 . 9 6 a 0 . 8 4 6 . 6 4 1 . 1 2 1 . 2 2 ) % ( V C 8 . MATERIAL AND METHODS Al- though water availability during the cycle was higher in the first year (Table 2), the occurrence of dry and hot weather spells, especially during the stage just be- fore flowering and during the maturation stage (Fig- ure 1), may have affected perfect grain development in the rainfed system, since the irrigation effect on grain development occurs, in some cases, well before that stage, promoting increased root growth and im- proved nutritional status, affecting grain filling dynam- ics. Rice chalkiness ratio and percentage of unfilled grains significantly increase when water deficits oc- cur during the panicle emission and grain filling stages (Stermer, 1968). Well-developed grains show higher head rice yield, due to higher resistance to shocks and vibrations caused by harvest and industrial process- ing (Arf et al., 2002). The cultivars showed similar head rice yield values, though these may vary due to genetic characteristics (Chen & Kunze, 1983; Srinivas & Bhashyam, 1985). Data were submitted to analysis of variance and the means compared by LSD (p ≤ 0.05) test. Means followed by different letters in the columns, for each factor, are different by the LSD test (p ≤ 0.05). ci. Agric. (Piracicaba, Braz.), v.65, n.5, p.468-473, September/October 2008 Sci. Agric. (Piracicaba, Braz.), v.65, n.5, p.468-473, September/October 2008 RESULTS AND DISCUSSION 4 9 . 0 1 . 2 2 3 . 1 1 0 . 2 3 4 . 1 2 r a vitlu C 1 0 2 C A I 6 . 5 6 4 . 8 6 8 . 4 4 4 . 5 4 b 9 . 0 2 0 . 3 2 s ája r a C 3 . 7 6 5 . 8 6 0 . 2 4 4 . 6 4 a 4 . 5 2 9 . 1 2 ) % ( V C 6 . 1 7 . 1 1 . 0 1 5 . 7 0 . 4 1 0 . 5 1 and sprinkler-irrigated systems. Selvíria, Mato Grosso do Sul state, Brazil. (1)Means followed by different letters in the columns, for each factor, are different by the LSD test (p ≤ 0.05). Sci. Agric. (Piracicaba, Braz.), v.65, n.5, p.468-473, September/October 2008 Sci. Agric. (Piracicaba, Braz.), v.65, n.5, p.468-473, September/October 2008 472 Crusciol et al. better conditions for nutrient absorption, especially during the grain filling stage. Similar results were verified by Crusciol et al. (2003a; b). Broken-grain yield was not influenced by crop- ping system in either experimental year (p ≤ 0.05) (Table 3). In the 1994/95 cropping season, cultivar IAC 201 presented lower broken-grain yield, a char- acteristic not influenced by cultivar in the second crop- ping year. Posphurus content in polished grains was affected by cropping system and by the interaction between cropping system and cultivar (Table 5 and 6). The rainfed treatments provided grains with higher P levels for both cultivars (Table 6). However, under rainfed conditions, culti- var IAC 201 showed increased P in grains, similarly to the Carajás cultivar in sprinkler-irrigated treatments. The variation in rice nutritional quality is mainly due to genetic characteristics of the cultivars and key environmental factors (soil and weather), and consequently is also influenced by irrigation (water availability) (Barbosa Filho & Fonseca, 1994; Nanda & Coffman, 1979). However, the rainfed treatments provided grains with increased pro- tein, N, Ca, Mg, Fe, and Zn (Table 5), which may be explained by a dilution effect, since productivity was higher for sprinkler-irrigated (3,727 kg ha -1) that in rainfed systems (2,516 kg ha -1). Crusciol et al. (2003a; b) also verified smaller contents of those nu- trients and protein in the polished grains of rice in sprinkler-irrigated system. RESULTS AND DISCUSSION Protein contents observed in this work are smaller than reported by Angladette (1969), however, larger than verified by Castro et al. (1999). Nutrient and protein contents in polished grains were similar for both cultivars, having differ- ence only for Mn content (Table 5). The protein con- tent average in polished grains of studied cultivars was of 80.0 g kg -1. Frei & Becker (2004), observed protein content in grains of upland rice cultivars ranging between 70 and 137 g kg-1 on a dry matter basis, with an average of 95 g kg -1, which is higher than the average of the high yielding varieties (89 g kg-1). The higher Fe contents in grains are probably related to the soil type, which contains higher content of this element. Evaluating the polished grains of 286 genotypes of rice, Yang et al. (1998) verified great variation in the micronutrients contents, with the me- dium contents varying of 4.0 to 29.5 mg kg-1 of Fe, 8.2 to 95.0 mg kg -1 of Zn, 2.5 to 143.5 mg kg -1 of Cu, and 9.0 to 30.5 mg kg -1 of Mn. Sulphur and copper levels were higher for grains obtained from sprinkler-irrigated systems, probably due to the fact that such treatment provides Table 4 - Cropping system versus cultivar interaction, partitioned from the analysis of variance for total milled rice (%). 1994/95 cropping season. Table 4 - Cropping system versus cultivar interaction, partitioned from the analysis of variance for total milled rice (%). 1994/95 cropping season. Although the cropping systems affected the levels of some nutrients, they did not disagree with re- gard to the nutrient content sequence found in the pol- ished portion. On average between cropping systems, upland rice polished grains produced the following rela- tive nutrient levels, in increasing order: N>Ca>P>S≥K>Mg>Zn>Fe>Mn>Cu, for both culti- vars. m ets y s g ni p p o r C r a vitlu C 1 0 2 C A I s ája r a C d ef nia R B b 3 . 2 6 ) 1 ( A b 5 . 5 6 d eta girri-r el k nir p S A a 0 . 9 6 A a 0 . 9 6 (1)Means followed by the same lower case letter in the columns, and upper case letters in the rows are different by the LSD test (p ≤ 0.05). Sci. Agric. RESULTS AND DISCUSSION (Piracicaba, Braz.), v.65, n.5, p.468-473, September/October 2008 stn e m ta e r T niet o r P N P K a C g M S e F n Z u C n M g k g ---------------------------- 1 - ---------------------------- g k g m --------------- 1 - --------------- m ets y s g ni p p o r C d ef nia R a 5 . 4 8 ) 1 ( a 2 . 4 1 a 8 2 . 1 8 4 . 0 a 8 . 4 a 8 4 . 0 b 9 3 . 0 a 6 . 1 5 a 5 . 4 0 1 b 6 . 1 8 . 6 d eta girri-r el k nir p S b 0 . 5 7 b 6 . 2 1 b 8 8 . 0 5 5 . 0 b 5 . 3 b 9 1 . 0 a 9 6 . 0 b 3 . 1 1 b 4 . 0 6 a 4 . 2 6 . 6 ) % ( V C 7 . 5 7 . 5 6 . 3 1 9 . 0 7 6 . 4 3 0 . 3 3 6 . 0 3 3 . 7 7 8 . 0 3 1 . 0 4 1 . 3 3 r a vitlu C 1 0 2 C A I 1 . 2 8 8 . 3 1 8 1 . 1 1 5 . 0 8 . 3 4 3 . 0 9 5 . 0 8 . 9 2 5 . 5 8 1 . 2 a 5 . 7 s ája r a C 9 . 7 7 1 . 3 1 7 9 . 0 2 5 . 0 5 . 4 3 3 . 0 9 4 . 0 1 . 3 3 4 . 9 7 0 . 2 b 9 . 5 ) % ( V C 1 . 1 1 2 . 1 1 3 . 5 1 6 . 6 4 3 . 7 3 8 . 0 6 7 . 8 2 8 . 6 8 9 . 3 2 4 . 6 1 7 . 8 2 Table 5 - Nutrients and protein content in polished grains obtained from upland rice grown under rainfed and sprinkler- irrigated systems. Selvíria, Mato Grosso do Sul state, Brazil. ci. Agric. (Piracicaba, Braz.), v.65, n.5, p.468-473, September/October 2008 RESULTS AND DISCUSSION (1)Means followed by different letters in the columns, for each factor, are different by the LSD test (p ≤ 0.05). tein content in polished grains obtained from upland rice grown under rainfed and sprinkler- Selvíria, Mato Grosso do Sul state, Brazil. Sci. Agric. (Piracicaba, Braz.), v.65, n.5, p.468-473, September/October 2008 Sci. Agric. (Piracicaba, Braz.), v.65, n.5, p.468-473, September/October 2008 473 Grain quality of upland rice cultivars Table 6 - Cropping system versus cultivar interaction, partitioned from the analysis of variance for P content (g kg-1) in polished grains. 1995/96 cropping season. Table 6 - Cropping system versus cultivar interaction, partitioned from the analysis of variance for P content (g kg-1) in polished grains. 1995/96 cropping season. CHEN, Y.L.; KUNZE, O.R. Effect of environmental changes of rice yield and particle size of broken kernels. Cereal Chemistry, v.60, p.238-241, 1983. CHEN, Y.L.; KUNZE, O.R. Effect of environmental changes of rice yield and particle size of broken kernels. Cereal y, , p , CRUSCIOL, C.A.C. Efeito de lâminas de água e da adubação mineral em dois cultivares de arroz-de-sequeiro sob irrigação por aspersão. Botucatu: UNESP/FCA 1998 129p Tese (Doutorado) ucatu: UNESP/FCA, 1998. 129p. Tese (Doutorado). m ets y s g ni p p o r C r a vitlu C 1 0 2 C A I s ája r a C d ef nia R A a 8 4 . 1 ) 1 ( B a 7 0 . 1 d eta girri-r el k nir p S A b 8 8 . 0 A b 8 8 . 0 (1)Means followed by the same lower case letter in the columns, and upper case letters in the rows are different by the LSD test (p ≤ 0.05). CRUSCIOL, C.A.C.; ARF, O.; SORATTO, R.P.; MACHADO, J.R. Qualidade industrial e teores de nutrientes dos grãos do arroz de terras altas sob diferentes lâminas de água e níveis de adubação mineral. Acta Scientiarum. Agronomy, v.25, p.409-415, 2003a. CRUSCIOL, C.A.C.; ARF, O.; SORATTO, R.P.; MACHADO, J.R. Rendimento de benefício e teores de nutrientes em grãos de arroz de terras altas em função de lâminas de água e adubação mineral. Científica, v.31, p.179-188-2003b. CRUSCIOL, C.A.C.; MACHADO, J.R.; ARF, O.; RODRIGUES, R.A.F. Rendimento de benefício e de grãos inteiros em função do espaçamento e da densidade de semeadura do arroz de sequeiro. Scientia Agricola, v.56, p.47-52, 1999a. ACKNOWLEDGEMENTS UNESP/FE, 1994. 41p. Monografia (Graduação). To CNPq for fellowships to the first and sec- ond authors. RAIJ, B. van; QUAGGIO, J.A. Métodos de análise de solo para fins de fertilidade. Campinas: Instituto Agronômico, 1983. 31p. (Boletim Técnico, 81). CONCLUSIONS CRUSCIOL, C.A.C.; MACHADO, J.R.; ARF, O.; RODRIGUES, R.A.F. Rendimento industrial de grãos de arroz irrigado por aspersão em razão do espaçamento entre fileiras e da densidade de semeadura. Científica, v.27, p.165-176, 1999b. For upland rice, sprinkler irrigation increased both milling and head rice yield, especially in years with the occurrence of moisture stress periods during the reproductive and maturation stages. Under lower wa- ter availability conditions, the Carajás cultivar presented higher milling yield as compared to cultivar IAC 201. Lower water availability promoted increased protein, N, P, Ca, Mg, Fe, and Zn levels and reduced S and Cu levels in polished grains. Cultivars IAC 201 and Carajás showed similar nutrient contents in the polished grains. p DOORENBOS, J.; PRUITT, W.O. Las necessidades de agua de los cultivos. Roma: FAO, 1976. 194p. (Estudos FAO: Riego e Drenaje, 24). FREI, M.; BECKER, K. Agro-biodiversity in subsistence-oriented farming systems in a Philippine upland region: nutritional considerations. Biodiversity and Conservation, v.13, p.1591-1610, 2004. NANDA, J.S.; COFFMAN, W.R. IRRI’s efforts to improve the protein content of rice. In: WORKSHOP ON CHEMICAL ASPECTS OF RICE GRAIN QUALITY, 1979, Los Banõs, 1979. Proceedings. Los Banõs: IRRI, 1979. p.33-47. OLIVEIRA, G.S. Efeito de densidades de semeadura no desenvolvimento de cultivares de arroz (Oryza sativa L.) em condições de sequeiro e irrigado por aspersão. Ilha Solteira: UNESP/FE 1994 41p Monografia (Graduação) REFERENCES REICHARDT, K. Relações solo-água-planta para algumas culturas. In: REICHARDT, K. A água em sistemas agrícolas. São Paulo: Manole, 1987. p.157-71. ANGLADETTE, A. El arroz. Barcelona: Editorial Blume, 1969. 867p. Á RODRIGUES, R.A.F. Efeitos do manejo de água nas características fenológicas e produtivas do arroz (Oryza sativa L.) cultivado em condições de sequeiro sob irrigação por aspersão. Botucatu: UNESP/FCA 1998 75p Tese (Doutorado) ARF, O.; RODRIGUES, R.A.F.; SÁ, M.E.; CRUSCIOL, C.A.C.; PEREIRA, J.C.R. Preparo do solo, irrigação por aspersão e rendimento de engenho do arroz de terras altas, Scientia Agricola, v.59, p.321-326, 2002. UNESP/FCA, 1998. 75p. Tese (Doutorado). SRINIVAS, T.; BHASHYAM, M.K. Effect of variety environment of milling quality of rice. In: INTERNATIONAL RICE RESEARCH INSTITUTE. Rice grain quality and marketing. Manila: IRRI, 1985. p.49-59. BARBOSA FILHO, M.P.; FONSECA, J.R. Importância da adubação na qualidade do arroz. In: SÁ, M.E.; BUZETTI, S. (Ed.) Importância da adubação na qualidade dos produtos agrícolas. São Paulo: Ícone, 1994. p.217-231. STERMER, R.A. Environmental conditions and stress cracks in milled rice. Cereal Chemistry, v.45, p.365-373, 1968. BATAGLIA, O.C.; FURLANI, A.M.C.; TEIXEIRA, J.P.F.; FURLANI, P.R.; GALLO, J.R. Métodos de análises químicas de plantas. Campinas: IAC, 1983. 48p. YANG, X.; YE, Z.Q.; SHI, C.H.; ZHU, M.L.; GRAHAM, R.D. Genotypic differences in concentrations of iron, manganese, copper, and zinc in polished rice grains. Journal of Plant Nutrition, v.21, p.1453-1462, 1998. BAZONI, R.; COSTA; C.A.; ARF, O.; MORAIS, O.P.; SANT’ANA, E.P.; MOURA NETO, F.P.; PITOL, C. Recomendação de cultivares de arroz de sequeiro para o ano agrícola de 1995/96, em Mato Grosso do Sul. Campo Grande: EMPAER, 1995. p.1-5. (Comunicado Técnico, 17). CASTRO, E.M.; VIEIRA, N.R.A.; RABELO, R.R.; SILVA, S.A. Qualidade de grãos de arroz. Santo Antonio de Goiás: Embrapa Arroz e Feijão, 1999. 30p. (Circular Técnica, 34). Received April 19, 2006 Accepted April 14, 2008 Sci. Agric. (Piracicaba, Braz.), v.65, n.5, p.468-473, September/October 2008
https://openalex.org/W2100130564	https://link.springer.com/content/pdf/10.1007/s10342-014-0801-y.pdf	English	null	The anatomical traits of trunk wood and their relevance to oak (Quercus robur L.) vitality	European journal of forest research	2,014	cc-by	8,783	Eur J Forest Res (2014) 133:845–855 DOI 10.1007/s10342-014-0801-y Eur J Forest Res (2014) 133:845–855 DOI 10.1007/s10342-014-0801-y ORIGINAL PAPER ORIGINAL PAPER The anatomical traits of trunk wood and their relevance to oak (Quercus robur L.) vitality Mirela Tulik Received: 26 September 2013 / Revised: 3 March 2014 / Accepted: 7 March 2014 / Published online: 26 March 2014 The Author(s) 2014. This article is published with open access at Springerlink.com Abstract Oaks’ decline in vitality is attributed to a complex process that involves interactions of several fac- tors leading to increased trees’ mortality. This study investigates the structure of trunk wood of oaks with ref- erence to its physiological role in hydraulic conductivity. On the basis of the crown condition, the oaks were clas- siﬁed into three health groups: healthy trees, declining trees and dead trees. Anatomical traits of wood, such as annual ring width, vessel density, vessel diameter of earlywood and theoretical hydraulic conductivity, were measured and calculated. The narrowest annual rings formed by the cambium were observed in dead oaks. These trees were also characterized by the smallest diameter of earlywood vessels, not only in the period of occurrence of dieback symptoms, but also during their whole life. It is suggested that the formation of narrow annual rings and earlywood vessels of small diameter increases susceptibility of a tree to decay. A reduced vessel diameter implies changes in hydraulic conductivity of oak trunks and thus impairs the water transport, which affects the health of trees. The process of oak decline is considered to have characteristics of natural selection and leads to the elimination of the weakest trees. M. Tulik (&) Department of Forest Botany, Faculty of Forestry, Warsaw University of Life Sciences (WULS), Nowoursynowska St. 159, 02-776 Warsaw, Poland e-mail: mirela.tulik@wl.sggw.pl Communicated by M. Meincken. Introduction Trees, the long-lived and largest land plants, are organisms of highly specialized cell systems, whose structure and functions were, in the course of evolution, adapted to the environment they lived in. The current forms and shapes that trees assume are the result of the ability of their cells to respond to the interaction of intracellular and extracellular factors (Kozlowski et al. 1991; Dobbertin 2005). Since the 1970s of the nineteenth century, the process of decline of oaks has been progressing in Europe on a regular basis (Thomas and Boza 1984; Oleksyn and Przybył 1987; Thomas 2008). It does not apply to dying specimens of this species, but to a massive elimination of trees from the forest stand (Kaus et al. 1995). Among the European spe- cies of oak, Quercus robur is more susceptible to decay than Quercus petrea (Ufnalski 2006). This is probably due to its lower resistance to drought (Dickson and Tomlinson 1996), which could be related to changes in groundwater availability which have occurred in the recent decades. The oak dieback phenomenon is also observed in Canada and the United States with reference to Quercus agrifolia and Quercus kellogii (Oak et al. 1996; Rizzo et al. 2005). There are also reports of its presence in Asia (Mosca et al. 2007). While diagnosing the phenomenon of declining oaks, models were developed to describe it, like the model of the three-step dying process, where ‘‘predisposing’’, ‘‘contrib- uting’’, and ‘‘inciting’’ factors, respectively, (Manion 1981) play an essential role. The predisposing factors include, among others, the phenotype and unfavourable habitat changes (Thomas and Hartmann 1996; Szewczyk et al. 2011). The second group can include frequent and long- lasting droughts, irruptive decrease in groundwater level, strong frost and tree damages caused by insects from the group of primary pests as well as fungal diseases. The Keywords Tree decline Hydraulic conductivity Oak trees Wood annual increment Vessel diameter Communicated by M. Meincken. 123 12 3 3 Eur J Forest Res (2014) 133:845–855 846 factors described as ‘‘inciting’’ include mainly secondary pests (e.g. insects from Argillus and Scolytus genera), as well as fungi causing root rot (e.g. Armillaria spp.) (Bugała 2006). early stage of water deﬁcit, followed by a reduction in the number of cells produced. Eckstein et al. (1977) indicated also that size of water-conducting elements in broadleaved trees reﬂects the availability of water during their forma- tion. Introduction Wodzicki (1971) grouped the environmental factors into (1) basic conditions for forma- tion of wood, such as temperature, moisture, nutrients in the soil, gravity and photoperiod and (2) occasional factors such as wind, ﬁre, frost, ﬂoods, defoliation, forest man- agement and air pollution. Among these factors, the con- tent of water in the soil is one of the most relevant factors affecting the cambium activity and the formation of new wood cells (Larson 1994). Abe and Nakai (1999) showed that cell diameter of the tracheids decreased during the The phenomenon of oak vitality decline is likely caused by speciﬁc sequential stress factors, differing in duration and intensity, that interfere with physiological processes (e.g. photosynthetic CO2 uptake). This can cause severe structural anomalies which could lead to the elimination of this tree species (Wargo 1996; Thomas et al. 2002). On the other hand, oak species are known to show morphological and physiological adaptations that enable them to delay the impact of unfavourable factors (e.g. drought). The mor- phological adaptations are, among others, a deep-reaching root system or the ability of mature trees to shed twigs (cladoptosis) (Klugmann and Roloff 1999; Rust and Roloff 2004). The physiological mode that contributes to delaying drought mainly concerns transpiration (Maseda and Fern- a´ndez 2006). This process is effectively controlled by regulation of the stomatal closure. Oak trees represent two opposite water use strategies, i.e. isohydric and anisohydric (Dickson and Tomlinson 1996). Q. robur demonstrates a more isohydric behaviour than Quercus rubra due to the fact that it is more sensitive to water stress (Thomsen et al. 2013). This isohydric response protects wood from cavi- tation through avoidance of severe low water potentials, but it can cause eventual carbon starvation as stomatal closure shuts down photosynthesis, while respiration costs continue to deplete carbon stores (McDowell et al. 2008). Oak wood is made up of vessels, tracheids, ﬁbres and parenchyma cells. Earlywood vessels are mainly respon- sible for the transport of water in trees, which is assumed to take place in the outermost tree ring only (type of ring- porous wood, Fonti et al. 2009). The transport efﬁciency depends on the size of vessels’ lumen (Cochard and Tyree 1990, Sperry and Sullivan 1992) because according to the Hagen–Poiseuille formula, water conductivity is propor- tional to the fourth power of the tracheary element diam- eter (Tyree and Zimmermann 2002; McElrone et al. 2004). Introduction The aim of this study was, therefore, a retrospective analysis of earlywood vessels’ diameter liable for hydraulic conductivity. The working hypothesis of the study was that any reduction in vessel lumen inﬂuences oak tree (Q. robur L.) vitality because of water transport reduction. Oak wood is made up of vessels, tracheids, ﬁbres and parenchyma cells. Earlywood vessels are mainly respon- sible for the transport of water in trees, which is assumed to take place in the outermost tree ring only (type of ring- porous wood, Fonti et al. 2009). The transport efﬁciency depends on the size of vessels’ lumen (Cochard and Tyree 1990, Sperry and Sullivan 1992) because according to the Hagen–Poiseuille formula, water conductivity is propor- tional to the fourth power of the tracheary element diam- eter (Tyree and Zimmermann 2002; McElrone et al. 2004). The aim of this study was, therefore, a retrospective analysis of earlywood vessels’ diameter liable for hydraulic conductivity. The working hypothesis of the study was that any reduction in vessel lumen inﬂuences oak tree (Q. robur L.) vitality because of water transport reduction. Materials and methods The study was conducted on oak (Q. robur L.) trees from two areas located in the central part of Poland, i.e. in the Chojno´w Forest District (Masovian Voivodship) and Kro- toszyn Forest District (Wielkopolskie Voivodship, Table 1), where the process of oak decline has been observed for several years (Siwecki and Ufnalski 1998). The forest within each study site was dominated by Q. robur. The material was collected from the area of Cho- jno´w Forest District in September 2008 and from Kro- toszyn Forest District in June 2009. Thirty trees (15 from each study site) at the age of 30–40 years and the com- parable total height (representing the highest biosocial positions—class II according to the Kraft’s classiﬁcation) were selected for the study. Oaks growing in the Chojno´w Forestry District area will be referred to in this study as oaks from Chojno´w, while those growing in the Krotoszyn Forestry District—as oaks from Krotoszyn. The selection of the trees for the study within each site was made on the basis of the crown condition (i.e. the degree of its defoli- ation, the presence of dead branches) and classiﬁed as (i) healthy, (ii) declining and (iii) dead trees. Healthy oaks were deﬁned as a reference. Defoliation of their crowns is estimated at around 20 %. Trees with crown defoliation of 70–90 % were considered declining. Trees with no leaves and withered branches constituted the group of dead oaks. These oaks died in the year preceding the collection of the p ( ) Wood is a source of information about environmental events that took place during its formation (Sass and Eckstein 1995; Tulik 2001; Fonti et al. 2009; Martin- Benito et al. 2013). The process of wood formation includes post-cambial cell growth, which determines the diameter of cells, deposition of the secondary cell wall and in case of vessels, tracheids and ﬁbres—also ligniﬁcation. It is ended by programmed cell death (Plomion et al. 2001). The successive phases of wood formation are not prede- termined but are plastic end-products of interactions between genotype, hormones and the environment (Sav- idge 2000). The external factors are considered to affect the onset, the end and the rate of the phases of cambial cell derivatives’ differentiation and thus determine the mor- phology of woody cells. Introduction Reduced size of conductive wood cells is known to be a result of the turgor pressure loss in expanding cambial cell derivatives in limited water growth conditions (Abe et al. 2003). 2006). The phenomenon of oak vitality decline is likely caused by speciﬁc sequential stress factors, differing in duration and intensity, that interfere with physiological processes (e.g. photosynthetic CO2 uptake). This can cause severe structural anomalies which could lead to the elimination of this tree species (Wargo 1996; Thomas et al. 2002). On the other hand, oak species are known to show morphological and physiological adaptations that enable them to delay the impact of unfavourable factors (e.g. drought). The mor- phological adaptations are, among others, a deep-reaching root system or the ability of mature trees to shed twigs (cladoptosis) (Klugmann and Roloff 1999; Rust and Roloff 2004). The physiological mode that contributes to delaying drought mainly concerns transpiration (Maseda and Fern- a´ndez 2006). This process is effectively controlled by regulation of the stomatal closure. Oak trees represent two opposite water use strategies, i.e. isohydric and anisohydric (Dickson and Tomlinson 1996). Q. robur demonstrates a more isohydric behaviour than Quercus rubra due to the fact that it is more sensitive to water stress (Thomsen et al. 2013). This isohydric response protects wood from cavi- tation through avoidance of severe low water potentials, but it can cause eventual carbon starvation as stomatal closure shuts down photosynthesis, while respiration costs continue to deplete carbon stores (McDowell et al. 2008). Wood is a source of information about environmental events that took place during its formation (Sass and Eckstein 1995; Tulik 2001; Fonti et al. 2009; Martin- Benito et al. 2013). The process of wood formation includes post-cambial cell growth, which determines the diameter of cells, deposition of the secondary cell wall and in case of vessels, tracheids and ﬁbres—also ligniﬁcation. It is ended by programmed cell death (Plomion et al. 2001). The successive phases of wood formation are not prede- termined but are plastic end-products of interactions between genotype, hormones and the environment (Sav- idge 2000). The external factors are considered to affect the onset, the end and the rate of the phases of cambial cell derivatives’ differentiation and thus determine the mor- phology of woody cells. Materials and methods Wodzicki (1971) grouped the environmental factors into (1) basic conditions for forma- tion of wood, such as temperature, moisture, nutrients in the soil, gravity and photoperiod and (2) occasional factors such as wind, ﬁre, frost, ﬂoods, defoliation, forest man- agement and air pollution. Among these factors, the con- tent of water in the soil is one of the most relevant factors affecting the cambium activity and the formation of new wood cells (Larson 1994). Abe and Nakai (1999) showed that cell diameter of the tracheids decreased during the 123 123 Eur J Forest Res (2014) 133:845–855 847 Table 1 Characteristics of study sites Locationa Forest habitat typea Species compositiona Soil typea Mean annual temperature (C)b Annual precipitation sum (mm)b Relative humidity (%)c Chojno´w Forest District 520101300N, 210501600E Fresh mixed broadleaved forest Quercus, Betula, Carpinus Proper pseudogley soil, strongly fresh, developed from stony loamy sand shallow overlying on silty loam, ﬂat area without slopes 8.3 (SD = 1) 498.9 (SD = 75) 79 Krotoszyn Forest District 514103700N, 172505400E Fresh broadleaved forest Quercus, Larix, Pine Brownish rusty soil, developed from sand on sandy loam underlying on loamy sand, ﬂat area without slopes 8.9 (SD = 0.9) 534.1 (SD = 79) 77 SD standard deviation a Forest Management Plan of Chojno´w Forest District and Krotoszyn Forest District for 2008–2017 b Data from Climate Research Unit and Tyndall Centre for 1970–2002 (Mitchell and Jones 2005) c Documentation of Forest Research Institute in Se˛kocin, Warsaw, Poland, data for 2008–2012 Table 1 Characteristics of study sites a Forest Management Plan of Chojno´w Forest District and Krotoszyn Forest District for 2008–2017 b Data from Climate Research Unit and Tyndall Centre for 1970–2002 (Mitchell and Jones 2005) c Documentation of Forest Research Institute in Se˛kocin, Warsaw, Poland, data for 2008–2012 least 20,400 earlywood vessels was calculated. Data regarding the earlywood vessel diameter were used to calculate hydraulically weighted diameter (Ds) and potential hydraulic conductivity using the Hagen– Poiseuille formula. plant material. Each group was represented by ﬁve oaks. The bark of dead oaks form Chojno´w was affected by Agrilus biguttatus. During sampling, Operophtera bru- mata, Erannis defoliaria, Tortix viridana and Euproctis chrysorrhoea were observed on the foliage of oaks from Krotoszyn. Hydraulically weighted diameter was computed using an estimator deﬁned by Sperry and Sullivan (1992): y The material for the study consisted of wood samples taken from the tree trunk at 1.3 m height from the ground. Materials and methods Wood blocks covering all of the annual growth rings were obtained from the collected discs, along the radial direc- tion. The blocks were then boiled in a solution of water, alcohol and glycerol (1:1:1 in a volume ratio), which allowed softening and air release in the wood. The material prepared this way was cut on an HM 440E sliding micro- tome in order to obtain cross sections through the wood at a thickness of 30 lm. The sections were moved onto glass slides and sealed in glycerol. These were studied under an Olympus BX61 light microscope, equipped with a motor- ized table, colour DP70 digital camera and Cell P, a soft- ware for archiving photos and computer image analysis. On the basis of microscopic analysis and photographic docu- mentation of the wood sections, the following parameters were measured: Ds ¼ RD5=RD4 ð1Þ ð1Þ Ds ¼ RD5=RD4 Data concerning the hydrostatic pressure gradient that pulls water from roots were not available at the time of tree sampling. Therefore, potential (theoretical) hydraulic con- ductivity (Kp) was calculated according to the Hagen– Poiseuille formula (Sterck et al. 2008) as follows: Kp ¼ ðpqw=128gÞ VD D4 s ð2Þ ð2Þ Kp ¼ ðpqw=128gÞ VD D4 s where Kp is the potential speciﬁc stem conductivity (in kg m-1 MPa-1 s-1), g is the viscosity coefﬁcient of water at 20 C (1.002 9 10-3 Pa s at 20 C), qw is the density of water at 20 C (998.2 kg m-3 at 20 C), VD is the vessel density and Ds is the hydraulically weighted diameter (in m). where Kp is the potential speciﬁc stem conductivity (in kg m-1 MPa-1 s-1), g is the viscosity coefﬁcient of water at 20 C (1.002 9 10-3 Pa s at 20 C), qw is the density of water at 20 C (998.2 kg m-3 at 20 C), VD is the vessel density and Ds is the hydraulically weighted diameter (in m). The determined wood parameters were averaged for ﬁve oaks from each health group and for 5-year periods to show general developmental tendencies more clearly. In total, the data covered the last 30 years of trees’ growth, corre- sponding to the calendar years from 1978 to 2007 in case of oaks growing at Chojno´w and from 1979 to 2008 for oaks from Krotoszyn. Materials and methods Vertical bars denote SEs (a) 0 1 2 3 4 5 6 1978-1982 1983-1987 1988-1992 1993-1997 1998-2002 2003-2007 Mean width of annual wood ring [mm] Calendar year intervals healthy declining dead (b) 0 1 2 3 4 5 6 1979-1983 1984-1988 1989-1993 1994-1998 1999-2003 2004-2008 Mean width of annual wood ring [mm] Calendar year intervals healthy declining dead of of ent of to ng r (a) 0 1 2 3 4 5 6 1978-1982 1983-1987 1988-1992 1993-1997 1998-2002 2003-2007 Mean width of annual wood ring [mm] Calendar year intervals healthy declining dead Calendar year intervals (b) 0 1 2 3 4 5 6 1979-1983 1984-1988 1989-1993 1994-1998 1999-2003 2004-2008 Mean width of annual wood ring [mm] Calendar year intervals healthy declining dead means, a Tukey’s test was performed. Signiﬁcance was determined at p \ 0.05. All statistical calculations were done with SAS software, version 9.0. means, a Tukey’s test was performed. Signiﬁcance was determined at p \ 0.05. All statistical calculations were done with SAS software, version 9.0. formed in healthy oaks during 1993–1997, in declining oaks during 1988–1992, and in dead oaks during 1983–1987. The narrowest annual increments for all oaks appeared in the last 5-year period; their width reached 2.77 mm (SE = 0.11) for healthy oaks, 1.01 mm (SE = 0.06) for declining oaks and 0.55 mm (SE = 0.04) for dead oaks. Similar variations in the average width of annual rings appeared in the oaks from Krotoszyn (Fig. 1b). However, unlike the oaks from Chojno´w, in the ﬁrst 5-year period (1979–1983), the average width of the annual ring of healthy oaks reached 3.20 mm (SE = 0.18). This value was not signiﬁcantly greater than one for declining oaks, i.e. 2.74 mm (SE = 0.23), or for dead trees, i.e. 2.98 mm (SE = 0.32). During the second 5-year span, and later on, signiﬁcantly wider annual rings were formed by the cambium of healthy oaks, compared to dead oaks. In the last phase (2004–2008), the width of the annual ring of dead oak wood reached its lowest, i.e. 1.05 mm (SE = 0.1). The ring was more than 3 times narrower than Materials and methods The samples are thus matched according to the year of ring formation but not necessarily of the tree age or the age at breast height. 1. The width of all annual rings (covering early and latewood zones cumulatively) deposited by the cam- bium of oaks growing at Chojno´w and Krotoszyn. The width of a total of 1,020 annual wood rings was measured. 2. The density of vessels in all annual rings of oak wood from Chojno´w and Krotoszyn. The results were calculated in mm2 of annual wood growth. 3. The diameters (D) of at least 20 earlywood vessels in all annual wood rings of oaks from Chojno´w and Krotoszyn (all in each annual ring in the ﬁeld of vision at magniﬁcation of 409). In total, the diameter of at To examine differences between oak groups in ana- tomical properties of wood and hydraulic conductivity, the datasets were ﬁrst tested for normal distribution and the homogeneity of variance. Then, in order to compare the 12 3 Eur J Forest Res (2014) 133:845–855 848 means, a Tukey’s test was performed. Signiﬁcance was determined at p \ 0.05. All statistical calculations were done with SAS software, version 9.0. formed in healthy oaks during 1993–1997, in declining oaks during 1988–1992, and in dead oaks during 1983–1987. The narrowest annual increments for all oaks appeared in the last 5-year period; their width reached 2 77 mm (SE = 0 11) for healthy oaks 1 01 mm (a) 0 1 2 3 4 5 6 1978-1982 1983-1987 1988-1992 1993-1997 1998-2002 2003-2007 Mean width of annual wood ring [mm] Calendar year intervals healthy declining dead (b) 0 1 2 3 4 5 6 1979-1983 1984-1988 1989-1993 1994-1998 1999-2003 2004-2008 Mean width of annual wood ring [mm] Calendar year intervals healthy declining dead Fig. 1 Change in the width of wood increments in the stem of oak trees, representing different health conditions at the time of sampling. Wood increment width was calculated as the mean for ﬁve trees belonging to the group of healthy, declining and dead oaks from Chojno´w (a) and Krotoszyn (b) and for 5-year periods. Density of earlywood vessels In the ﬁrst two 5-year periods, there were no signiﬁcant differences in the density of earlywood vessels, i.e. in their number per 1 mm2 of annual wood increment, among the analysed groups of oak from Chojno´w (Fig. 4a). The intergroup differences emerged during the years 1988–1992 and in the subsequent time phases analysed. Since 1993, the healthy oak wood has been always char- acterized by the highest density of earlywood vessels and the dead oaks by the lowest one. The number of earlywood vessels per 1 mm2 of wood in dead oaks was more than 7 times lower than in healthy oaks in the last 5-year period. It was observed that in all the analysed groups of oak trees, the ﬁrst annual rings of wood were characterized by the highest density of earlywood vessels. However, in 1988–1992, when the density of earlywood vessels per 1 mm2 of wood of declining and dead oaks was dimin- ishing, an upward trend appeared in the wood of healthy oak trees. Only starting from the next 5-year interval, a reduction in the value of this parameter occurred, which continued until the last time interval. Fig. 3 Transverse section through wood of declining oaks from Krotoszyn. Within the youngest and incessantly forming annual increment, the lumen of vessels ﬁlled with tyloses is indicated by arrows. Observation was performed under a light microscope Olympus BX61 equipped with an Olympus DP70 camera and Cell P software Similarly to the oaks from Chojno´w, there were no signiﬁcant differences in the distribution of earlywood vessels per 1 mm2 of wood in the three groups of oak from Krotoszyn, during 1979–1983 (Fig. 4b). Differences with p \ 0.05 appeared between the groups of healthy and dead oak trees and the healthy and declining ones in all the subsequent 5-year periods analysed. The density of early- wood vessels in healthy oak wood has been always sig- niﬁcantly higher, with its maximum occurring during 1999–2003. In 2004–2008, the vessel density in the annual growth of healthy oak wood was signiﬁcantly higher, compared with the declining and dead oaks. In the last 5-year period, the wood of healthy oaks contained about 16 earlywood vessels per mm2 (SE = 1.66), with 5/mm2 (SE = 0.4) for declining oak trees and 3/mm2 (SE = 0.48) for dead oak trees. The highest density of earlywood Fig. 3 Transverse section through wood of declining oaks from Krotoszyn. Width of annual rings Width of annual rings In the ﬁrst 5 years (1978–1982), the average width of annual ring in healthy oaks from Chojno´w was 4.11 mm (SE = 0.23), which was signiﬁcantly higher than the one in dead oaks, i.e. 2.82 mm (SE = 0.23, Fig. 1a). The ring width in trees of different groups did not vary between the groups of healthy and declining oaks during the next 5-year span (1983–1987) and in the following period (1988–1992). In the next 5-year periods, there were sig- niﬁcant differences in the width of annual increments between the groups of oaks. The widest annual rings were 123 123 Eur J Forest Res (2014) 133:845–855 849 Fig. 2 Cross section through wood of dead oaks from Chojno´w observed under an Olympus SZX9 stereomicroscope system equipped with an Olympus ColorView IIIu camera and getIT software at 963. Close to the bark, a sequence of a few annual rings comprising very narrow increments is marked by brackets. The abatement of latewood zone is intrinsic to the annual ring width decrease, while the earlywood zone is almost unchanged. The radial arrangement of latewood vessels within wider annual increments is showed with arrows and declining oaks, but in the case of healthy oaks, they were signiﬁcantly wider, i.e. more than 2 times, in com- parison with declining oaks. The share of earlywood and latewood within the annual wood increment was not measured during this study. However, the relation between the width of annual incre- ment and the share of earlywood and latewood could be seen clearly on microscopic pictures. Regardless of the health condition of trees, a simple relation appeared in respect to the last ﬁve rings: the narrower the increment, the lower the share of latewood (Fig. 2). It was also observed that in last incessantly forming ring in declining oaks from Krotoszyn, the lumen of earlywood vessels was ﬁlled with tyloses (Fig. 3). Fig. 2 Cross section through wood of dead oaks from Chojno´w observed under an Olympus SZX9 stereomicroscope system equipped with an Olympus ColorView IIIu camera and getIT software at 963. Close to the bark, a sequence of a few annual rings comprising very narrow increments is marked by brackets. The abatement of latewood zone is intrinsic to the annual ring width decrease, while the earlywood zone is almost unchanged. The radial arrangement of latewood vessels within wider annual increments is showed with arrows Density of earlywood vessels Within the youngest and incessantly forming annual increment, the lumen of vessels ﬁlled with tyloses is indicated by arrows. Observation was performed under a light microscope Olympus BX61 equipped with an Olympus DP70 camera and Cell P software in healthy oaks. The differences in the width of annual wood increments between the groups of healthy and declining oaks occurred in years: 1984–1988, 1994–1998 and during the next two 5-year spans. The width of the annual ring for healthy oaks attained its highest value in the years 1999–2003; this was signiﬁcantly higher than in the other two groups of oaks. Starting from 2004, the average width of annual increments was reduced for both healthy 123 12 Eur J Forest Res (2014) 133:845–855 850 (a) 0 5 10 15 20 25 30 35 1978-1982 1983-1987 1988-1992 1993-1997 1998-2002 2003-2007 Earlywood vessels mean density [no/mm2] Calendar year intervals healthy declining dead (b) 0 5 10 15 20 25 30 35 1979-1983 1984-1988 1989-1993 1994-1998 1999-2003 2004-2008 Earlywood vessels mean density [no/mm2] Calendar year intervals healthy declining dead Fig. 4 Variation in mean density of earlywood vessels’ distribution in the trunk of oak trees, representing different health conditions at the time of sampling. Density of vessels was deﬁned as the number of earlywood vessels per mm2 of annual increments and calculated as a mean for ﬁve trees representing the group of healthy, declining and dead oaks from Chojno´w (a) and Krotoszyn (b), respectively, and for 5-year periods. Vertical bars denote SEs (a) 0 5 10 15 20 25 30 35 1978-1982 1983-1987 1988-1992 1993-1997 1998-2002 2003-2007 Earlywood vessels mean density [no/mm2] Calendar year intervals healthy declining dead 2003-2007 Calendar year intervals (b) 0 5 10 15 20 25 30 35 1979-1983 1984-1988 1989-1993 1994-1998 1999-2003 2004-2008 Earlywood vessels mean density [no/mm2] Calendar year intervals healthy declining dead healthy declining dead vessels in dead and declining oak trees was observed in annual increments in the ﬁrst 5 years. In the subsequent years, it decreased. In the wood of declining oak trees, the number of earlywood vessels per 1 mm2 of the annual growth decreased by more than 2.5 times, when comparing the period of 1979–1983 with that of 2004–2008, and it was 4.9 times lower than in the corresponding period for dead oaks. (SE = 0.011), while for the dead oaks, it reached the value of 0.19 mm (SE = 0.013). Density of earlywood vessels In the years 1988–1987, there occurred differences in the earlywood vessel diameter among the three groups of trees. Signiﬁcantly, the greatest diameter of the earlywood vessels was found in the healthy oak wood and the smallest (by 70 lm smaller compared to the healthy oak wood) in the dead oak wood. From the year 1993 onwards, the diameter began to decrease in the declining and dead oaks, while the upward trend still con- tinued in healthy oaks. However, in the years 1993–1997, the vessel diameter in healthy oak wood did not differ (p \ 0.05) in comparison with declining oaks. In the last phase (2003–2007), the vessel diameter was the largest, i.e. 0.39 mm (SE = 0.07) in the healthy oak wood, whereas in declining oaks, it was 0.34 lm (SE = 0.005) and in dead oaks—0.27 mm (SE = 0.004), i.e. nearly 1.5 times smaller than in healthy oaks. An increase in the diameter of early- wood vessels corresponding to the age of the tree was also observed in all groups of oak trees from Krotoszyn, being Diameter of earlywood vessels In all analysed groups of oaks from Chojno´w, the earlywood vessel diameter increased together with the age of the tree during the ﬁrst 20 years, and the later changes were only a minor ﬂuctuation (Fig. 5a). In the ﬁrst 5-year period (1978–1982), there occurred differences in the earlywood vessel diameter between the healthy and dead oak trees. The healthy oak wood was characterized by signiﬁcantly larger diameter of earlywood vessels, that is 0.23 mm 123 123 123 Eur J Forest Res (2014) 133:845–855 851 (a) 0,00 0,05 0,10 0,15 0,20 0,25 0,30 0,35 0,40 0,45 1978-1982 1983-1987 1988-1992 1993-1997 1998-2002 2003-2007 Earlywood mean vessels diameter [mm] Calendar year intervals healthy declining dead (b) 0,00 0,05 0,10 0,15 0,20 0,25 0,30 0,35 0,40 0,45 1979-1983 1984-1988 1989-1993 1994-1998 1999-2003 2004-2008 Earlywood mean vessels diameter [mm] Calendar year intervals healthy declining dead Fig. 5 Change in the average earlywood vessels’ diameters of the trunk oak trees, representing different health conditions at the time of sampling. The mean diameter was calculated for ﬁve trees representing the group of healthy, declining and dead oaks from Chojno´w (a) and Krotoszyn (b), respectively, and for 5-year periods. Vertical bars denote SEs (a) 0,00 0,05 0,10 0,15 0,20 0,25 0,30 0,35 0,40 0,45 1978-1982 1983-1987 1988-1992 1993-1997 1998-2002 2003-2007 Earlywood mean vessels diameter [mm] Calendar year intervals healthy declining dead 1978-1982 1983-1987 1988-1992 1993-1997 1998-2002 2003-2007 Calendar year intervals (b) 0,00 0,05 0,10 0,15 0,20 0,25 0,30 0,35 0,40 0,45 1979-1983 1984-1988 1989-1993 1994-1998 1999-2003 2004-2008 Earlywood mean vessels diameter [mm] Calendar year intervals healthy declining dead most prominent in the wood of healthy oak trees (Fig. 5b). In the ﬁrst 5-year period (1979–1983), the wood of healthy and declining oaks was characterized by a signiﬁcantly larger diameter of the earlywood vessels, i.e. 0.32 mm, while it attained the value of 0.27 mm in dead oak trees. In the penultimate period of 5 years (1999–2003), there was a reduction in earlywood vessel diameter in the declining and dead oak trees, but it increased in the wood of healthy oak trees. In the years 2004–2008, that is the last 5-year period, the diameter of earlywood vessels was signiﬁcantly higher in healthy oak trees than in other two, reaching 0.41 mm (SE = 0.01) for healthy, 0.34 mm (SE = 0.008) for declining and 0.31 mm (SE = 0.007) for dead oaks. Diameter of earlywood vessels from Chojno´w and Krotoszyn were similar in shape, especially for healthy oaks, and showed a clear upward trend during 25-year time span (Fig. 6a, b). The maximum value of this variable for the healthy oaks from Chojno´w and Krotoszyn was recorded in the penultimate phase. On both sites, the hydraulic conductivity remained the mini- mum for dead oaks. During the whole period of study, the group of healthy oaks from Chojno´w was signiﬁcantly (with p \ 0.05) different from the group of dead trees in terms of potential water conductivity volume. In the ﬁrst 5-year period, the wood of healthy oak trunks was almost 6 times more effective in water conduction, but in the last phase, it was 30 times more effective. In the last three 5-year intervals, there were differences among the three groups of oaks. The hydraulic conductivity of healthy oaks was signiﬁcantly higher (1.6 times) in 1993–1997, almost 5.8 times higher during 1998–2002 and 6.8 times in the last 5-year period in comparison with the group of declining oaks. However, in recent years, the water transport Potential hydraulic conductivity The curves drawn to illustrate changes in the values of the theoretical hydraulic conductivity of the oak trunk wood 12 3 3 3 852 Eur J Forest Res (2014) 133:845–855 efﬁciency of oak tree trunks in all groups of trees showed a for healthy oaks remained constant, while a clear reduc- (a) 0,000 0,002 0,004 0,006 0,008 0,010 0,012 0,014 0,016 0,018 0,020 1978-1982 1983-1987 1988-1992 1993-1997 1998-2002 2003-2007 Theoretical hydraulic conductivity [kg x m-1x s-1 x MPa-1] Calendar year intervals healthy declining dead (b) 0,000 0,002 0,004 0,006 0,008 0,010 0,012 0,014 0,016 0,018 0,020 1979-1983 1984-1988 1989-1993 1994-1998 1999-2003 2004-2008 Theoretical hydraulic conductivity [kg x m-1 x s-1 x MPa-1] Calendar year intervals healthy declining dead Fig. 6 The potential hydraulic conductivity of earlywood vessels in the trunk of oak trees, representing different health conditions at the time of sampling. It was calculated for ﬁve trees representing the group of healthy, weakened and dead oaks from Chojno´w (a) and Krotoszyn (b), respectively, and for 5-year periods. Vertical bars denote SEs (a) 0,000 0,002 0,004 0,006 0,008 0,010 0,012 0,014 0,016 0,018 0,020 1978-1982 1983-1987 1988-1992 1993-1997 1998-2002 2003-2007 Theoretical hydraulic conductivity [kg x m-1x s-1 x MPa-1] Calendar year intervals healthy declining dead 1998-2002 2003-2007 Calendar year intervals (b) 0,000 0,002 0,004 0,006 0,008 0,010 0,012 0,014 0,016 0,018 0,020 1979-1983 1984-1988 1989-1993 1994-1998 1999-2003 2004-2008 Theoretical hydraulic conductivity [kg x m-1 x s-1 x MPa-1] Calendar year intervals healthy declining dead healthy declining dead efﬁciency of oak tree trunks in all groups of trees showed a downward trend. for healthy oaks remained constant, while a clear reduc- tion in the value of this parameter was observed for the declining and dead oaks. In the ﬁrst 5-year period, no signiﬁcant differences in the hydraulic conductivity of wood trunks were found among the three groups of oak trees from Krotoszyn. The efﬁciency of water conduction in healthy oak trees started increasing from the second 5-year period as compared with the groups of declining and dead oaks, for which the hydraulic conductivity in the last 5-year period was sig- niﬁcantly (more than 16 times) smaller in comparison with the healthy oak group. Also for the declining oak trees, there was no clear upward trend for the hydraulic conductivity efﬁciency of the trunk wood during the period analysed, just like in the case of the healthy oaks. Potential hydraulic conductivity Even in the period of 1983–1993, it did not differ sig- niﬁcantly from the dead oak group. The largest value of the theoretical hydraulic conductivity for the declining oak trees was obtained in the penultimate period of 5 years, and this was 1.8 times higher than for the dead oak group and 3.7 times lower than for the healthy oaks. After this period, the value of the hydraulic conductivity Discussion The elements conducting water most efﬁciently are the earlywood vessels (Tyree and Ewers 1991), and their development is controlled by genetic factors (Zobel and van Buijtenene 1989) and also by environmental ones, like the availability of water (Hsiao 1973; Fritts 2001). The enlargement of cambial cell derivatives that undergo differentiation towards vessel elements is a turgor-driven process which depends strictly on solute accumulation and cellular water uptake (Langer et al. 2002). As in other species with ring-porous wood, the variation in the diameter of the earlywood vessels in three health groups of oak took the shape of a sigmoid curve (Helin´ska- Raczkowska 1994; Helin´ska-Raczkowska and Fabisiak 1999), which means that the diameter increased along the radius, i.e. with the distance from the pith or the age of the cambium until reaching a value around which it ﬂuctuated. It should be emphasized that the widest vessels were always in the wood of trees representing the group of healthy oaks, whereas the wood of dead oak trees was characterized by the smallest diameter of vessels. A con- comitant decrease in earlywood vessels’ density per unit of the annual ring surface was noted: the lowest for the trees from the group of dead oaks and the highest for the healthy ones, contributing to the reduction in the potential hydraulic conductivity of oak trees represented particularly in the groups of declining and dead trees. The increase in the vessel diameter and the consequent improved water conductivity possibly constitute a mechanism of adaptation to changing environmental conditions and a response to stress factors affecting the growth potential and reproduc- tion capacity of the tree (Drew et al. 2009). This is in line with the Tyree’s hypothesis (2003), which assumes that the growth rates are hydraulically mediated. Thus, the adverse changes in structural wood parameters important for hydraulic conductivity, such as the diameter of tracheary elements, may indicate a lack of adaptability to changing environmental conditions and may result in intensiﬁcation of the decline symptoms. Levanicˇ et al. (2011), on the other hand, by analysing the inﬂuence of drought on xylem hydraulic parameters and mortality of Q. robur, proposed that ‘‘the ability for hydraulic wood rebuild’’ can determine the successful survival of trees. It appears that the ability for ‘‘hydraulic reconstruction’’ is essential for the survival of an individual under the conditions of water stress. It should be noted, however, that Levanicˇ et al. Discussion The observed changes in the structure of wood in oak trees with different health status in the year of the study are a function of the tree age and environmental factors. The width of annual rings measured at the breast height shows an overall downward trend corresponding to the increasing tree age, except for the juvenile growth phase and juvenile wood (Zobel and Sprague 1998). A similar downward trend was observed for oak trees, whereas the average width of the ring was always the highest for oaks classiﬁed as healthy and the lowest for those classiﬁed as dead. It seems that the persistence of difference in the width of the ring for trees of similar age and growing in similar envi- ronmental conditions (in reference to oak from Chojno´w and Krotoszyn, respectively) may indicate the growth 123 123 853 Eur J Forest Res (2014) 133:845–855 ﬁrst mechanism is channelled by plants during moderate water stress when the reduction in vessel size may have an adaptive character. The second one is induced by a short and more severe water stress, which leads to vessels’ embolism. Reduced lumen of vessels in wood of oaks from the declining group, subjected to different types of unfa- vourable factors, may contribute to control of water transport and to reduction in vulnerability to cavitation and embolism. It is known that the large earlywood vessels contribute to the greater part of water ﬂow throughout the shoot/trunk, but due to their large lumen diameter, these vessels are vulnerable to embolization (Cochard et al. 1997). Due to the fact that the plants function at constant risk with regard to hydraulic conductance (Carlquist 1988; Hacke et al. 2006), the transport of water is a matter of compromises between safety and efﬁciency. The method of the safe transport in oaks from declining group is consid- ered to have not been sufﬁcient under the impact of factors of both biotic and abiotic nature. potential and the consequent longevity of particular trees, which in turn may determine their susceptibility to decay. Survival of a tree is dependent, however, on its efﬁciency of water conduction rather than on the width of growth increments, especially in ring-porous species (Carlquist 1975; Zimmermann 1983). Discussion (2011) analysed the effect of only one factor, namely water stress. Dieback of oak trees, observed in the forest districts, is probably an outcome of the interaction of abiotic factors, including water avail- ability, with defoliating insects, borer attack and patho- genic fungi (according to information from the staff from Chojno´w Forest District and Krotoszyn Forest District). The presence of the tyloses in the earlywood vessels of the incessantly emerging annual wood increment is considered to have had impact on the reduction of the hydraulic con- ductance in declining oaks from Krotoszyn. The tyloses and gel substances were observed in the wood of weakened oaks by Woz´ny and Siwecki (1991). The authors postulated that modiﬁcations in the structure of conductive elements were caused by fungi belonging to Ceratocystis genus, which resulted in wood dysfunction. In the case of oaks from Krotoszyn, it could be also defoliation, which is known as a factor altering the water transport due to the fact that the lumen of its vessels was ﬁlled with tyloses (Fajvan et al. 2008). A similar response concerning the increase in the ves- sels’ lumen under stress factors causes water-limited growth conditions. Corcuera et al. (2004a) observed that the size of earlywood vessels in ring-porous oaks increased together with increasing water deﬁcit. However, conﬂicting results were also showed by Corcuera et al. (2004b) or Eilmann et al. (2006). Lovisolo and Schubert (1998) sug- gested two possible mechanisms concerning the inﬂuence of water stress on hydraulic conductance of shoots. The On the other hand, it is known that the cambial activity and wood formation are processes dependent on the accessibility of photoassimilates (Eilmann et al. 2009). The crown of a declining oak tree was defoliated by approximately 80 %. It can therefore be assumed that the photosynthetic efﬁciency of declining oaks was lower, compared with the group of healthy trees, which could 12 3 3 Eur J Forest Res (2014) 133:845–855 854 Corcuera L, Camarero JJ, Gil-Pelegrin E (2004b) Effects of a severe drought on Quercus ilex growth and xylem anatomy. Trees 18:83–92 affect the meristematic activity of the cambium and consequently the wood formation process during growth seasons. Thus, the results of this study may have con- ﬁrmed that the individuals best adapted to the changes of the external environment survive in the ‘‘struggle for existence’’, in accordance with the Darwin’s law of nat- ural selection. Conclusions Eckstein D, Frisse E, Quiehl F (1977) Wood anatomical studies of a beech tree to demonstrate anthropogenic inﬂuences on its environment. Angew Bot 51:47–56 1. Formation of narrow growth rings and vessels of small diameter over the course of a tree’s life is relevant to decline in tree vitality. 1. Formation of narrow growth rings and vessels of small diameter over the course of a tree’s life is relevant to decline in tree vitality. Eilmann B, Weber P, Rigling A, Eckstein D (2006) Growth reactions of Pinus sylvestris L. and Quercus pubescens Willd. to drought years at a xeric site in Valais, Switzerland. Dendrochronologia 23:121–132 2. Hydraulic conductivity decreases in response to reduc- tion in the vessel diameter as well as vessels’ density, which during the complex process of oak decline contributes to the diminishing of the vigour of trees and to their death. 2. Hydraulic conductivity decreases in response to reduc- tion in the vessel diameter as well as vessels’ density, which during the complex process of oak decline contributes to the diminishing of the vigour of trees and to their death. Eilmann B, Zweifel R, Buchmann N, Fonti P, Rigling A (2009) Drought-induced adaptation of the xylem in Scots pine and pubescent oak. 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https://openalex.org/W2970084525	http://www.uco.es/ucopress/az/index.php/az/article/download/4197/2502	Portuguese	null	Caracterização Biométrica dos Caprinos da República de Cabo Verde	Archivos de zootecnia	2,019	cc-by-sa	9,975	Palavras chave adicionais Palavras chave adicionais Aptidão zootécnica. Biometria. Conservação animal. Recursos genéticos. SUMMARY Information Cronología del artículo. Recibido/Received: 16.04.2018 Aceptado/Accepted: 03.10.2019 On-line: 15.07.2019 Correspondencia a los autores/Contact e-mail: luanna.ufsb@gmail.com Additional keywords Zootechnical aptitude. Biometry. Animal conservation. Genetic resources. Additional keywords Zootechnical aptitude. Biometry. Animal conservation. Genetic resources. The aims in this study was test the genetic discernment among five goats populations of the Republic of Cape Verde (n=533) by biometric data and statistical analysis. It were analysed 16 cha racteristics in adult goats, through descriptive statistics, analysis of variance, test for multicollinearity, Mahalanobis distance (D²) and UPGMA algorithm (Unweighted Pair Group Method Arithmetic Mean). After the test of multicollinearity was identified and discarded the variable thoracic depth. The D² were calculated using 15 biometric measurements. The maximum value of D² was between the populations of the islands of Fire and São Nicolau (22.73), and D² was lower between Santo Antão and São Vicente (3.71). The dendrogram based on biometric data in five populations put the goats on the Fire Island in other branch. Clustered in one the branch the goats from the islands of Santo Antão and São Vicente. This result is consistent with the geographic distance among the islands of Cape Verde and the recent history of exchange of animals among them. Information Cronología del artículo. Recibido/Received: 16.04.2018 Aceptado/Accepted: 03.10.2019 On-line: 15.07.2019 Correspondencia a los autores/Contact e-mail: luanna.ufsb@gmail.com cabra saheliana tenha entrado na constituição da cabra destas ilhas (Pires et al. 2012, p.777). Segundo a docu mentação da época da colonização, as ilhas achavam-se despovoadas e sem vestígios de presença humana na altura da chegada dos portugueses, pelo que foi neces sário trazer gente, gado e plantas para dar início a um processo de permanência dos habitantes iniciada em Caracterização Biométrica dos Caprinos da República de Cabo Verde Pires, L.C.1@; Machado, T.M.2; Fonseca, J. de D.3; Fonseca, J.F.4; Pile, E.5 e Brandão, F.Z.6 ¹Universidade Federal do Sul da Bahia. Brasil. ²Universidade Federal de Viçosa. Minas Gerais. Brasil. ³Ministério do Ambiente, do Desenvolvimento Rural e dos Recursos Marinhos da República de Cabo Verde. Brasil. 4Empresa Brasileira de Pesquisa Agropecuária. Brasil. 5Universidade do Panama. Brasil. 6Universidade Federal Fluminense. Brasil. Arch. Zootec. 68 (263): 384-394. 2019. RESUMO Objetivou-se discernir populações caprinas de cinco ilhas da República de Cabo Verde (n=533) por meio de dados biométricos e análises estatísticas. Foram avaliadas 16 características de fêmeas adultas, através da estatística descritiva simples, análise de variância, teste de multicolinearidade, distância gene ralizada de Mahalanobis (D²) e algoritmo UPGMA (Unweighted Pair Group Method Arithmetic Mean). Após o teste de multicolinearidade foi identificada e descartada a variável profundidade torácica. As D² foram calculadas com base nas 15 medidas biométricas. O maior valor da D² foi entre as populações das ilhas do Fogo e São Nicolau (22,73), e a menor D² foi entre Santo Antão e São Vicente (3,71). O dendrograma a partir de 15 variáveis em cinco populações colocou as cabras da ilha de Fogo em ramo a parte das demais. Agruparam-se num ramo as cabras das ilhas de Santo Antão e São Vicente. Este resultado está de acordo com a distância geográfica entre as ilhas de Cabo Verde e o histórico recente de intercâmbio de animais entre elas. MATERIAL E MÉTODOS Os dados foram provenientes de 533 caprinos aci ma de dois anos de idade de cinco das dez principais ilhas e em 18 diferentes localidades. Na ilha do Fogo foram coletadas amostras em oito diferentes localida des (Figura 1, Tabela I). Em cada rebanho se preencheu um formulário de pesquisa que reúne informações do fenótipo de cada animal amostrado, da propriedade e proprietário. Foram também feitas anotações quanto ao trânsito de animais, às raças exóticas utilizadas na caprinocultura e ao consumo de produtos de origem caprina. As características biométricas, como peso, altura, comprimento, perímetro escrotal, que precisam ser mensuradas com uso de instrumentos de medição, permitem caracterizar, classificar ou agrupar os in divíduos de uma mesma espécie em raças ou tipos distintos. Tais características podem ser definidas como uma particularidade individual, que em maior ou me nor grau de variação, determina o tipo de raça ou tipo étnico a qual pertence (Rodero et al. 1992, pp.519-30; Bedotti et al. 2004, pp.261-71). A amostragem em Santiago foi reduzida em com paração às outras ilhas em estudo. Apesar do efetivo caprino ser maior na Ilha de Santiago (República de CaboVerde/MADRRM, 2004), seus rebanhos são me nores. Em algumas das propriedades eles são com postos por um único animal. Nesta ilha, as unidades de produção possuem mais machos que fêmeas em idade reprodutiva, devido à tradição local de consumir carne de cabritos e bodes em datas festivas. Nas ilhas do Fogo e Santo Antão, há rebanhos mais numerosos que nas demais, o que possibilitou maior número de animais amostrados nestas ilhas. As medidas biométricas são tomadas com base no conhecimento que há grupos genéticos caprinos per naltas e anões, caprinos de orelhas longas e caídas e outros de orelhas eretas e curtas. Além disso, o baixo custo na coleta e processamento dos dados biométri cos, juntamente com o poder discriminante da análise multivariada, permitem preconizá-las como técnicas auxiliares no discernimento de populações caprinas. Estas medidas variam entre grupos genéticos e tam bém se prestam à identificação racial, sendo utilizadas para classificação de caprinos desde a segunda metade do século passado. Para aprimorar estes estudos pas sou-se a realizar a mensuração simultânea de várias características e estabelecer índices entre medidas cor porais (Bourzat et al. 1993, pp.667-74; Bouchel et al. 1997, pp.37-46). CARACTERIZAÇÃO BIOMÉTRICA DOS CAPRINOS DA REPÚBLICA DE CABO VERDE de distintividade (Alderson, 2018), a caracterização fenotípica é uma ferramenta acessível e fácil de ser rea lizada em programas de conservação e melhoramento e os criadores são promotores de diversidade genética, e devem, portanto, serem consultados nos programas de conservação e melhoramento da raça. Devido à falta de informações sobre os caprinos de Cabo Verde, objetivou-se caracterizar e discernir fenotipicamente as populações caprinas de Cabo Verde por meio de marcadores biométricos. 1462 (Carvalho & Sá 2007, pp.124-5). Após a conquis ta, as ilhas tornaram-se uma importante estação de negociação marítima através da qual as raças caprinas foram introduzidas e, posteriormente, influenciaram as populações caprinas locais. O grau de influência varia de uma ilha para outra. Com o passar dos séculos, os caprinos cabo-verdianos, em cada ilha, evoluíram, em grande parte, de forma isolada. Estes animais não são reconhecidos oficialmente como uma raça. 1462 (Carvalho & Sá 2007, pp.124-5). Após a conquis ta, as ilhas tornaram-se uma importante estação de negociação marítima através da qual as raças caprinas foram introduzidas e, posteriormente, influenciaram as populações caprinas locais. O grau de influência varia de uma ilha para outra. Com o passar dos séculos, os caprinos cabo-verdianos, em cada ilha, evoluíram, em grande parte, de forma isolada. Estes animais não são reconhecidos oficialmente como uma raça. Cabo Verde possui aproximadamente 215,9 mil ca beças caprinas (FAOSTAT, 2017). As ilhas de Santiago (40,43%), Fogo (17,74%) e Santo Antão (16,05%) pos suem maior efetivo caprino (República de Cabo Verde/ MADRRM, 2004). Embora a criação de caprinos seja algo comum nas famílias rurais cabo-verdianas, espe cialmente nas ilhas do Fogo, Santo Antão, São Nicolau, Santiago e São Vicente, ela é feita ainda em pequena escala e de forma rudimentar (Pecuária, 2011). INTRODUÇÃO A existência de caprinos nas Ilhas de Cabo Verde tem sido conhecida desde o século 15, quando as ilhas foram colonizadas pelos portugueses. Os caprinos ca bo-verdianos foram oriundos, no período colonial, de Portugal (Machado 2013, pp.11-8). É possível que a Arch. Zootec. 68 (263): 384-394. 2019. CARACTERIZAÇÃO BIOMÉTRICA DOS CAPRINOS DA REPÚBLICA DE CABO VERDE Archivos de zootecnia vol. 68, núm. 263, p. 379. MATERIAL E MÉTODOS Apesar de que hoje a caracterização genética é a principal fonte de informação nas análises Para obter as medidas lineares corporais foi uti lizada uma fita métrica, enquanto os caprinos eram mantidos em posição correta de aprumos. As 16 ca racterísticas avaliadas foram: altura de cernelha (AC), distância da parte mais alta da cernelha ao chão; altura das patas (AP); altura da garupa ao chão (AG); com primento corporal (CC), distância entre a parte cranial da tuberosidade maior do úmero e a tuberosidade isquiática; comprimento de orelha (CO); circunferência Tabela I. Caprinos amostrados em cinco ilhas da República de Cabo Verde (Goats sampled on five islands in the Re public of Cape Verde). Ilha No de animais Concelho Localidades Fogo 195 São Filipe Vicente Dias, Genebra, Salto, Rocha Branca, Salto Acima, Monte Grande Santa Catarina do Fogo Achada Furna, Cabeça Fundão Santiago 28 Tarrafal São Miguel Tarrafal Achada Pizarra São Nicolau 64 Ribeira Brava Boqueirão, Caleijão São Vicente 64 São Vicente Ribeira do Calhau, Curral Tortolho, Ribeira de Vinha e Ribeira de Julião Santo Antão 182 Porto Novo Ponte Sul e Aureliano TOTAL 533 18 Tabela I. Caprinos amostrados em cinco ilhas da República de Cabo Verde (Goats sampled on five public of Cape Verde). 18 PIRES, MACHADO, FONSECA, FONSECA, PILE E BRANDÃO Figura 1. Localização das principais ilhas da República de Cabo Verde e distribuição geográfica das locali dades dentro das ilhas de Cabo Verde onde foi realizada a amostragem (Location of the main islands of the Republic of Cape Verde and geographical distribution of the localities within the islands of Cape Verde where the sampling was carried out). Figura 1. Localização das principais ilhas da República de Cabo Verde e distribuição geográfica das locali dades dentro das ilhas de Cabo Verde onde foi realizada a amostragem (Location of the main islands of the Republic of Cape Verde and geographical distribution of the localities within the islands of Cape Verde where the sampling was carried out). rais. As demais 15 variáveis foram padronizadas e sub metidas às análises de agrupamento e de componentes principais. O critério para descarte de variáveis na análise de componentes principais (ACP) seguiu as recomen dações de Jolliffe (1973, pp.21-31). Estabeleceu-se que o número de variáveis descartadas devia ser igual ao de componentes cuja variância (autovalor) é inferior a 0,7. Archivos de zootecnia vol. 68, núm. 263, p. 380. MATERIAL E MÉTODOS Resumo da análise descritiva e de variância para medidas corporais (cm) em cinco populações caprinas da República de Cabo Verde (Summary of the descriptive and variance analysis for body measurements (cm) in five goat populations of the Republic of Cape Verde). AC AP PT CC CO AG CT CCAB LR LCAB LIL LIS CGA PCA TCH DCH Ilhas * * * * *** * * * * * * * * * ns CV 5,90 9,61 10,31 7,50 9,44 6,09 7,11 6,26 8,11 7,36 8,94 9,64 7,25 8,22 22,85 12,28 Média 66,16 35,60 30,55 65,18 13,92 68,73 78,84 19,82 15,62 14,06 15,39 16,42 14,83 8,57 20,29 9,01 Desvio- padrão 3,90 3,42 3,15 4,89 1,31 4,18 5,60 1,24 1,27 1,04 1,38 1,58 1,07 0,70 4,64 1,11 AC: altura de cernelha; AP: altura das patas; PT: profundidade torácica; CC: comprimento corporal; CO: comprimento da orelha; AG: altura da garupa; CT: circunferência torácica; CCAB: comprimento da cabeça; LR: longitude do rosto; LCAB: largura da cabeça; LIL: largura da garupa entre ílios; LIS: largura da garupa entre ísquios; CGA: comprimento de garupa; PCA: perímetro da canela; TCH: tamanho do chifre; DHC: diâmetro do chifre; CV: coeficiente de variação; * P < 0,05; P < 0,01; * P < 0,001; e ns: não-significativo. Tabela II. Resumo da análise descritiva e de variância para medidas corporais (cm) em cinco populações caprinas da República de Cabo Verde (Summary of the descriptive and variance analysis for body measurements (cm) in five goat populations of the Republic of Cape Verde). Após estes investimentos, a pecuária na ilha do Fogo deixou de ser uma atividade tradicional, para ser uma atividade econômica sustentável (Inforpress, 2011). Pelas anotações realizadas em campo, verificou-se que em praticamente todos os rebanhos avaliados, os reprodutores são oriundos do próprio rebanho, o que está de acordo com Ayantunde et al. (2007, p.143), analisando os sistemas de criação ovinos, caprinos e bovinos no Oeste da África, em que observaram que cerca de 75% dos animais advêm do próprio rebanho, prática que contribui para aumento de endogamia. Observou-se que os criadores das diferentes ilhas de Cabo Verde possuem preferência por reproduto res da raça Majorera oriundos das ilhas Canárias ou cruzamentos destes, devido, na opinião deles, terem as melhores características físicas para um bom repro dutor e por possuírem muitas características comuns entre os tipos locais. MATERIAL E MÉTODOS torácica (CT), circunferência externa do tórax, ao nível da cernelha; comprimento da cabeça (CCAB), medida desde o occipital até o lábio inferior; longitude do rosto (LR), medida entre a linha imaginária que une o ângulo interno dos olhos e o ponto mais rostral da parte nasal; largura da cabeça (LCAB), medida entre os arcos zigo máticos; largura da garupa entre ílios (LIL), distância entre as protuberâncias ilíacas; largura da garupa entre ísquios (LIS), distância entre as pontas dos ísquios; comprimento da garupa (CGA), distância entre a tu berosidade coxal do ílio e a tuberosidade isquiática ou anca-jarrete; perímetro da canela (PCA), medida do ter ço médio da região metacarpiana do membro posterior esquerdo; tamanho do chifre (TCH); diâmetro do chifre (DCH). A profundidade torácica (PT) foi calculada pela diferença entre duas medidas (AC - AP). A análise de agrupamento foi conduzida adotando-se a distância generalizada de Mahalanobis (D2) como me dida de dissimilaridade, e foi empregado o método de agrupamento hierárquico Unweighted Pair Group Method With Arithmetic Mean (UPGMA) para gerar os agrupa mentos e, consequentemente, o dendrograma. Para a for mação de grupos mutuamente exclusivos empregou-se o método de otimização de Tocher. Avaliou-se, também, a importância relativa das características para a divergên cia segundo metodologia de Singh (1981, pp.237-45), o coeficiente de correlação cofenética (CCC) para avaliar a distorção provocada pelo agrupamento das populações e, realizou-se o bootstrap com 1000 repetições para avaliar a acurácia dos agrupamentos. Os dados biométricos foram tabulados em planilhas do Excel e posteriormente analisados por da estatísti ca descritiva, análise da variância, correlações entre as variáveis e o teste Student Newman Keuls para compara ção das médias das diferentes populações (raças e tipos caprinos). Na análise de variância foi examinado o efeito da população sobre as medidas biométricas. Preliminar mente, foi realizado o teste do número de condições (NC), de acordo Montgomery, Peck &Vining (2012, p.528), para diagnóstico do efeito da multicolinearidade. Após essa análise, foi identificada e descartada a variável PT, que provocou forte multicolinearidade nas medidas corpo As análises foram realizadas pelos programas SAS System for Windows NT, versão 8.0, licenciado pela Uni versidade Federal de Viçosa (SAS, 1999) e GENES-versão 6.0 (Cruz, 2008). RESULTADOS E DISCUSSÃO CARACTERIZAÇÃO BIOMÉTRICA DOS CAPRINOS DA REPÚBLICA DE CABO VERDE Tabela II. MATERIAL E MÉTODOS Na ilha do Fogo observou-se grande variabilida de dos caprinos quanto à sua forma e aptidão física, promovida pelo sistema de criação baseado no uso comum de recursos forrageiros locais, que facilita a conexão natural entre rebanhos próximos, proporcio nando fluxo gênico. Verificou-se que nas localidades com maiores altitudes nesta ilha, os sistemas de cria ção apresentaram melhor aptidão dos rebanhos. Estes rebanhos caprinos são visados para produção leiteira e, no próprio local, os criadores produzem queijos de sabores peculiares, provavelmente devido às pastagens de campos altos com espécies nativas, ou mesmo en dêmicas. O projeto de Proteção dos Recursos Naturais na Ilha do Fogo proporcionou este melhor desenvol vimento da caprinocultura na ilha, principalmente nas áreas de maior altitude, através da construção de currais melhorados, bebedouros, laticínios familiares, recuperação de pastagens degradadas e melhoria dos animais com introdução de bodes das ilhas Canárias. Na análise descritiva e de variância realizada para as diferentes medidas corporais nas cabras cabo-ver dianas (Tabela II), observou-se que os coeficientes de variação (CV), para todas as variáveis, variaram de 5,90 a 22,85%. As medidas que apresentaram os maiores CV foram: tamanho do chifre (22,85%) e diâmetro do chifre (12,28%). Os CV, para a maioria das medidas corporais foram próximos a 10%. Estes CV indicam grau de va riação fenotípica entre os indivíduos coletados e a exis tência de variabilidade genética para as características estudadas, passíveis de melhoria a partir de programas de melhoramento genético intra-populacional. O resultado da análise de variância das diferentes medidas corporais (Tabela II) mostrou diferença sig nificativa entre as populações das cinco ilhas, para a maioria das medidas avaliadas, exceto para diâmetro Tabela III. Médias estimadas pelos mínimos quadrados (lsmeans) para medidas corporais em cinco popula ções caprinas da República de Cabo Verde (Medium estimated by least squares (lsmeans) for body measurements in five goat populations of the Republic of Cape Verde). V. Coeficientes de correlação de Pearson entre medidas corporais (cm) em cinco populações caprinas da República de Cabo Verde (Pearson correlation between body measurements (cm) in five goat populations of the Republic of Cape Verde). AC AP PT CC CO AG CT CCAB LR LCAB LIL LIS CGA PCA TCH DCH 1 00 Assim, elas se caracterizam por serem as mais profundas (PT = 32,59 cm), com a maior circunferência torácica (CT = 80,67 cm) e com o maior tama nho dos chifres (TCH = 22,16 cm) dentre as ilhas em estudo. Como a CT está correlacionada com maior peso corporal, pode-se concluir que as cabras de São Nicolau possuem maior peso à idade adulta em com paração as demais. Na ilha de Santiago, as cabras são as mais baixas (AC = 61,90 cm) e possuem as menores: altura das patas (AP = 32,08 cm); altura da garupa (AG = 67,20 cm); circun ferência torácica (CT = 75,65 cm); comprimento da cabeça (CCAB = 18,98 cm); e largura da cabeça (LCAB = 13,33 cm) em relação às demais ilhas. As cabras da ilha de Santo Antão possuem as meno res médias para as características comprimento da garupa (CGA = 14,28 cm), tamanho do chifre (TCH = 18,82 cm) e diâmetro do chifre (DCH = 8,83 cm). Para AC e PT es tas cabras obtiveram valores próxi mos aos encontrados por Bouchel et al. (1997, pp.37-46) em caprinos do Rove, França, com média para As cabras da ilha de São Nico lau apresentam as maiores médias para as variáveis PT, CT, LR, LCAB, LIL, LIS, PCA e TCH, e as menores médias para CC e CO. Assim, elas se caracterizam por serem as mais profundas (PT = 32,59 cm), com a maior circunferência torácica (CT = 80,67 cm) e com o maior tama nho dos chifres (TCH = 22,16 cm) dentre as ilhas em estudo. Como a CT está correlacionada com maior peso corporal, pode-se concluir que as cabras de São Nicolau possuem maior peso à idade adulta em com paração as demais. Na ilha de Santiago, as cabras são as mais baixas (AC = 61,90 cm) e possuem as menores: altura das patas (AP = 32,08 cm); altura da garupa (AG = 67,20 cm); circun ferência torácica (CT = 75,65 cm); comprimento da cabeça (CCAB = 18,98 cm); e largura da cabeça (LCAB = 13,33 cm) em relação às demais ilhas. As cabras da ilha de Santo Antão possuem as meno res médias para as características comprimento da garupa (CGA = 14,28 cm), tamanho do chifre (TCH = 18,82 cm) e diâmetro do chifre (DCH = 8,83 cm). Archivos de zootecnia vol. 68, núm. 263, p. 382. MATERIAL E MÉTODOS Ilha AC AP PT CC CO AG CT CCAB LR LCAB LIL LIS CGA PCA TCH DCH Fogo 64,77b 37,23a 27,53c 68,27a 14,62a 68,37ab 78,30ab 19,03c 15,68ab 14,50a 16,04a 15,95c 15,67a 8,37b 20,79ab 9,05a São Nicolau 67,79a 35,21b 32,59a 61,09c 12,89b 68,82ab 80,67a 19,86b 16,01a 14,66a 16,07a 19,05a 14,41b 9,14a 22,16a 9,11a Santiago 61,90c 32,08c 29,83b 61,55c 12,90b 67,20b 75,65b 18,98c 15,33b 13,33b 15,38ab 17,53b 14,70b 8,68b 21,89a 9,08a Santo Antão 66,76ab 34,55b 32,21a 64,58b 13,51b 68,72ab 78,76a 20,25ab 15,62ab 13,68b 14,99b 15,97c 14,28b 8,61b 18,82b 8,83a São Vicente 67,95a 35,82ab 32,13a 63,65bc 14,55a 70,13a 79,83a 20,86a 15,21b 13,66b 14,21c 16,16c 14,60b 8,41b 20,57ab 9,24a Teste de Student Newman Keuls para as medidas corporais. Letras diferentes na mesma coluna indicam diferença significativa entre as medidas. AC: altura de cernelha; AP: altura das patas; PT: profundidade torácica; CC: comprimento corporal; CO: comprimento da orel ha; AG: altura da garupa; CT: circunferência torácica; CCAB: comprimento da cabeça; LR: longitude do rosto; LCAB: largura da cabeça; LIL: largura da garupa entre ílios; LIS: largura da garupa entre ísquios; CGA: comprimento de garupa; PCA: perímetro da canela; TCH: tamanho do chifre; e DHC: diâmetro do chifre. Archivos de zootecnia vol. 68, núm. 263, p. 381. PIRES, MACHADO, FONSECA, FONSECA, PILE E BRANDÃO PIRES, MACHADO, FONSECA, FONSECA, PIL coefficients between body measurements (cm) in five goat populations of the Republic of Cape Verde). MATERIAL E MÉTODOS LCAB 0,17 0,14 0,04 0,34 0,19 0,21 0,33 0,10 0,33 1,00 LIL 0,20* 0,12 0,10* 0,41* 0,18* 0,33* 0,42* 0,06ns 0,20* 0,45* 1,00 LIS 0,25* -0,02ns 0,29* 0,12 -0,05ns 0,20* 0,27* 0,16* 0,12 0,28* 0,38* 1,00 CGA 0,18* 0,21* -0,01ns 0,52* 0,29* 0,28* 0,32* 0,03ns 0,01ns 0,31* 0,43* 0,10* 1,00 PCA 0,37* 0,04ns 0,36* 0,23*** 0,09* 0,29* 0,38* 0,42* 0,18* 0,21* 0,28* 0,35* 0,21* 1,00 TCH 0,22* 0,03ns 0,21* 0,30* 0,04ns 0,29* 0,53* 0,18* 0,26* 0,38* 0,42* 0,22* 0,29* 0,16 1,00 DCH 0,32* 0,04ns 0,29* 0,35* 0,14 0,40* 0,53* 0,30* 0,30* 0,32* 0,36* 0,24* 0,30* 0,29* 0,58* 1,00 AC: altura de cernelha; AP: altura das patas; PT: profundidade torácica; CC: comprimento corporal; CO: comprimento da orelha; AG: altura da garupa; CT: circunferência torácica; CCAB: comprimento da cabeça; LR: longitude do rosto; LCAB: largura da cabeça; LIL: largura da garupa entre ílios; LIS: largura da garupa entre ísquios; CGA: comprimento de garupa; PCA: perímetro da canela; TCH: tamanho do chifre; DHC: diâmetro do chifre; * P < 0,05; P < 0,01; * P < 0,001; e ns: não-significativo. MATERIAL E MÉTODOS , , PT 0,58* -0,39* 1,00 CC 0,29* 0,28* 0,04ns 1,00 CO 0,18* 0,25* -0,04ns 0,39* 1,00 AG 0,59* 0,32* 0,33* 0,33* 0,20* 1,00 CT 0,40* -0,01ns 0,44* 0,40* 0,14 0,40* 1,00 CCAB 0,45* -0,03ns 0,51* 0,14 0,11* 0,32* 0,43* 1,00 LR 0,13** 0,06ns 0,09* 0,19* 0,03ns 0,14 0,34* 0,38* 1,00 LCAB 0,17* 0,14 0,04ns 0,34* 0,19* 0,21* 0,33* 0,10* 0,33* 1,00 LIL 0,20* 0,12** 0,10* 0,41* 0,18* 0,33* 0,42* 0,06ns 0,20* 0,45* 1,00 LIS 0,25* -0,02ns 0,29* 0,12 -0,05ns 0,20* 0,27* 0,16* 0,12 0,28* 0,38* 1,00 CGA 0,18* 0,21* -0,01ns 0,52* 0,29* 0,28* 0,32* 0,03ns 0,01ns 0,31* 0,43* 0,10* 1,00 PCA 0,37* 0,04ns 0,36* 0,23*** 0,09* 0,29* 0,38* 0,42* 0,18* 0,21* 0,28* 0,35* 0,21* 1,00 TCH 0,22* 0,03ns 0,21* 0,30* 0,04ns 0,29* 0,53* 0,18* 0,26* 0,38* 0,42* 0,22* 0,29* 0,16 1,00 DCH 0,32* 0,04ns 0,29* 0,35* 0,14 0,40* 0,53* 0,30* 0,30* 0,32* 0,36* 0,24* 0,30* 0,29* 0,58* 1,00 AC: altura de cernelha; AP: altura das patas; PT: profundidade torácica; CC: comprimento corporal; CO: comprimento da orelha; AG: altura da garupa; CT: circunferência torácica; CCAB: comprimento da cabeça; LR: longitude do rosto; LCAB: largura da cabeça; LIL: largura da garupa entre ílios; LIS: largura da garupa entre ísquios; CGA: comprimento de garupa; PCA: perímetro da canela; TCH: tamanho do chifre; DHC: diâmetro do chifre; * P < 0,05; P < 0,01; * P < 0,001; e ns: não-significativo. do chifre (DCH). De acordo com as médias das características das diferentes populações caprinas comparadas pelo teste de Student Newman Keuls (Tabela III), ob servou-se a formação de até três grupos de médias. As cabras da ilha do Fogo obtiveram as maiores médias para AP, CC, CO, e CGA e as menores médias para PT, LIS e PCA. Assim, elas se caracterizam por serem compridas (CC = 68,27 cm), pouco profundas (PT = 27,53 cm), pernaltas (AP = 37,23 cm), de altura mediana (AC = 64,77 cm), de orelhas de comprimento mediano (CO = 14,62 cm), linha dorsal não retilínea, ou seja, possui a garupa mais alta que a cernelha. Poder-se ia supor a possibilidade de uma maior deposição de carne na garu pa devido ao maior comprimento da garupa (CGA = 15,67 cm) com parado aos caprinos das demais ilhas. MATERIAL E MÉTODOS AC AP PT CC CO AG CT CCAB LR LCAB LIL LIS CGA PCA TCH DCH AC 1,00 AP 0,52* 1,00 PT 0,58* -0,39* 1,00 CC 0,29* 0,28* 0,04ns 1,00 CO 0,18* 0,25* -0,04ns 0,39* 1,00 AG 0,59* 0,32* 0,33* 0,33* 0,20* 1,00 CT 0,40* -0,01ns 0,44* 0,40* 0,14 0,40* 1,00 CCAB 0,45* -0,03ns 0,51* 0,14** 0,11* 0,32* 0,43* 1,00 LR 0,13** 0,06ns 0,09* 0,19* 0,03ns 0,14 0,34* 0,38* 1,00 LCAB 0,17* 0,14 0,04ns 0,34* 0,19* 0,21* 0,33* 0,10* 0,33* 1,00 LIL 0,20* 0,12** 0,10* 0,41* 0,18* 0,33* 0,42* 0,06ns 0,20* 0,45* 1,00 LIS 0,25* -0,02ns 0,29* 0,12 -0,05ns 0,20* 0,27* 0,16* 0,12 0,28* 0,38* 1,00 CGA 0,18* 0,21* -0,01ns 0,52* 0,29* 0,28* 0,32* 0,03ns 0,01ns 0,31* 0,43* 0,10* 1,00 PCA 0,37* 0,04ns 0,36* 0,23*** 0,09* 0,29* 0,38* 0,42* 0,18* 0,21* 0,28* 0,35* 0,21* 1,00 TCH 0,22* 0,03ns 0,21* 0,30* 0,04ns 0,29* 0,53* 0,18* 0,26* 0,38* 0,42* 0,22* 0,29* 0,16 1,00 DCH 0,32* 0,04ns 0,29* 0,35* 0,14 0,40* 0,53* 0,30* 0,30* 0,32* 0,36* 0,24* 0,30* 0,29* 0,58* 1,00 AC: altura de cernelha; AP: altura das patas; PT: profundidade torácica; CC: comprimento corporal; CO: comprimento da orelha; AG: altura da garupa; CT: circunferência torácica; CCAB: comprimento da cabeça; LR: longitude do rosto; LCAB: largura da cabeça; LIL: largura da garupa entre ílios; LIS: largura da garupa entre ísquios; CGA: comprimento de garupa; PCA: perímetro da canela; TCH: tamanho do chifre; DHC: diâmetro do chifre; * P < 0,05; P < 0,01; * P < 0,001; e ns: não-significativo. V. Coeficientes de correlação de Pearson entre medidas corporais (cm) em cinco populações caprinas da República de Cabo Verde (Pearson correlation between body measurements (cm) in five goat populations of the Republic of Cape Verde). AC AP PT CC CO AG CT CCAB LR LCAB LIL LIS CGA PCA TCH DCH 1 00 , , , CC 0,29* 0,28* 0,04ns 1,00 CO 0,18* 0,25* -0,04ns 0,39* 1,00 AG 0,59* 0,32* 0,33* 0,33* 0,20* 1,00 CT 0,40* -0,01ns 0,44* 0,40* 0,14 0,40* 1,00 CAB 0,45* -0,03ns 0,51* 0,14 0,11* 0,32* 0,43* 1,00 LR 0,13** 0,06ns 0,09* 0,19* 0,03ns 0,14 0,34* 0,38* 1,00 CAB 0,17* 0,14 0,04ns 0,34* 0,19* 0,21* 0,33* 0,10* 0,33* 1,00 LIL 0,20* 0,12** 0,10* 0,41* 0,18* 0,33* 0,42* 0,06ns 0,20* 0,45* 1,00 LIS 0,25* -0,02ns 0,29* 0,12 -0,05ns 0,20* 0,27* 0,16* 0,12 0,28* 0,38* 1,00 GA 0,18* 0,21* -0,01ns 0,52* 0,29* 0,28* 0,32* 0,03ns 0,01ns 0,31* 0,43* 0,10* 1,00 CA 0,37* 0,04ns 0,36* 0,23*** 0,09* 0,29* 0,38* 0,42* 0,18* 0,21* 0,28* 0,35* 0,21* 1,00 CH 0,22* 0,03ns 0,21* 0,30* 0,04ns 0,29* 0,53* 0,18* 0,26* 0,38* 0,42* 0,22* 0,29* 0,16 1,00 CH 0,32* 0,04ns 0,29* 0,35* 0,14 0,40* 0,53* 0,30* 0,30* 0,32* 0,36* 0,24* 0,30* 0,29* 0,58* 1,00 altura de cernelha; AP: altura das patas; PT: profundidade torácica; CC: comprimento corporal; CO: comprimento da orelha; AG: altura da garupa; CT: circunferência torácica; CCAB: comprimento cabeça; LR: longitude do rosto; LCAB: largura da cabeça; LIL: largura da garupa entre ílios; LIS: largura da garupa entre ísquios; CGA: comprimento de garupa; PCA: perímetro da canela; TCH: anho do chifre; DHC: diâmetro do chifre; * P < 0,05; P < 0,01; * P < 0,001; e ns: não-significativo. do chifre (DCH). De acordo com as médias das características das diferentes populações caprinas comparadas pelo teste de Student Newman Keuls (Tabela III), ob servou-se a formação de até três grupos de médias. As cabras da ilha do Fogo obtiveram as maiores médias para AP, CC, CO, e CGA e as menores médias para PT, LIS e PCA. Assim, elas se caracterizam por serem compridas (CC = 68,27 cm), pouco profundas (PT = 27,53 cm), pernaltas (AP = 37,23 cm), de altura mediana (AC = 64,77 cm), de orelhas de comprimento mediano (CO = 14,62 cm), linha dorsal não retilínea, ou seja, possui a garupa mais alta que a cernelha. Poder-se ia supor a possibilidade de uma maior deposição de carne na garu pa devido ao maior comprimento da garupa (CGA = 15,67 cm) com parado aos caprinos das demais ilhas. As cabras da ilha de São Nico lau apresentam as maiores médias para as variáveis PT, CT, LR, LCAB, LIL, LIS, PCA e TCH, e as menores médias para CC e CO. V. Coeficientes de correlação de Pearson entre medidas corporais (cm) em cinco populações caprinas da República de Cabo Verde (Pearson correlation between body measurements (cm) in five goat populations of the Republic of Cape Verde). AC AP PT CC CO AG CT CCAB LR LCAB LIL LIS CGA PCA TCH DCH 1 00 Para AC e PT es tas cabras obtiveram valores próxi mos aos encontrados por Bouchel et al. (1997, pp.37-46) em caprinos do Rove, França, com média para AC de 66,5 cm e para PT de 32,3 cm. Na ilha de São Vicente, as ca Archivos de zootecnia vol. 68, núm. 263, p. 382. CARACTERIZAÇÃO BIOMÉTRICA DOS CAPRINOS DA REPÚBLICA DE CABO VERDE bras possuem maior altura da garupa (AG = 70,13 cm), comprimento da cabeça (CCAB = 20,86 cm), diâmetro do chifre (DCH = 9,24 cm) e altura de cernelha (AC = 67,95 cm), caracterizando como as mais altas. Possuem também as menores médias para longitude do rosto (LR = 15,21 cm) e largura de garupa entre ílios (LIL = 14,21 cm). servadas foram não-significativas, a única negativa e significativa foi entre AP e PT (-0,39), uma vez que AC é a soma AP e PT. As maiores correlações foram entre: AC e AG (0,59); AC e PT (0,58); e TCH e DCH (0,58). Já as menores correlações significativas foram entre: CO e PCA (0,09); e PT e LR (0,09). As correlações ob servadas foram, geralmente, inferiores as encontradas por Teixeira et al. (2000, p. 184) e Ribeiro et al. (2004, p. 339). A correlação entre AC e CC observado nas cabras cabo-verdianas foi similar ao encontrado por Ribeiro et al. 2004, p. 339 para Graúna (0,28) e Moxotó (0,21), e foi inferior ao observado para raça Canindé (0,47). As cabras de Cabo Verde avaliadas, em relação aos caprinos das ilhas Canárias estudados por Capote et al. (1998, pp.168-69), obtiveram as menores médias para as características: comprimento corporal (CC), altura de garupa (AG) e circunferência torácica (CT). Para altura das patas (AP), altura de cernelha (AC), largura (LCAB) e comprimento da cabeça (CCAB), as cabras de ambos arquipélagos foram similares entre si. O TCH das cabras cabo-verdianas variaram de 18,82 a 22,15 cm e foi, aproximadamente, similar ao encontrado por Ma chado et al. (1998, p.363) nos caprinos norte-africanos (20,5±5,30 cm).As cabras de Cabo Verde são pequenas se comparadas com os valores de 68 a 78 cm de altura de cernelha das raças espanholas (Herrera et al. 1996, p.41; Capote et al. 1998, p.168).Contudo, os valores da AC foram similares aos das cabras do Rove e das mexicanas (Bouchel et al. 1997, p.40; Lauvergne et al. 1997, p.503; Vargas et al. 2005, p.531). V. Coeficientes de correlação de Pearson entre medidas corporais (cm) em cinco populações caprinas da República de Cabo Verde (Pearson correlation between body measurements (cm) in five goat populations of the Republic of Cape Verde). AC AP PT CC CO AG CT CCAB LR LCAB LIL LIS CGA PCA TCH DCH 1 00 Em comparação com os caprinos do Norte de Camarões avaliados por Bourzat et al. (1993, p. 671), as cabras cabo-verdianas apresentaram maior estatura e foram mais profundas, pois o AC e PT os caprinos africanos foram, respecti vamente, 50,2 cm e 23,5 cm. As características intrínsecas a cada população, ou seja, as características mais relacionadas com as popu lações, foram CCAB, LR, LCAB, CO e PC. Isso porque elas possuem de baixa à média correlação com outras medidas, já que são mais influenciadas pela população, do que pela criação ou ambiente, fato também observa do por Carneiro et al. (2010, pp. 58-65) em ovinos e por Silva (2007, pp.65-75) em caprinos, onde as medidas corporais AC, PT, AG e CT apresentaram as maiores correlações entre si, pois sofrem maior influência do ambiente, regime alimentar e sistema de criação. Médias e correlações observadas entre as variáveis (Tabelas II, III e IV) justificam o uso de componentes principais para averiguar o grau de importância das características para a variabilidade total nos dados e para discriminação dos indivíduos no diagrama de dispersão. De acordo com os resultados obtidos para os com ponentes principais, seus respectivos autovalores e porcentagens da variância explicada (Tabela V), obser va-se que dos 15 componentes principais, nove (60%) apresentaram variância (autovalor) inferior a 0,7. Por As correlações de Pearson entre as característi cas avaliadas foram em grande parte significativas (P<0,05), positivas e de baixa à média magnitude (Tabela IV). A maioria das correlações negativas ob Figura 2. Dispersão gráfica dos escores das cinco populações de cabras na República de Cabo Verde, em re lação aos componentes principais (CP) 1 (abscissa) e 2 (coordenada). Populações: F: Ilha do Fogo; N: Ilha de São Nicolau; T: Santiago; A: Santo Antão; V São Vicente (Graphical dispersion of the five goat populations in the Republic of Cape Verde, in relation to the main components (CP) 1 (abscissa) and 2 (coordinate). Populations: F: Ilha do Fogo; N: St. Nicholas Island; T: Santiago; A: Santo Antão; V São Vicente). Figura 2. Dispersão gráfica dos escores das cinco populações de cabras na República de Cabo Verde, em re lação aos componentes principais (CP) 1 (abscissa) e 2 (coordenada). V. Coeficientes de correlação de Pearson entre medidas corporais (cm) em cinco populações caprinas da República de Cabo Verde (Pearson correlation between body measurements (cm) in five goat populations of the Republic of Cape Verde). AC AP PT CC CO AG CT CCAB LR LCAB LIL LIS CGA PCA TCH DCH 1 00 Portanto, a menor variação acumulada nos componentes prin cipais, no presente estudo, se deve as baixas e médias correlações entre as variáveis. No diagrama de dispersão gráfica (Figura 2) obser vou-se que as cabras do Fogo encontraram-se predo minantemente nos dois quadrantes superiores, espe cialmente direito, enquanto as cabras de São Nicolau encontram-se em ambos quadrantes inferiores e pre dominaram no inferior direito. As cabras de Santo Antão se posicionaram nos quadrantes inferiores. Os tanto, as nove variáveis que apresentarem maiores coeficientes, em valor absoluto, a partir do último com ponente principal, são passíveis de descarte, conforme apresentado na Tabela VI. Para os rebanhos estudados, os seis primeiros componentes principais foram se tanto, as nove variáveis que apresentarem maiores coeficientes, em valor absoluto, a partir do último com ponente principal, são passíveis de descarte, conforme apresentado na Tabela VI. Para os rebanhos estudados, os seis primeiros componentes principais foram se Tabela VI. Coeficientes de ponderação das variáveis com os nove componentes principais (CP’s) menos importantes para explicar a variação total das características mensuradas em caprinos de Cabo Verde (Weight ing coefficients of the variables with the nine major components (CPs) less important to explain the total variation of the characteristics measured in Cape Verde goats). V. Coeficientes de correlação de Pearson entre medidas corporais (cm) em cinco populações caprinas da República de Cabo Verde (Pearson correlation between body measurements (cm) in five goat populations of the Republic of Cape Verde). AC AP PT CC CO AG CT CCAB LR LCAB LIL LIS CGA PCA TCH DCH 1 00 Populações: F: Ilha do Fogo; N: Ilha de São Nicolau; T: Santiago; A: Santo Antão; V São Vicente (Graphical dispersion of the five goat populations in the Republic of Cape Verde, in relation to the main components (CP) 1 (abscissa) and 2 (coordinate). Populations: F: Ilha do Fogo; N: St. Nicholas Island; T: Santiago; A: Santo Antão; V São Vicente). Archivos de zootecnia vol. 68, núm. 263, p. 383. Archivos de zootecnia vol. 68, núm. 263, p. 383. Archivos de zootecnia vol. 68, núm. 263, p. 383. Archivos de zootecnia vol. 68, núm. 263, p. 383. PIRES, MACHADO, FONSECA, FONSECA, PILE E BRANDÃO lecionados, explicando 73,15% da variação total dos dados avaliados (Tabela V). Tabela V. Componentes Principais (CP), au tovalores (λi) e porcentagem da variância ex plicada pelos componentes das característi cas mensuradas em caprinos da República de Cabo Verde (Main Components (CP) eigenvalues (λi) and percentage of variance explained by the components of the cha racteristics measured in goats of the Republic of Cape Verde). As características em ordem de menor importância para explicar a variação total, foram: AC, TCH, CT, CC, CGA, PCA, DCH, LIL e CO (Tabela VI). Conside rando os resultados, verifica-se que as variáveis mais importantes para discriminar os indivíduos neste es tudo foram: AP, AG, CCAB, LR, LCAB e LIS. Entre as 15 variáveis avaliadas, apenas seis contribuíram para uma maior discriminação das populações. Para outro conjunto de populações, diferente desta amostragem, os resultados das características mais ou menos discri minantes, podem diferir deste resultado, necessitando investigar cada situação. CP λi Variação simples (%) Variação acumulada (%) CP1 4,64 30,96 30,96 CP2 1,71 11,43 42,39 CP3 1,58 10,56 52,95 CP4 1,17 7,81 60,75 CP5 0,96 6,42 67,17 CP6 0,90 5,98 73,15 CP7 0,65 4,31 77,46 CP8 0,53 3,54 81,00 CP9 0,52 3,47 84,47 CP10 0,49 3,27 87,74 CP11 0,47 3,13 90,87 CP12 0,41 2,76 93,63 CP13 0,38 2,51 96,14 CP14 0,35 2,33 98,48 CP15 0,23 1,52 100,00 Observa-se que as características de menor impor tância para explicar a variação total apresentaram cor relações lineares simples e significativas com as demais (Tabelas IV e VI). Manly (2008, pp.89-104) ressalta que, quanto menor a correlação existente entre as variáveis, mais componentes serão necessários para explicar a va riação total dos dados e, se tais correlações forem muito baixas, esse tipo de estudo não faz sentido. Archivos de zootecnia vol. 68, núm. 263, p. 384. AC: altura de cernelha; AP: altura das patas; CC: comprimento corporal; CO: comprimento da orelha; AG: altura da garupa; CT: circunfe rência torácica; CCAB: comprimento da cabeça; LR: longitude do rosto; LCAB: largura da cabeça; LIL: largura da garupa entre ílios; LIS: largura da garupa entre ísquios; CGA: comprimento de garupa; PCA: perímetro da canela; TCH: tamanho do chifre; e DHC: diâmetro do chifre. AC: altura de cernelha; AP: altura das patas; CC: comprimento corporal; CO: comprimento da orelha; AG: altura da ga rência torácica; CCAB: comprimento da cabeça; LR: longitude do rosto; LCAB: largura da cabeça; LIL: largura da garu largura da garupa entre ísquios; CGA: comprimento de garupa; PCA: perímetro da canela; TCH: tamanho do chifre; e chifre. AC: altura de cernelha; AP: altura das patas; CC: comprimento corporal; CO: comprimento da orelha; AG: altura da ga CARACTERIZAÇÃO BIOMÉTRICA DOS CAPRINOS DA REPÚBLICA DE CABO VERDE CARACTERIZAÇÃO BIOMÉTRICA DOS CAPRINOS DA REPÚBLICA DE CABO VERDE Figura 3. Dispersão gráfica dos escores da população de cabras de oito localidades da ilha do Fogo, em rela ção aos componentes principais (CP) 1 (abscissa) e 2 (coordenada). Populações: V=Vicente Dias; G=Genebra; S=Salto; R=Rocha Branca; A=Salto Acima; M=Monte Grande; F=Achada Furna; C=Cabeça Fundão (Graphic dispersion of goat population scores from eight localities of Fogo Island, relative to the main components (CP) 1 (abscissa) and 2 (coordi nate). Populations: V=Vicente Dias; G=Genebra; S=Salto; R=Rocha Branca; A=Salto Acima; M=Monte Grande; F=Achada Furna; C=Cabeça Fundão.) Figura 3. Dispersão gráfica dos escores da população de cabras de oito localidades da ilha do Fogo, em rela ção aos componentes principais (CP) 1 (abscissa) e 2 (coordenada). Populações: V=Vicente Dias; G=Genebra; S=Salto; R=Rocha Branca; A=Salto Acima; M=Monte Grande; F=Achada Furna; C=Cabeça Fundão (Graphic dispersion of goat population scores from eight localities of Fogo Island, relative to the main components (CP) 1 (abscissa) and 2 (coordi nate). Populations: V=Vicente Dias; G=Genebra; S=Salto; R=Rocha Branca; A=Salto Acima; M=Monte Grande; F=Achada Furna; C=Cabeça Fundão.) lhoramento genético, o que gera risco de perda da diversidade e do patrimônio genético. Diante destes fatos e também do distanciamento destes caprinos com os das demais ilhas em estudo, analisou-se, em particular, caprinos da ilha do Fogo com o objetivo de saber se eles são melhor ou pior conservados. caprinos de Santiago encontram-se no quadrante in ferior esquerdo em maior proximidade com as cabras das ilhas de Barlavento (São Nicolau, Santo Antão e São Vicente). As cabras de Santiago foram biometrica mente mais similares as cabras das ilhas do norte, ou Barlavento, que a vizinha ilha do Fogo, ou Sotavento. Isso deve ser explicado por outros fatores que possi bilitaram o intercâmbio entre estas ilhas. Mesmo com maior proximidade geográfica entre Santiago e Fogo observou-se maior distanciamento entre estas cabras por meio deste estudo com dados biométricos e ACP. As cabras de São Vicente ocuparam os quadrantes inferiores, especialmente o direito. Caprinos oriundos das ilhas de Barlavento (Santo Antão, São Vicente e São Nicolau) agruparam-se na Figura 2 à semelhança da proximidade física das ilhas que povoam. De acordo com o diagrama de dispersão gráfica (Figura 3), observa-se que os rebanhos da ilha do Fogo agruparam-se de acordo com a proximidade geográfica entre eles. Estes rebanhos possuem vasta variabilidade, o que pode ser observado devido à dispersão deles em vários quadrantes. Archivos de zootecnia vol. 68, núm. 263, p. 385. V. Coeficientes de correlação de Pearson entre medidas corporais (cm) em cinco populações caprinas da República de Cabo Verde (Pearson correlation between body measurements (cm) in five goat populations of the Republic of Cape Verde). AC AP PT CC CO AG CT CCAB LR LCAB LIL LIS CGA PCA TCH DCH 1 00 CP7 CP8 CP9 CP10 CP11 CP12 CP13 CP14 CP15 AC 0,03 -0,18 -0,07 0,19 -0,04 0,00 0,04 -0,16 -0,71 AP -0,14 -0,14 0,24 0,10 -0,20 0,14 0,00 -0,03 0,49 CC -0,36 -0,08 0,14 0,06 -0,05 -0,71 -0,10 0,16 -0,06 CO 0,65 0,11 0,16 0,11 -0,04 0,18 -0,11 0,07 -0,05 AG 0,11 0,47 -0,32 -0,39 0,30 -0,11 -0,16 0,27 0,15 CT -0,09 -0,07 -0,31 0,25 -0,28 0,16 -0,59 -0,30 0,22 CCAB 0,01 -0,05 -0,12 0,31 0,23 -0,12 0,50 -0,05 0,35 LR -0,24 0,39 0,23 0,01 0,08 0,16 -0,18 0,10 -0,20 LCAB 0,26 -0,40 -0,44 -0,16 0,23 -0,16 -0,01 -0,10 0,02 LIL 0,02 0,51 -0,17 0,07 -0,47 -0,04 0,43 -0,21 -0,03 LIS 0,17 0,11 0,36 0,35 0,29 -0,13 -0,26 0,06 0,08 CGA -0,43 -0,04 -0,05 0,05 0,48 0,51 0,15 -0,06 -0,07 PCA -0,12 -0,25 0,10 -0,51 -0,33 0,20 0,00 0,26 0,01 TCH 0,15 -0,23 0,08 0,21 -0,13 0,15 0,18 0,63 -0,03 DCH 0,17 -0,09 0,51 -0,40 0,12 -0,07 0,11 -0,49 0,04 AC: altura de cernelha; AP: altura das patas; CC: comprimento corporal; CO: comprimento da orelha; AG: altura da garupa; CT: circunfe rência torácica; CCAB: comprimento da cabeça; LR: longitude do rosto; LCAB: largura da cabeça; LIL: largura da garupa entre ílios; LIS: largura da garupa entre ísquios; CGA: comprimento de garupa; PCA: perímetro da canela; TCH: tamanho do chifre; e DHC: diâmetro do chifre. Tabela VI. Coeficientes de ponderação das variáveis com os nove componentes principais (CP’s) menos importantes para explicar a variação total das características mensuradas em caprinos de Cabo Verde (Weight ing coefficients of the variables with the nine major components (CPs) less important to explain the total variation of the characteristics measured in Cape Verde goats). Archivos de zootecnia vol. 68, núm. 263, p. 384. CARACTERIZAÇÃO BIOMÉTRICA DOS CAPRINOS DA REPÚBLICA DE CABO VERDE A e R predominaram no quadrante superior esquerdo e não foram cruzadas com a raça Majorera. O dendrograma a partir de 15 variáveis em cinco populações (Figura 4) colocou as cabras da ilha de Fogo em ramo a parte das demais com 100% de acu rácia. Agruparam-se num ramo as cabras das ilhas de Santo Antão e São Vicente com 85% de acurácia, enquanto as de São Nicolau e Santiago apresentaram agrupamento incerto entre si, mas distintas das de mais. Os agrupamentos das populações nas ilhas de Santo Antão e São Vicente denota que a proximidade geográfica de ambas foi importante para seu agrupa mento, com suposta relação de parentesco entre estas populações devido ao trânsito de animais entre estas ilhas. As medidas corporais consideradas nas análises de agrupamento foram AC, AP, CC, CO, AG, CT, CCAB, LR, LCAB, LIL, LIS, CGA, PCA, TCH e DHC que apre sentaram fraca multicolinearidade (NC<100) na amos tra. Resultado semelhante foi encontrado em rebanhos espanhóis de caprinos nativos (Herrera et al. 1996, p. 46) em que a profundidade torácica não foi discri minante, não devendo, portanto, ser considerada na elaboração de estudos de diferenças raciais, e também por Pires et al. (2013), com caprinos brasileiros e mar roquinos. As cabras da ilha do Fogo são as mais distintas das demais, isto devido a sua distância geográfica. Elas apresentam melhor conformação corporal por esta rem mais direcionados para a produção leiteira e são submetidas a um melhor manejo nutricional que as demais. Além disso, houve recentemente, a introdução de bodes das ilhas Canárias em certas localidades da ilha do Fogo. A análise de agrupamento, a partir das 15 medi das corporais das fêmeas adultas (acima de dois anos de idade), foi conduzida adotando-se a distância de Mahalanobis (D2) como medida de dissimilaridade e o método UPGMA. Verificou-se na matriz da distância generalizada de Mahalanobis (D2) que o valor máxi mo D2 foi entre as populações das ilhas do Fogo e São Nicolau (22,73), as mais divergentes, enquanto o valor mínimo foi entre Santo Antão e São Vicente (3,71), as mais similares (Tabela VII). Em ambos os casos a distância geográfica entre as ilhas pode explicar os resultados encontrados. CARACTERIZAÇÃO BIOMÉTRICA DOS CAPRINOS DA REPÚBLICA DE CABO VERDE Em outros contextos, como dos caprinos mexicanos, a análise discriminante com variáveis canônicas canônica tem oferecido também excelentes resultados nos agrupamentos (Sánchez Gu tiérrez et al., 2018) No dendrograma (Figura 5) para as diferentes loca lidades da ilha do Fogo, observa-se que o agrupamento da localidade de Vicente Dias foi o mais divergente de todos. As localidades Monte Grande, Achada Furna e Cabeça Fundão agruparam-se formando um segundo ramo. Este agrupamento foi concordante com o gráfico de dispersão gráfica utilizando a ACP (Figura 3). A localidade Genebra formou um terceiro ramo. Salto Acima formou um agrupamento incerto com as locali dades Salto e Rocha Branca. Os resultados dos dendrogramas foram facilmente interpretáveis, mas não foi possível saber ao certo o nú mero ótimo de grupos. Para contornar esta deficiência utilizou-se o método de otimização de Tocher que pos sibilitou verificar a formação de dois grupos distintos utilizando as D2. Um composto pelas populações das ilhas de Santo Antão, São Vicente, Santiago e São Ni colau; outro grupo composto somente pela população do Fogo. A formação dos grupos está de acordo com o dendrograma obtido (Figura 4). Verificou-se, portanto, que o método de otimização de Tocher e o método hierárquico (UPGMA) foram concordantes na partição dos grupos. Com a utilização do método de agrupamento UPG MA, obteve-se CCC de 0,79 (Figura 4). A partir desse coeficiente, infere-se que a distância D2 foi adequada para resumir a informação do conjunto de dados e que o método UPGMA proporcionou uma fidedigna repre sentação gráfica dos agrupamentos. A acurácia obtida Figura 4. Dendrograma obtido a partir da distância generalizada de Mahalanobis e do algoritmo UPGMA, com base em caracteres biométricos evidenciando as relações entre cinco populações caprinas da República de Cabo Verde. As distâncias estão expressas em por centagens na linha abaixo ao dendrograma e os valores de bootstrap são indicados em cada nó (Dendrogram ob tained from the generalized distance of Mahalanobis and the algo rithm UPGMA, based on biometric characters evidencing the relations among five caprine populations of the Republic of Cape Verde. The dis tances are expressed in percentages in the line below the dendrogram and the bootstrap values are indicated on each node). Figura 5. CARACTERIZAÇÃO BIOMÉTRICA DOS CAPRINOS DA REPÚBLICA DE CABO VERDE O rebanho mais padronizado foi da localidade com maior altitude (cerca de 1064 m) chamada Cabeça Fundão. Este rebanho possui maior infusão de sangue Majorera tendo fêmeas F1 e F2 com Majorera. Em proximidade a estes animais, encontram -se as cabras oriundas de outras regiões com elevada altitude também, como Achada Furna. Este maior per centual de sangue Majorera nestas localidades se deve ao projeto de raças melhoradas que levou a pecuária praticada no Fogo a outros patamares, principalmente para os criadores de caprinos de Cabeça do Monte, Ribeira Filipe, Cabeça Fundão, Chã das Caldeiras e ou tras localidades das zonas altas do Fogo, que já expor tam raça caprina melhorada para as ilhas de Santiago, Boavista e Brava (A semana, 2011). Este tipo de resultado serve como subsídio para uma futura homologação, perante o Ministério da Agricultura de Cabo Verde, e conservação dos grupos genéticos caprinos existentes nas ilhas. Isso é impor tante para o Cabo Verde, que ainda não possui nenhum estudo da variabilidade genética molecular. Na ilha do Fogo foram amostradas localidades com características bem distintas segundo a altitude. Os rebanhos de áreas com maior altitude possuem, teo ricamente, melhores condições para a prática da ca prinocultura leiteira devido, provavelmente, ao maior percentual de sangue exótico observado neste grupo genético oriundo da introdução de bodes Majoreros das ilhas Canárias (Inforpress, 2011). Em algumas lo calidades destas ilhas, principalmente as com maior altitude, está tendo absorção dos genótipos locais pela raça Majorera devido ao plano governamental de me As cabras cabo-verdianas de M predominaram nos quadrantes superiores e as de S encontraram-se, praticamente, em mesmo número em cada quadrante (superior e inferior), ou seja, foram as mais dispersas; R encontraram-se em todos os quadrantes, mas pre dominaram no superior esquerdo; A é nitidamente majoritária no quadrante superior esquerdo; F está au PIRES, MACHADO, FONSECA, FONSECA, PILE E BRANDÃO nas junções do dendrograma foi igual e superior a 85% em dois dos três nós formados. nas junções do dendrograma foi igual e superior a 85% em dois dos três nós formados. sente no quadrante superior esquerdo e predomina no superior direito; C no quadrante direito inferior; V nos quadrantes inferiores; G particularmente no inferior direito (Figura 3). Duas (M e F) das três subpopulações da Ilha do Fogo onde foram realizados cruzamentos com a raça Majorera, ficaram predominantemente no quadrante superior direito, e C no inferior direito. CARACTERIZAÇÃO BIOMÉTRICA DOS CAPRINOS DA REPÚBLICA DE CABO VERDE Dendrograma obtido a partir da distância generalizada de Mahalanobis e do algoritmo UP GMA, com base em caracteres biométricos eviden ciando as relações entre caprinos de diferentes lo calidades da ilha do Fogo (Dendrogram obtained from the generalized distance of Mahalanobis and the algorithm UPGMA, based on biometric characters evidencing the relations among lo calities of Fogo Island). Figura 4. Dendrograma obtido a partir da distância generalizada de Mahalanobis e do algoritmo UPGMA, com base em caracteres biométricos evidenciando as relações entre cinco populações caprinas da República de Cabo Verde. As distâncias estão expressas em por centagens na linha abaixo ao dendrograma e os valores de bootstrap são indicados em cada nó (Dendrogram ob tained from the generalized distance of Mahalanobis and the algo rithm UPGMA, based on biometric characters evidencing the relations among five caprine populations of the Republic of Cape Verde. The dis tances are expressed in percentages in the line below the dendrogram and the bootstrap values are indicated on each node). Figura 4. Dendrograma obtido a partir da distância generalizada de Mahalanobis e do algoritmo UPGMA, com base em caracteres biométricos evidenciando as relações entre cinco populações caprinas da República de Cabo Verde. As distâncias estão expressas em por centagens na linha abaixo ao dendrograma e os valores de bootstrap são indicados em cada nó (Dendrogram ob tained from the generalized distance of Mahalanobis and the algo rithm UPGMA, based on biometric characters evidencing the relations among five caprine populations of the Republic of Cape Verde. The dis tances are expressed in percentages in the line below the dendrogram and the bootstrap values are indicated on each node). Figura 5. Dendrograma obtido a partir da distância generalizada de Mahalanobis e do algoritmo UP GMA, com base em caracteres biométricos eviden ciando as relações entre caprinos de diferentes lo calidades da ilha do Fogo (Dendrogram obtained from the generalized distance of Mahalanobis and the algorithm UPGMA, based on biometric characters evidencing the relations among lo calities of Fogo Island). Figura 5. Dendrograma obtido a partir da distância generalizada de Mahalanobis e do algoritmo UP GMA, com base em caracteres biométricos eviden ciando as relações entre caprinos de diferentes lo calidades da ilha do Fogo (Dendrogram obtained from the generalized distance of Mahalanobis and the algorithm UPGMA, based on biometric characters evidencing the relations among lo calities of Fogo Island). Archivos de zootecnia vol. 68, núm. 263, p. Archivos de zootecnia vol. 68, núm. 263, p. 386. CARACTERIZAÇÃO BIOMÉTRICA DOS CAPRINOS DA REPÚBLICA DE CABO VERDE 386. CONCLUSÃO Cruz, CD Programa Genes: aplicativo computacional em genética. Viçosa: UFV, versão 6.0, 2008. Existem diferentes tipos caprinos cabo-verdianos. As populações caprinas das ilhas do Fogo, Santiago, São Nicolau e São Vicente apresentam características biométricas diferenciadas entre si. As cabras do Fogo são um grupo a parte das demais cabras cabo-verdia nas. Era esperado que os grupos genéticos oriundos das ilhas de Sotavento se agrupassem, porém as cabras de Santiago são mais próximas das populações capri nas das ilhas de Barlavento. As populações caprinas das ilhas de Barlavento agrupam-se pelas distâncias geográficas. Os rebanhos da ilha do Fogo agrupam-se pela proximidade geográfica entre as localidades da ilha e dos locais onde são realizados cruzamentos com a raça Majorera de Canárias. Sugere-se aumentar o número de caprinos amostrados na ilha de Santiago e realizar estudos de caracterização genética por meio de marcadores moleculares para verificar, com maior acurácia, se a ocorrência destes agrupamentos com o uso de marcadores biométricos procede. Faostat. Live animals. Disponível em: <http://faostat.fao.org.>. Acesso em: 15 dez. 2017. Herrera, M, Rodero, E, Gutierrez, MJ, Peña, F, Rodero, JM 1996, ‘Ap plication of multifactorial discriminant analysis in the morphostructural differentiation of Andalusian caprine breeds’, Small Ruminant Research, vol. 22, no. 1, pp. 39-47. Inforpress 2011, Agência de Notícias de Cabo Verde, viewed 30 July 2011,<http://www.inforpress.publ.cv>. Jolliffe, IT1973 ‘Discarding variables in a principal component analysis. II. Real data’, Applied Statistics, vol.22, p.21-31. Lauvergne, JJ, Bouchel, D, Minvielle, F, Guibert, É 1997, ‘Étude mor pho-biométrique de la chèvre du Rove. II. Longueurd’oreille (LO) et indice auriculo-thoracique (IAt)’, Revue de Médecine Vétérinaire, vol.148, no. 6, pp.501-510. Machado, TMM, Lauvergne, JJ, Chakir, M, Souvenir-Zafindrajaona, P, Silva, HD 1998, ‘Morfo-biometria no estudo comparativo de po pulações caprinas’, Genetics and Molecular Biology, vol.21, no.3. (supplement), pp.363. Machado, TMM 2013 ‘Os pequenos ruminantes na história da pecuária brasileira’, Proceedings of the X Workshop de Produção de Caprinos na Região da Mata Atlântica, Embrapa, Juiz de Fora, pp. 11-45. AGRADECIMIENTOS Manly, BJF 2008, Métodos estatísticos multivariados: Uma introdução, 3rd edn, Bookman, Porto Alegre. Agradecimentos a CAPES, CNPq (Projeto 490488/2008-0), Empresa Brasileira de Pesquisa Agro pecuária (EMBRAPA-CNPC) e a Direção Geral da Agricultura Silvicultura e Pecuária (DGASP-Cabo Ver de) pelos auxílios concedidos. Montgomery, DC, Peck, EA & Vining, GG 2012, Introduction to Linear Regression Analysis, 5nd edn, John Wiley & Sons, Inc. Pecuária 2011, Criação de cabras de raça melhorada para produção de leite e derivados. Disponível em: <http://ie.ic.cv>. Acesso em:15 Julho 2011. Silva, NV, Fraga, AB, Filho, JTA., Neto, CCC, Silva, FL., Costa, PPS & Lira Junior, WB 2007, ‘Caracterização Morfométrica de ovinos deslanados Cabugi e Morada Nova’, Revista Científica de Produção Animal vol 9 n o 65-75 15July 2011 <http://ie ic cv> CARACTERIZAÇÃO BIOMÉTRICA DOS CAPRINOS DA REPÚBLICA DE CABO VERDE Bourzat, D,Souvenir-Zafindrajaona, P,Lauvergne, JJ, et al. 1993, ‘Comparaison morpho-biométrique dês chèvesau Nord Cameroun et au Tchad’, Revued ‘Élevage et de Médecine Vétérinaire des Pays Tropicaux, vol.46, no. 4, pp. 667-74.<http://revues.cirad.fr/index. php/REMVT/issue/view/792>. Todos os caracteres biométricos mensurados (AC, AP, CC, CO, AG, CT, CCAB, LR, LCAB, LIL, LIS, CGA, PCA, TCH e DHC) contribuíram para a determinação da divergência genética entre as populações, em maior ou menor proporção. Quanto à importância relativa das características para a divergência segundo a me todologia de Singh (1981), a medida da garupa entre ísquios apresentou maior contribuição relativa para a divergência (18,66%), seguida das características com primento da cabeça (12,90%), comprimento corporal (10,42%) e altura de cernelha (9,73%). Capote, J, Delado, JV, Fresno, M, Camacho, ME & Molina, A 1998, ‘Morphological variability in the Canary goat population’, Small Ruminant Research, vol.27, pp.167-72. Carneiro, H, Louvandini, H, Paiva, SR, Macedo, F, Mernies, B & McManus, C 2010, ‘Morphological characterization of sheep breeds in Brazil, Uruguay and Colombia’, Small Ruminant Research, vol.94, pp.58-65. Carvalho, IRTV & Sá, AJ 2007, ‘O retorno de emigrantes e o problema de reinserção em Cabo Verde’, Revista de Geografia, vol.24, no.2, pp.121-35. Archivos de zootecnia vol. 68, núm. 263, p. 387. BIBLIOGRAFIA Alderson, GLH 2008, ‘Conservation of breeds and maintenance of biodiversity: justification and methodology for the conservation of Animal Genetic Resources’, Archivos de Zootecnia, vol. 67, no. 258, pp. 300-309. Animal, vol. 9, n o. 65-75. 15July 2011, <http://ie.ic.cv>. Pires, LC, Machado, TMM, Araújo, AM, Olson, TA, Silva, JBL, Torres, RA & Costa, MS2012,‘Biometricvariability of goat populations revealed by means of principal component analysis’, Genetics and Molecular Biology, vol. 35, no.4, pp.777-782. A Semana 2011, Ministério da Agricultura apoia criadores com reprodu tores para melhorar raça de caprinos, Cape Verde, Jornal a Semana, viewed 28 July 2011, <http://www.asemana.publ.cv>. Pires, LC, Machado, TMM, Araújo, AM, Silva, JBL, Euclydes, RF, Costa, MS &Olson, TA 2013,‘Clusterevaluation of Brazilian and Moroccan goat populations using physical measurements’, Revista Brasileira de Zootecnia, vol.42, pp.713-720. y p p Ayantunde, AA, Kango, M, Hiernaux, P, Udo, HMJ & Tabo, R 2007, ‘Herders’ Perceptions on Ruminant Livestock Breeds and Breeding Management in Southwestern Niger’, Human Ecology, vol.35, no. 1, pp. 139–149.<https://doi.org/10.1007/s10745-006-9049-6>. República de Cabo Verde/MADRRM - Ministério Do Ambiente, do Desenvolvimento Rural e dos Recursos Marinhos. Recenseamento Geral 2004, Viewed 29 August 2011, <http://www.governo.cv>. Bedotti, D, Gómez Castro, AG, Sánchez Rodriguez, M, & Martos Peina do, J 2004, ‘Caracterización morfológica y faneróptica de la cabra colorada pampeana’, Archivos de Zootecnia, vol.53, no. 203-204, pp. 261-71. Ribeiro, MN, Silva, JV, Pimenta Filho, EC & Sereno, JRB2004, ‘Estúdio de las correlaciones entre características fenotípicas de caprinos na turalizados’, Archivos de Zootecnia, vol.53, pp.337-340. pp Bouchel, D, Lauvergne, JJ, Guibert, E & Minvielle, F 1997,‘Étudemor pho-biométrique de la chèvre du rove. Hauteurau garrot (HT), pro fondeur du thorax (PT), videsous-sternal (VSS) et índice de gracilités ous-sternale (IGs) chezfemelles’, Revue de Médecine Vétérinaire, vol. 148, no. 1, pp 37-46. Rodero, E, Herrera, M & Gutiérrez, MJ 1992, ‘Morphostructural evolu tion of the Blanca Serrana caprine breed based of their crossing for milking aptitude’, Archivos de Zootecnia, vol.41 (extra), pp.519-530. SAS/STAT. User´sguide. Versão 8.0. Cary: SAS Institut Inc., 1999. Archivos de zootecnia vol. 68, núm. 263, p. 387. PIRES, MACHADO, FONSECA, FONSECA, PILE E BRANDÃO Singh, D 1981, ‘The relative importance of characters affecting genetic divergence’, Indian Journal of Genetics, vol.41, no.2, pp. 237-245. Sánchez Gutiérrez, R.A. et al. 2018. ‘Caracterización morfológica de un rebaño de conservación de cabras criollas en Zacatecas, México’, Archivos de Zootecnia, vol. 67, no.257, pp.73-79. pp Silva, NV, Fraga, A., Filho, JTA, Neto, CCC, Silva, FL, Costa, PPS & Lira Junior, WB 2007,‘Caracterização Morfométrica de ovinos deslanados Cabugi e Morada Nova’, Revista Científica de Produção Animal, vol. 9, pp. 65-75. Vargas, S, Sánchez, M, Rodríguez, JJ, et al. 2005, ‘La cabra para carne y su respuesta a la mejora del manejo’, Archivos de Zootecnia, vol.54, pp.529-534. Archivos de zootecnia vol. 68, núm. 263, p. 388 Teixeira, MPB, Barros, NN, Araújo, AM, Villarroel, AS 2000, ‘Relação entre medidas corporais e peso vivo em caprinos das raças Saanen e Anglo-Nubiana’, Revista Científica de Produção Animal, vol.2, no.2, pp.178-189. PIRES, MACHADO, FONSECA, FONSECA, PILE E BRANDÃO Teixeira, MPB, Barros, NN, Araújo, AM, Villarroel, AS 2000, ‘Relação entre medidas corporais e peso vivo em caprinos das raças Saanen e Anglo-Nubiana’, Revista Científica de Produção Animal, vol.2, no.2, pp.178-189. Teixeira, MPB, Barros, NN, Araújo, AM, Villarroel, AS 2000, ‘Relação entre medidas corporais e peso vivo em caprinos das raças Saanen e Anglo-Nubiana’, Revista Científica de Produção Animal, vol.2, no.2, pp.178-189. Singh, D 1981, ‘The relative importance of characters affecting genetic divergence’, Indian Journal of Genetics, vol.41, no.2, pp. 237-245. Archivos de zootecnia vol. 68, núm. 263, p. 388
https://openalex.org/W3192336951	https://hull-repository.worktribe.com/preview/3815663/Cancers%202021%20%28CE%29.pdf	English	null	De-Palmitoylation of Tissue Factor Regulates Its Activity, Phosphorylation and Cellular Functions	Cancers	2,021	cc-by	13,787	De-Palmitoylation of Tissue Factor Regulates Its Activity, Phosphorylation and Cellular Functions Camille Ettelaie 1,* , Sophie Featherby 1, Araci M. R. Rondon 2 , John Greenman 1 , Henri H. Versteeg 2 and Anthony Maraveyas 3 Camille Ettelaie 1,* , Sophie Featherby 1, Araci M. R. Rondon 2 , John Greenman 1 , Henri H. Versteeg 2 and Anthony Maraveyas 3 , Sophie Featherby 1, Araci M. R. Rondon 2 , John Greenman 1 , Henri H. Versteeg 2 and as 3 Camille Ettelaie 1,* , Anthony Maraveyas 3 1 Biomedical Section, University of Hull, Cottingham Road, Hull HU6 7RX, UK; y g S.Featherby-2016@hull.ac.uk (S.F.); j.greenman@hull.ac.uk (J.G.) 2 Einthoven Laboratory for Vascular and Regenerative Medicine, Division of Thrombosis and Hemostasis, Department of Internal Medicine, Leiden University Medical Center, 2333 ZA Leiden, The Netherlands; A.M.da_Rocha_Rondon@lumc.nl (A.M.R.R.); H.H.Versteeg@lumc.nl (H.H.V.) 3 Division of Cancer-Hull York Medical School, University of Hull, Cottingham Road, Hull HU6 7RX, UK; anthony.maraveyas@hey.nhs.uk * Correspondence: C.Ettelaie@hull.ac.uk; Tel.: +44-(0)1482-465528; Fax: +44-(0)1482-465458 * Correspondence: C.Ettelaie@hull.ac.uk; Tel.: +44-(0)1482-465528; Fax: +44-(0)1482-465458 Simple Summary: The relationship between cancer and blood clotting has been well established. The activation of blood coagulation proteins regulates the fate of cells and is known to be used by cancer cells to enhance survival and proliferation. Cells strictly regulate the initiation of coagulation through controlling the action of the protein “tissue factor (TF)”. In addition to initiating clotting, TF also acts as a deciding factor to determine the extent of damage and instructs cells to proliferate and repair or, when severely damaged, to die. Therefore, normal cells keep TF in a dormant state, achieved through mechanisms called “TF encryption”. Understanding the mechanisms by which the cells control the activity of TF is crucial, especially since cancer cells bypass these regulatory mechanisms, ensuring survival and tumour growth. This study has elucidated essential molecular mechanisms by which cells regulate TF clotting activity, and also the cellular signals arising from these. Citation: Ettelaie, C.; Featherby, S.; Rondon, A.M.R.; Greenman, J.; Versteeg, H.H.; Maraveyas, A. De-Palmitoylation of Tissue Factor Regulates Its Activity, Phosphorylation and Cellular Functions. Cancers 2021, 13, 3837. https://doi.org/10.3390/cancers 13153837 Academic Editor: Florian Langer Received: 15 June 2021 Accepted: 26 July 2021 Published: 30 July 2021 Citation: Ettelaie, C.; Featherby, S.; Rondon, A.M.R.; Greenman, J.; Versteeg, H.H.; Maraveyas, A. De-Palmitoylation of Tissue Factor Regulates Its Activity, Phosphorylation and Cellular Functions. Cancers 2021, 13, 3837. De-Palmitoylation of Tissue Factor Regulates Its Activity, Phosphorylation and Cellular Functions https://doi.org/10.3390/cancers 13153837 Academic Editor: Florian Langer Received: 15 June 2021 Accepted: 26 July 2021 Published: 30 July 2021 cancers cancers cancers 1. Introduction Uncontrolled coagulation arising from tissue factor (TF) activity has been implicated in both arterial and venous thrombosis with potentially fatal outcomes [1,2]. TF encryption refers to the post-translational suppression of TF procoagulant activity on the surface of cells. Unperturbed or non-activated cells present little TF activity despite the presence of TF antigen on the cell surface. Interestingly, encrypted TF may form a complex with fVIIa without promoting signiﬁcant coagulation, and the kinetics of this interaction have previously been studied [3]. However, it is suggested that the afﬁnity between these two proteins is ampliﬁed following decryption. The mechanism of TF decryption is not currently understood, although at least four mechanisms have been suggested [4–8]. However, none of these alone is sufﬁcient to explain the observed tight regulation of TF activity. y TF has a small cytoplasmic domain that has no kinase activity and is not thought to be required for its procoagulant activity [9,10]. However, Ser253 and Ser258 within the cytoplasmic domain of TF are phosphorylated following de-palmitoylation of the cytoplasmic domain [11,12] and are capable of regulating the incorporation of TF into microvesicles [13]. The role of phosphorylation in the regulation of incorporation of TF into microvesicles has been characterised [13–17]. Phosphorylation of Ser253 per- mits the interaction of TF with the cytoskeletal protein ﬁlamin-A, which is required for the trafﬁcking of TF to the site of microvesicle formation [14–16]. Palmitoylation of Cys245 within the cytoplasmic domain of human TF was one of the earliest post- translational modiﬁcations reported [18] and was suggested to be a means of anchoring TF in cholesterol-rich lipid rafts [5,6] containing procoagulant phospholipids [4,11,19]. How- ever, experimental data indicated that the procoagulant activity of TF is reduced following palmitoylation [11,12,19]. It has also been shown that de-palmitoylation of TF enhances and precedes TF phosphorylation [11]. We have recently shown that subsequent TF phos- phorylation at Ser253 results in increased TF procoagulant activity [14] as well as TF release within cell-derived microvesicles [13]. Other studies have shown that the association of TF with lipid-raft occurs as part of the mechanism of TF release into microvesicles [14,20] and control of TF procoagulant activity [21–23]. In this study, we hypothesised that the de-palmitoylation of TF alters the orientation of the transmembrane domain of TF within the plasma membrane. Abstract: In this study, the role of de-palmitoylation of tissue factor (TF) in the decryption of its activity was explored. TF-tGFP constructs were prepared by mutagenesis-substitution at Cys245 to prevent or mimic palmitolyation. Additionally, to reduce TF de-palmitoylation, the expression of palmitoyl-protein thioesterases (PPT) was suppressed. Other TF mutants were prepared with altered ﬂexibility, hydrophobicity or length of the transmembrane domain. The outcome of these alterations on fXa-generation, fVIIa binding, Ser253 phosphorylation and TF-microvesicle release were assessed in endothelial cells, and the inﬂuence on endothelial and MCF-7 cell proliferation and apoptosis was analysed. Preventing TF palmitoylation (TFSer245-tGFP), increasing the hydropho- bicity (TFPhe241-tGFP) or lengthening (TFLongTM-tGFP) of the transmembrane domain enhanced fXa-generation in resting cells compared to cells expressing TFWt-tGFP, but fXa-generation was not further increased following PAR2 activation. Extending the available length of the transmembrane domain enhanced the TF-tGFP release within microvesicles and Ser253 phosphorylation and in- creased cell proliferation. Moreover, prevention of PKCα-mediated Ser253 phosphorylation with Gö6976 did not preclude fXa-generation. Conversely, reducing the hydrophobicity (TFSer242-tGFP), shortening (TFShortTM-tGFP) or reducing the ﬂexibility (TFVal225-tGFP) of the transmembrane domain suppressed fXa-generation, fVIIa-HRP binding and Ser253 phosphorylation following PAR2 activa- tion. PPT knock-down or mimicking palmitoylation (TFPhe245-tGFP) reduced fXa-generation without affecting fVIIa binding. This study has for the ﬁrst time shown that TF procoagulant activity is regulated through de-palmitoylation, which alters the orientation of its transmembrane domain and is independent of TF phosphorylation. However, Ser253 phosphorylation is facilitated by changes in the orientation of the transmembrane domain and can induce TF-cellular signalling that inﬂuences cellular proliferation/apoptosis. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. 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/). 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/ /) creativecommons.org/licenses/by/ https://www.mdpi.com/journal/cancers Cancers 2021, 13, 3837. https://doi.org/10.3390/cancers13153837 Cancers 2021, 13, 3837 2 of 20 Keywords: tissue factor; factor VIIa; encryption; palmitoylation; transmembrane-domain; palmitoyl- protein thioesterase 1. Introduction This in turn could permit the transfer and association of TF with thicker membrane regions such as cholesterol-rich microdomains, which contain lipids that support TF activity. We also explored the association of these events with TF-mediated proliferative and pro-apoptotic signals. 2.1. Site-Directed Mutagenesis The pCMV6-Ac-TF-tGFP plasmid DNA (OriGene/Insight Biotechnology, Wembley, UK) was used to express wild-type or variant forms of TF, as previously described [13,24,25]. Preparations of pCMV6-Ac-tGFP (control) and pCMV6-Ac-TFAla253-tGFP were described previously [13]. Single amino acid-substitutions or deletions were carried out to obtain the mutants, as illustrated in Table 1, using the primer pairs indicated. All procedures were carried out using the Q5-site directed mutagenesis kit (New England Biolabs, Hitchin, UK) according to the manufacturer’s procedure. All mutants were veriﬁed by sequencing (Euroﬁn MWG, Wolverhampton, UK). 3 of 20 Cancers 2021, 13, 3837 Table 1. The list of TF variants and DNA primers used for mutagenesis. Plasmid Mutation Primer Set pCMV-Ac-TFSer245-tGFP Cys245→Ser 5′-TCTACACAAGAGTAGAAAGGCAG 5′-GATATAGCCAGGATGATG pCMV-Ac-TFPhe245-tGFP Cys245→Phe 5′-CTACACAAGTTTAGAAAGGCAG 5′-AGATATAGCCAGGATGATG pCMV-Ac-TFPhe241-tGFP Ser241→Phe 5′-GTGGCTATATTTCTACACAAGTG 5′GATGATGACAAGGATGATG pCMV-Ac-TFSer242-tGFP Leu242→Ser 5′-GGCTATATCTTCACACAAGTGTAG 5′-AGGATGATGACAAGGATG pCMV-Ac-TFShortTM-tGFP Ser241-Leu242-del 5′-CACAAGTGTAGAAAGGCAG 5′-TATAGCCAGGATGATGAC pCMV-Ac-TFLongTM-tGFP Ser243-Leu244-add 5′-TCCCTGCACAAGTGTAGAAAGGCAG 5′-TAGAGATATAGCCAGGATG pCMV-Ac-TFVal225-tGFP Gly225→Val 5′-TACATCATTGTAGCTGTGGTATTTG 5′-GAATATTTCTCTGAATTCCC Table 1. The list of TF variants and DNA primers used for mutagenesis. 2.2. Cell Culture, Transfection and Cellular Activation 2.2. Cell Culture, Transfection and Cellular Activation Human dermal blood primary endothelial cells (HDBEC) and coronary artery endothe- lial cells (HCAEC) devoid of endogenous TF were cultured in MV media containing 5% (v/v) foetal calf serum (FCS) and growth supplements (PromoCell, Heidelberg, Germany). MCF-7 and MDA-MB-231 breast cancer cell lines (ATCC, Teddington, UK) expressing low and high levels of TF were cultured in EMEM and DMEM, respectively, containing 10% (v/v) FCS. Cells (5 × 105) were seeded out into 48-well plates and transfected with 0.5 µg of pCMV6-Ac-TF-tGFP plasmid DNA or, alternatively, mutant forms of the plasmid, as shown in Table 1. Transfection of the cells was carried out using TransIT-2020 (Geneﬂow, Lichﬁeld, UK) according to the manufacturer’s instructions. Cells were permitted to express the proteins for 48 h, and the expression of the TF variants was conﬁrmed by ﬂow cytometry as before [13,14]. Prior to experiments, cells were pre-adapted to respective serum-free media for 1 h and then activated by incubation with protease-activated receptor 2-activating peptide (PAR2-AP); SLIGKV; (20 µM). In some experiments, cells were transfected with a speciﬁc set of SilencerSelect®-siRNA (10 pmol; Life Technologies, Paisley, UK) to suppress the expression of palmitoyl–protein thioesterase (PPT) 1 and 2 concurrently, or transfected with a comparable set of control siRNA (10 pmol; Life Technologies) for 48 h prior to activa- tion. The concentration of siRNA to knock-down PPT1 and PPT2 expression was optimised by western blot. 2.1. Site-Directed Mutagenesis To block the phosphorylation of TF at Ser253, cells were pre-incubated with the PKC inhibitor Gö6976 (100 nM; R&D Systems, Abingdon, UK) for 40 min, prior to activation. 2.3. Cell Proliferation and Apoptosis Assays Cell numbers were determined by staining with crystal violet as previously de- scribed [26,27] and calculated from a standard curve. In addition, cellular apoptosis was quantiﬁed using the TiterTACS™Colorimetric Apoptosis Detection Kit (AMS Biotech- nology, Abingdon, UK) according to the manufacturer’s instructions [28,29]. 2.4. Determination of Microvesicle Density and TF Antigen 2.5. Cell-Based Factor Xa-Generation Assay Cell surface TF-fVIIa activity was measured by modiﬁcation of previously described procedures [3,24,31]. Cells (5 × 104) were seeded out into 48-well plates and transfected with the appropriate wild-type or mutant TF plasmids, as shown in Table 1. The cells were washed with phosphate-buffered saline (PBS) pH 7.4 and then pre-adapted to serum-free medium. The cells were incubated with PAR2-AP for up to 60 min and then washed and incubated with fVIIa (20 nM; Enzyme Research Labs, Swansea, UK) in HEPES-buffered saline (HBS) pH 7.4, containing 1% (w/v) bovine serum albumin (BSA) and 5 mM CaCl2 (100 µL) for an additional 10 min. Finally, the samples were supplemented with fX (100 nM) together with fXa substrate (0.2 mM; Hyphen) diluted in the same buffer (100 µL). The samples were incubated for 60 min to develop the colour. Aliquots (150 µL) were then transferred to a 96-well plate containing 2% (v/v) acetic acid (50 µL) and the absorptions were measured immediately at 405 nm. The amount of fXa generated was determined using a standard curve prepared using fXa (Enzyme Research Labs). To inhibit cell surface TF activity, cells were pre-incubated with the inhibitory anti-TF antibody HTF1 (40 µg/mL; eBioscience/Thermo Scientiﬁc, Warrington, UK) prior to the addition of fVIIa [15]. 2.4. Determination of Microvesicle Density and TF Antigen The release of microvesicles was examined using the Zymuphen MP-assay kit (Hyphen BioMed/Quadratech, Epsom, UK) and the microvesicle density was determined from the standards provided. The properties of the MV were previously conﬁrmed [29,30]. The Cancers 2021, 13, 3837 4 of 20 released microvesicle-associated TF antigen was measured using the Quantikine TF-ELISA kit (R & D Systems) according to the manufacturer’s instructions. Cell surface TF antigen was measured in situ using an ELISA-based procedure as previously described [15,24]. 2.8. Confocal Microscopy HDBEC (5 × 104) were seeded out into 35 mm glass-based µ-dishes (InVitro Scien- tiﬁc/Cellvis, Sunnyvale, CA, USA) and transfected to express wild-type or variants of TF-tGFP [14,31]. In some experiments, the cells were co-transfected with siRNA to silence the expression of PPT1 and PPT2, concurrently. Sets of cells were activated with PAR2-AP for 30 min and then ﬁxed and labelled with DAPI (2 µg/mL) [14,31]. The samples were then analysed by confocal microscopy at room temperature using a Zeiss LSM 710 confocal microscope with a ×63 water immersion objective. Images were acquired using the ZEN software (Carl Zeiss Ltd., Welwyn Garden City, UK) and analysis of the aggregate sizes was carried out using the ImagePro Plus software (Media Cybernetics, Bethesda, MD, USA). 2.9. Statistical Analysis All data represent the calculated mean values from the number of experiments stated in each ﬁgure legend ± the calculated standard error of the mean. Statistical analysis was carried out using the Statistical Package for the Social Sciences (SPSS Inc., Chicago, IL, USA). Signiﬁcance was determined using one-way ANOVA (analysis of variance) and Tukey’s honest signiﬁcance test or, where appropriate, by paired t-test. 2.6. Factor VIIa Binding Assay Analysis of PPT1 Cancers 2021, 13, 3837 5 of 20 and PPT2 expression in cell lysates with and without siRNA-mediated silencing of PPT was carried out using rabbit anti-human PPT1 and PPT2 antibodies (CloudClone/Insight Biotechnology, Wembley, UK) diluted 1:2000 (v/v) in TBST and detected as above. and PPT2 expression in cell lysates with and without siRNA-mediated silencing of PPT was carried out using rabbit anti-human PPT1 and PPT2 antibodies (CloudClone/Insight Biotechnology, Wembley, UK) diluted 1:2000 (v/v) in TBST and detected as above. 3.1. Validation of the fVIIa-HRP Conjugation and fVIIa Activity Prior to usage, the ability of the fVIIa-HRP conjugate preparation to bind to recom- binant TF (Supplementary Figure S1A) and cell surface TFWt-tGFP (Supplementary Fig- ure S1B) was conﬁrmed in human primary endothelial cells. Additionally, fVIIa-HRP and non-conjugated fVIIa exhibited similar fXa-generation activities (Supplementary Fig- ure S1C). 2.6. Factor VIIa Binding Assay In order to measure the binding of fVIIa to cell-surface TF, fVIIa (30 µg) was conjugated to horseradish peroxidase (HRP) using the Innova Lightning-link HRP kit (Expedeon, Cambridge, UK). A similar control was also prepared by conjugating BSA (30 µg) to HRP. After neutralisation of excess HRP, the speciﬁcity of the fVIIa to interact with TF was conﬁrmed as follows; 96-well plates were coated with recombinant Innovin TF (13 ng/mL; Dade Behring, Deerﬁeld, MA, USA) in PBS containing BSA (1% w/v), or coated with the vehicle solution alone by incubating overnight at 4 ◦C. In parallel experiments, sets of endothelial cells (5 × 104) were plated out into 48-well plates and transfected to express TF-tGFP or tGFP, along with non-transfected cells. All plates were washed with PBS and incubated with fVIIa-HRP (20 nM) (100 µL) for 15 min. The plates were then washed three times with PBS, and HRP activity was determined using the TMB One-solution HRP substrate (200 µL). Aliquots (150 µL) of the substrate were then transferred into fresh wells containing 2 M H2SO4 (50 µL), and the absorptions were determined at 450 nm. The amount of bound fVIIa-HRP was determined from a standard curve prepared with a range of concentrations of fVIIa-HRP (100 µL). To conﬁrm the fVIIa activity of the HRP- conjugated preparations, fXa-generation potential was determined on both endothelial cells transfected to express TF and also immobilised-recombinant TF using a range of fVIIa-HRP concentrations (0–10 nM) and compared to fXa generation using unlabelled-fVIIa. 2.7. Immunoprecipitation of TF and Western Blot Analysis of TF Phosphorylation TF-tGFP was immunoprecipitated from cells lysed in PhosphoSafe buffer (Novagen/ Merck Millipore, Watford, UK) using anti-tGFP-magnetic beads (clone 2H8) (25 µL, Ori- Gene/Insight Biotechnology) or, in some cases, using anti-TF antibody (HTF1) followed by protein A-magnetic bead puriﬁcation, as previously described [24,25]. The samples were separated by SDS-PAGE, transferred onto nitrocellulose membranes and probed with a rabbit anti-TF antibody (FL295) (Santa Cruz Biotechnology, Heidelberg, Germany) diluted 1:4000 (v/v) in TBST. The membranes were developed with goat anti-rabbit al- kaline phosphatase-conjugated antibody (Santa Cruz), diluted 1:4000 (v/v), and bands were visualised using the Western Blue stabilised alkaline phosphatase-substrate (Promega, Southampton, UK). Phosphorylation of TF on Ser253 was detected in the immunoprecipi- tated samples using a rabbit anti-phospho-PKC-substrate motif antibody (Cell Signalling Technology) diluted 1:2000 (v/v) in TBST buffer and detected as above. 3.2. Non-Active/Encrypted TF Is Capable of Binding fVIIa without Increasing Procoagulant Activity (A) The ability of HDBEC (5 × 104) expressing either TF-tGFP or tGFP to support fXa-generation was measured before and after activation of the cells with PAR2-AP (20 µM) for up to 60 min. (n = 10, * = p < 0.05 vs. non-activated cells expressing tGFP; # = p < 0.05 TFWt-tGFP samples vs. non-activated cells expressing TFWt-tGFP.) (B) HDBEC were incubated with fVIIa-HRP (20 nM) for 10 min and washed, and the amount of bound fVII-HRP was determined using HRP substrate. (n = 3, * = p < 0.05 vs. respective cells expressing tGFP.) (C) HDBEC were pre-incubated with the inhibitory TF antibody HTF1 (40 µg/mL) prior to analysis using the fXa-generation assay. (n = 3, * = p < 0.05 vs. respective controls expressing tGFP.) (D) The expression of TF variants on the surface of the transfected HDBEC was quantiﬁed using an ELISA-based procedure. (n = 4, * = p < 0.05 vs. cells expressing TFWt-tGFP.) gure 1. Analysis of TF-tGFP activity and fVIIa-HRP binding to TF. (A) The ability of HDBEC (5 × 104) expr tGFP o tGFP to u o t fXa e e atio a ea u ed befo e a d afte a ti atio of the ell ith PAR2 gure 1. Analysis of TF-tGFP activity and fVIIa-HRP binding to TF. (A) The ability of HDBEC (5 × 104) expr Table 2. Analysis of the interaction of fVIIa-HRP with TF-tGFP variants expressed on the cell surface. TF Variant Non-Activated (Fmol/Million Cells) PAR2-Activated (Fmol/Million Cells) tGFP 11.81 ± 1.19 * 10.2 ± 1.28 * TFWt-tGFP 20.15 ± 2.56 20.7 ± 2.33 TFSer245-tGFP + PPT siRNA 19.33 ± 1.03 19.51 ± 0.92 TFSer245-tGFP + control siRNA 19.70 ± 0.64 20.01 ± 0.38 TFSer245-tGFP 18.29 ± 2.12 19.6 ± 0.99 TFPhe245-tGFP 17.04 ± 1.59 14.4 ± 1.01 * TFShortTM-tGFP 18.13 ± 4.04 8.8 ± 2.87 * TFLongTM-tGFP 20.80 ± 1.50 21.0 ± 1.51 TFPhe241-tGFP 20.00 ± 1.00 20.0 ± 0.82 TFSer242-tGFP 20.00 ± 2.00 19.0 ± 2.50 TFVal225-tGFP 17.00 ± 2.05 12.0 ± 3.10 * HDBEC were co-transfected to express TFWt-tGFP with PPT or control siRNA. Alternatively, cells were transfected to express the TF variants as indicated in the table as well as a control set expressing tGFP. The cells were incubated with fVIIa-HRP for 10 min and washed, and the amount of bound fVII-HRP was determined against a standard curve. 3.2. Non-Active/Encrypted TF Is Capable of Binding fVIIa without Increasing Procoagulant Activity 3.2. Non-Active/Encrypted TF Is Capable of Binding fVIIa without Increasing Procoagulant Activity The activation of cells by incubation with the PAR2-agonist peptide is a reproducible procedure [13,14] that permits the accurate measurement of changes in fXa-generation in a short period, which was imperative to the success of this study. Non-activated cells transfected to express TFWt-tGFP exhibited a low level of fXa-generation (Figure 1A). Fur- thermore, cells expressing tGFP (Figure 1A) or non-transfected cells (not shown) exhibited little fXa-generation potential. Activation of PAR2 resulted in increased fXa-generation in HDBEC expressing TF-tGFP, reaching a peak at around 20 min post-activation. The interaction between TF and fVIIa was robust, and the fXa-generation did not change by washing out excess fVIIa. Interestingly, no increase in cell-surface TF-tGFP antigen was observed in endothelial cells at 20 min post-activation (not shown). Moreover, fVIIa-HRP was capable of binding to cell surface TF-tGFP at similar magnitudes in both non-activated cells and following PAR2 activation for 20 min (Figure 1B and Table 2). Pre-incubation of cells with the inhibitory HTF1 antibody signiﬁcantly reduced fXa-generation (Figure 1C), indicating the requirement for the TF-fVIIa complex. Together, these data indicate that the observed elevation in fXa-generation following cell activation was not due to change in the availability of TF for fVIIa binding. Furthermore, any variations in fXa-generation and fVIIa-HRP binding did not arise from differences in cell surface expression of the TF-tGFP variants, demonstrated by the comparable cell-surface TF antigen (Figure 1D). 6 of 20 g sion of Fi Cancers 2021, 13, 3837 ) Figure 1. Analysis of TF-tGFP activity and fVIIa-HRP binding to TF. (A) The ability of HDBEC (5 × 104) expressing either TF-tGFP or tGFP to support fXa-generation was measured before and after activation of the cells with PAR2-AP (20 µM) for up to 60 min. (n = 10, * = p < 0.05 vs. non-activated cells expressing tGFP; # = p < 0.05 TFWt-tGFP samples vs. non- activated cells expressing TFWt-tGFP.) (B) HDBEC were incubated with fVIIa-HRP (20 nM) for 10 min and washed, and the amount of bound fVII-HRP was determined using HRP substrate. (n = 3, * = p < 0.05 vs. respective cells expressing tGFP.) (C) HDBEC were pre-incubated with the inhibitory TF antibody HTF1 (40 µg/mL) prior to analysis using the fXa- generation assay. (n = 3, * = p < 0.05 vs. 3.2. Non-Active/Encrypted TF Is Capable of Binding fVIIa without Increasing Procoagulant Activity respective controls expressing tGFP.) (D) The expression of TF variants on the surface of the transfected HDBEC was quantified using an ELISA-based procedure. (n = 4, * = p < 0.05 vs. cells expressing TFWt-tGFP.) Figure 1. Analysis of TF-tGFP activity and fVIIa-HRP binding to TF. (A) The ability of HDBEC (5 × 104) expressing either TF-tGFP or tGFP to support fXa-generation was measured before and after activation of the cells with PAR2-AP (20 µM) for up to 60 min. (n = 10, * = p < 0.05 vs. non-activated cells expressing tGFP; # = p < 0.05 TFWt-tGFP samples vs. non-activated cells expressing TFWt-tGFP.) (B) HDBEC were incubated with fVIIa-HRP (20 nM) for 10 min and washed, and the amount of bound fVII-HRP was determined using HRP substrate. (n = 3, * = p < 0.05 vs. respective cells expressing tGFP.) (C) HDBEC were pre-incubated with the inhibitory TF antibody HTF1 (40 µg/mL) prior to analysis using the fXa-generation assay. (n = 3, * = p < 0.05 vs. respective controls expressing tGFP.) (D) The expression of TF variants on the surface of the transfected HDBEC was quantiﬁed using an ELISA-based procedure. (n = 4, * = p < 0.05 vs. cells expressing TFWt-tGFP.) (A) h b l f EC ( 4) h Figure 1. Analysis of TF-tGFP activity and fVIIa-HRP binding to TF. (A) The ability of HDBEC (5 × 104) expressing either TF-tGFP or tGFP to support fXa-generation was measured before and after activation of the cells with PAR2-AP (20 µM) for up to 60 min. (n = 10, * = p < 0.05 vs. non-activated cells expressing tGFP; # = p < 0.05 TFWt-tGFP samples vs. non- activated cells expressing TFWt-tGFP.) (B) HDBEC were incubated with fVIIa-HRP (20 nM) for 10 min and washed, and the amount of bound fVII-HRP was determined using HRP substrate. (n = 3, * = p < 0.05 vs. respective cells expressing tGFP.) (C) HDBEC were pre-incubated with the inhibitory TF antibody HTF1 (40 µg/mL) prior to analysis using the fXa- generation assay. (n = 3, * = p < 0.05 vs. respective controls expressing tGFP.) (D) The expression of TF variants on the surface of the transfected HDBEC was quantified using an ELISA-based procedure. (n = 4, * = p < 0.05 vs. cells expressing TFWt-tGFP.) Figure 1. Analysis of TF-tGFP activity and fVIIa-HRP binding to TF. 3.2. Non-Active/Encrypted TF Is Capable of Binding fVIIa without Increasing Procoagulant Activity (n = 4, * = p < 0.05 vs. cells expressing TFWt-tGFP.) Table 2. Analysis of the interaction of fVIIa-HRP with TF-tGFP variants expressed on the cell surface. HDBEC were co-transfected to express TFWt-tGFP with PPT or control siRNA. Alternatively, cells were transfected to express the TF variants as indicated in the table as well as a control set expressing tGFP. The cells were incubated with fVIIa-HRP for 10 min and washed, and the amount of bound fVII-HRP was determined against a standard curve. (n = 4, * = p < 0.05 vs. cells expressing TFWt-tGFP.) 7 of 20 Cancers 2021, 13, 3837 3.3. De-Palmitoylation of Cys245 Is Required for TF-fVIIa Activity but Not Complex Formation 3. De-Palmitoylation of Cys245 Is Required for TF-fVIIa Activity but Not Complex Formation Two approaches were employed in order to examine the role of de-palmitoylation in the decryption of TF activity. The only palmitoylation site on TF is at Cys245, and PPT1 and PPT2 are the two identiﬁed enzymes shown to be responsible for the de-palmitoylation of cellular proteins, although controversies exist regarding such proteins [32]. Therefore, siRNA-mediated knock-down of both PPT1 and PPT2 was optimised to reduce the expres- sion of these two genes concurrently (Figure 2A). Analysis of PAR2-activated cells previ- ously transfected to express TFWt-tGFP, together with the PPT-siRNA, showed signiﬁcantly reduced fXa-generation following PAR2 activation of HDBEC (Figure 2B,C) and HCAEC (Supplementary Figure S2) compared to cells transfected with control siRNA. However, the ability of fVIIa-HRP to interact with cell surface TF-tGFP remained unhindered by the suppression of PPT expression (Table 2). To conﬁrm these data, the substitution of Cys245 with serine was carried out to prevent the palmitoylation of TF. Conversely, the substitution of this residue to phenylalanine was used to increase the hydrophobicity of the residue at position 245 and encourage its incorporation into the membrane. Prevention of TF palmi- toylation by expressing TFSer245-tGFP signiﬁcantly increased fXa-generation in resting cells, compared to TFWt-tGFP (Figure 2D and Supplementary Figure S2). However, the level of activities converged following activation of cells with PAR2-AP. The ability of fVIIa-HRP to bind to the cells remained unaffected by Cys245→Ser substitution (Table 2). In contrast, the expression of TFPhe245-tGFP prevented the increase in fXa-generation following cell activation but did not alter the residual activity in resting cells (Figure 2E). Additionally, the ability of fVIIa-HRP to interact with cell surface TFPhe245-tGFP was partially impaired following activation (Table 2). In agreement with the data generated with transfected- endothelial cells, siRNA-mediated knock-down of PPT1 and PPT2 in the breast cancer cell line MDA-MB-231, which constitutively expresses TF, also reduced fXa-generation (Figure 2F). EW 8 of 21 Figure 2. Cont. Figure 2. Cont. Figure 2. Cont. 8 of 20 Cancers 2021, 13, 3837 Figure 2. Analysis of TF-tGFP procoagulant activity and fVIIa-HRP binding in the presence and absence of TF pal- mitoylation. (A) HDBEC (5 × 104) were transfected with PPT-siRNA (0–15 pmol) and the expression of PPT1 and -2 meas- ured by western blot. 3.3. De-Palmitoylation of Cys245 Is Required for TF-fVIIa Activity but Not Complex Formation the respective cells expressing TFWt-tGFP.) (F) MDA-MB-231 (5 × 104) cells expressing endogenous TF were transfected with PPT-siRNA or control siRNA and fXa-generation measured (n = 3, * = p < 0.05 vs. cells without siRNA). g y p g y g p p mitoylation. (A) HDBEC (5 × 104) were transfected with PPT-siRNA (0–15 pmol) and the expression of PPT1 and -2 meas- ured by western blot. (B) HDBEC were co-transfected with combinations of pCMV-Ac-TF-tGFP and PPT-siRNA or control siRNA, and fXa-generation was measured in resting and PAR2-activated cells. (n = 5, * = p < 0.05 vs. the respective non- activated sample.) (C) HDBEC were co-transfected to express TF-tGFP, with PPT siRNA or control siRNA. Factor Xa- generation was measured before and after PAR2 activation for up to 60 min. (n = 4, * = p < 0.05 vs. the respective control siRNA sample.) HDBEC were transfected to express (D) TFSer245-tGFP or (E) TFPhe245-tGFP and fXa-generation were meas- ured as above. (n = 6, * = p < 0.05 vs. the respective cells expressing TFWt-tGFP.) (F) MDA-MB-231 (5 × 104) cells expressing endogenous TF were transfected with PPT-siRNA or control siRNA and fXa-generation measured (n = 3, * = p < 0.05 vs. cells without siRNA). 3 4 D P l it l ti f C 245 P t th A ti f TF th C ll M b Figure 2. Analysis of TF tGFP procoagulant activity and fVIIa HRP binding in the presence and absence of TF palmitoylation. (A) HDBEC (5 × 104) were transfected with PPT-siRNA (0–15 pmol) and the expression of PPT1 and -2 measured by western blot. (B) HDBEC were co-transfected with combinations of pCMV-Ac-TF-tGFP and PPT-siRNA or control siRNA, and fXa-generation was measured in resting and PAR2-activated cells. (n = 5, * = p < 0.05 vs. the respective non-activated sample.) (C) HDBEC were co-transfected to express TF-tGFP, with PPT siRNA or control siRNA. Factor Xa-generation was measured before and after PAR2 activation for up to 60 min. (n = 4, * = p < 0.05 vs. the respective control siRNA sample.) HDBEC were transfected to express (D) TFSer245-tGFP or (E) TFPhe245-tGFP and fXa-generation were measured as above. (n = 6, * = p < 0.05 vs. 3.3. De-Palmitoylation of Cys245 Is Required for TF-fVIIa Activity but Not Complex Formation the respective cells expressing TFWt-tGFP.) (F) MDA-MB-231 (5 × 104) cells expressing endogenous TF were transfected with PPT-siRNA or control siRNA and fXa-generation measured (n = 3, * = p < 0.05 vs. cells without siRNA). 3.3. De-Palmitoylation of Cys245 Is Required for TF-fVIIa Activity but Not Complex Formation Analysis of TF-tGFP procoagulant activity and fVIIa-HRP binding in the presence and it l ti (A) HDBEC (5 104) t f t d ith PPT iRNA (0 15 l) d th i f Figure 2. Analysis of TF-tGFP procoagulant activity and fVIIa-HRP binding in the presence and absence o Figure 2. Analysis of TF-tGFP procoagulant activity and fVIIa-HRP binding in the presence and absence of TF pal- mitoylation. (A) HDBEC (5 × 104) were transfected with PPT-siRNA (0–15 pmol) and the expression of PPT1 and -2 meas- ured by western blot. (B) HDBEC were co-transfected with combinations of pCMV-Ac-TF-tGFP and PPT-siRNA or control siRNA, and fXa-generation was measured in resting and PAR2-activated cells. (n = 5, * = p < 0.05 vs. the respective non- activated sample.) (C) HDBEC were co-transfected to express TF-tGFP, with PPT siRNA or control siRNA. Factor Xa- generation was measured before and after PAR2 activation for up to 60 min. (n = 4, * = p < 0.05 vs. the respective control siRNA sample.) HDBEC were transfected to express (D) TFSer245-tGFP or (E) TFPhe245-tGFP and fXa-generation were meas- ured as above. (n = 6, * = p < 0.05 vs. the respective cells expressing TFWt-tGFP.) (F) MDA-MB-231 (5 × 104) cells expressing endogenous TF were transfected with PPT-siRNA or control siRNA and fXa-generation measured (n = 3, * = p < 0.05 vs. cells without siRNA). 3 4 D P l it l ti f C 245 P t th A ti f TF th C ll M b Figure 2. Analysis of TF-tGFP procoagulant activity and fVIIa-HRP binding in the presence and absence of TF palmitoylation. (A) HDBEC (5 × 104) were transfected with PPT-siRNA (0–15 pmol) and the expression of PPT1 and -2 measured by western blot. (B) HDBEC were co-transfected with combinations of pCMV-Ac-TF-tGFP and PPT-siRNA or control siRNA, and fXa-generation was measured in resting and PAR2-activated cells. (n = 5, * = p < 0.05 vs. the respective non-activated sample.) (C) HDBEC were co-transfected to express TF-tGFP, with PPT siRNA or control siRNA. Factor Xa-generation was measured before and after PAR2 activation for up to 60 min. (n = 4, * = p < 0.05 vs. the respective control siRNA sample.) HDBEC were transfected to express (D) TFSer245-tGFP or (E) TFPhe245-tGFP and fXa-generation were measured as above. (n = 6, * = p < 0.05 vs. 3.3. De-Palmitoylation of Cys245 Is Required for TF-fVIIa Activity but Not Complex Formation (B) HDBEC were co-transfected with combinations of pCMV-Ac-TF-tGFP and PPT-siRNA or control siRNA, and fXa-generation was measured in resting and PAR2-activated cells. (n = 5, * = p < 0.05 vs. the respective non- activated sample.) (C) HDBEC were co-transfected to express TF-tGFP, with PPT siRNA or control siRNA. Factor Xa- generation was measured before and after PAR2 activation for up to 60 min. (n = 4, * = p < 0.05 vs. the respective control siRNA sample.) HDBEC were transfected to express (D) TFSer245-tGFP or (E) TFPhe245-tGFP and fXa-generation were meas- ured as above. (n = 6, * = p < 0.05 vs. the respective cells expressing TFWt-tGFP.) (F) MDA-MB-231 (5 × 104) cells expressing endogenous TF were transfected with PPT-siRNA or control siRNA and fXa-generation measured (n = 3, * = p < 0.05 vs. cells without siRNA). 3 4 De-Palmitoylation of Cys245 Promotes the Aggregation of TF on the Cell Membrane Figure 2. Analysis of TF-tGFP procoagulant activity and fVIIa-HRP binding in the presence and absence of TF palmitoylation. (A) HDBEC (5 × 104) were transfected with PPT-siRNA (0–15 pmol) and the expression of PPT1 and -2 measured by western blot. (B) HDBEC were co-transfected with combinations of pCMV-Ac-TF-tGFP and PPT-siRNA or control siRNA, and fXa-generation was measured in resting and PAR2-activated cells. (n = 5, * = p < 0.05 vs. the respective non-activated sample.) (C) HDBEC were co-transfected to express TF-tGFP, with PPT siRNA or control siRNA. Factor Xa-generation was measured before and after PAR2 activation for up to 60 min. (n = 4, * = p < 0.05 vs. the respective control siRNA sample.) HDBEC were transfected to express (D) TFSer245-tGFP or (E) TFPhe245-tGFP and fXa-generation were measured as above. (n = 6, * = p < 0.05 vs. the respective cells expressing TFWt-tGFP.) (F) MDA-MB-231 (5 × 104) cells expressing endogenous TF were transfected with PPT-siRNA or control siRNA and fXa-generation measured (n = 3, * = p < 0.05 vs. cells without siRNA). sis of TF-tGFP procoagulant activity and fVIIa-HRP binding in the presence and absence of TF pal- HDBEC (5 104) t f t d ith PPT iRNA (0 15 l) d th i f PPT1 d 2 is of TF-tGFP procoagulant activity and fVIIa-HRP binding in the presence and absence of TF palmitoylation. Figure 2. It has been reported that the procoagulant activity of TF is strongly influenced 3.4. De-Palmitoylation of Cys245 Promotes the Aggregation of TF on the Cell Membrane It has been reported that the procoagulant activity of TF is strongly influenced 3.4. De-Palmitoylation of Cys245 Promotes the Aggregation of TF on the Cell Membrane membrane microenvironment [21–23]. We hypothesised that de-palmitoylation of TF fol- lowing cellular activation may sequester TF molecules into specific microdomains within the cell membrane. Therefore, the aggregation of TF-tGFP variants before and after cellu- lar activation was examined by confocal microscopy. These were compared to cells ex- pressing TFWt-tGFP in which the expression of PPT1 and PPT2 was silenced. Examination It has been reported that the procoagulant activity of TF is strongly inﬂuenced by the membrane microenvironment [21–23]. We hypothesised that de-palmitoylation of TF following cellular activation may sequester TF molecules into speciﬁc microdomains within the cell membrane. Therefore, the aggregation of TF-tGFP variants before and after cellular activation was examined by confocal microscopy. These were compared to cells expressing TFWt-tGFP in which the expression of PPT1 and PPT2 was silenced. Examination of the cells indicated an outspread cellular distribution of TFWt-tGFP (Figure 3), which then aggregated following activation, forming regions with a higher mean diameter (Table 3). Interestingly, TFSer245-tGFP aggregates were detected even prior to cell activation. In contrast, expression of TFPhe245-tGFP or suppression of the PPT expression reduced the ability of the TF protein to form aggregates following PAR2 activation. Table 3. Estimation of TF aggregate sizes. Sample Non-Activated (µm) PAR2-Activated (µm) tGFP 0.09 ± 0.03 0.09 ± 0.03 TFWt-tGFP 0.15 ± 0.06 0.43 ± 0.11 TFSer245-tGFP 0.41 ± 0.10 0.53 ± 0.12 TFPhe245-tGFP 0.09 ± 0.03 0.12 ± 0.05 TFWt-tGFP +PPT siRNA 0.12 ± 0.04 0.12 ± 0.04 HDBEC (5 × 104) were transfected to express TFWt-tGFP in the presence and absence of PPT-siRNA or to express TFSer245-tGFP or TFPhe245-tGFP as in Figure 3. Sets of cells were then activated for 20 min using PAR2-AP (20 µM), and the distribution of TF-tGFP was examined. Analysis of the aggregate sizes was carried out using the ImagePro Plus software. Table 3. Estimation of TF aggregate sizes. HDBEC (5 × 104) were transfected to express TFWt-tGFP in the presence and absence of PPT-siRNA or to express TFSer245-tGFP or TFPhe245-tGFP as in Figure 3. Sets of cells were then activated for 20 min using PAR2-AP (20 µM), and the distribution of TF-tGFP was examined. Analysis of the aggregate sizes was carried out using the ImagePro Plus software. It has been reported that the procoagulant activity of TF is strongly influenced 3.4. De-Palmitoylation of Cys245 Promotes the Aggregation of TF on the Cell Membrane 9 of 20 0 of 21 Cancers 2021, 13, 3837 Cancers 2021, 13, x FO Figure 3. Confocal microscopy analysis of TF-tGFP distribution within cells. HDBEC (5 × 104) were transfected to exp TFWt-tGFP in the presence and absence of PPT-siRNA or to express TFSer245-tGFP or TFPhe245-tGFP. Sets of cells were activated using PAR2-AP (20 µM), washed and fixed with 3% (v/v) formaldehyde. All cells were labelled with DAPI analysed using a Zeiss LSM 710 confocal microscope. gure 3. Confocal microscopy analysis of TF-tGFP distribution within cells. HDBEC (5 × 104) were transfected to e FWt-tGFP in the presence and absence of PPT-siRNA or to express TFSer245-tGFP or TFPhe245-tGFP. Sets of cells we tivated using PAR2-AP (20 µM), washed and ﬁxed with 3% (v/v) formaldehyde. All cells were labelled with DA alysed using a Zeiss LSM 710 confocal microscope. Figure 3. Confocal microscopy analysis of TF-tGFP distribution within cells. HDBEC (5 × 104) were transfected to express TFWt-tGFP in the presence and absence of PPT-siRNA or to express TFSer245-tGFP or TFPhe245-tGFP. Sets of cells were then activated using PAR2-AP (20 µM), washed and fixed with 3% (v/v) formaldehyde. All cells were labelled with DAPI and analysed using a Zeiss LSM 710 confocal microscope. Figure 3. Confocal microscopy analysis of TF-tGFP distribution within cells. HDBEC (5 × 104) were transfected to express TFWt-tGFP in the presence and absence of PPT-siRNA or to express TFSer245-tGFP or TFPhe245-tGFP. Sets of cells were then activated using PAR2-AP (20 µM), washed and ﬁxed with 3% (v/v) formaldehyde. All cells were labelled with DAPI and analysed using a Zeiss LSM 710 confocal microscope. Figure 3. Confocal microscopy analysis of TF-tGFP distribution within cells. HDBEC (5 × 104) were transfected to express TFWt-tGFP in the presence and absence of PPT-siRNA or to express TFSer245-tGFP or TFPhe245-tGFP. Sets of cells were then activated using PAR2-AP (20 µM), washed and fixed with 3% (v/v) formaldehyde. All cells were labelled with DAPI and analysed using a Zeiss LSM 710 confocal microscope. Figure 3. Confocal microscopy analysis of TF-tGFP distribution within cells. HDBEC (5 × 104) were transfected to express TFWt-tGFP in the presence and absence of PPT-siRNA or to express TFSer245-tGFP or TFPhe245-tGFP. Sets of cells were then activated using PAR2-AP (20 µM), washed and ﬁxed with 3% (v/v) formaldehyde. All cells were labelled with DAPI and analysed using a Zeiss LSM 710 confocal microscope. It has been reported that the procoagulant activity of TF is strongly influenced 3.4. De-Palmitoylation of Cys245 Promotes the Aggregation of TF on the Cell Membrane 10 of 20 10 of 20 Cancers 2021, 13, 3837 3.5. TF Activity Is Regulated by the Length and Orientation of the Transmembrane Domain 3.5. TF Activity Is Regulated by the Length and Orientation of the Transmembrane Domain Palmitoylation can adjust the orientation of transmembrane domains within the membrane by altering the length of the transmembrane domain [33]. This can inﬂuence the location of the protein within the membrane [34–36]. The peptide at the cytoplasmic- transmembrane interface of TF consists of a sequence of amino acids, which renders two of the amino acids (Ser241-Leu242) a neutral overall hydrophobicity. We envisaged that these amino acids may be accommodated equally well within the aqueous cytoplasm and within the hydrophobic membrane. Consequently, conformational changes within TF, arising from de-palmitoylation of Cys245, would permit the Ser241-Leu242 motif to be incorporated into the membrane and therefore lengthen the transmembrane domain by two amino acids. Deletion of Ser241-Leu242 to shorten the transmembrane domain (TFShortTM- tGFP) prevented the increase in fXa-generation following PAR2 activation (Figure 4A and Supplementary Figure S2). Interestingly, the interaction of fVIIa with TFShortTM-tGFP only declined following cellular activation (Table 2). In contrast, duplicating the motif to lengthen the transmembrane domain (TFLongTM-tGFP) increased the fXa-generation potential in resting cells (Figure 4B and Supplementary Figure S2) but had no outcome on fVIIa binding (Table 2). By substituting Ser241→Phe or alternatively Leu242→Ser, it was possible to manipulate the hydrophobicity of this region and potentially increase or decrease the length of the transmembrane region, respectively. Using this approach, a marginally enhanced level of fXa-generation was detected in resting cells expressing TFPhe241-tGFP compared to cells expressing TFWT-tGFP, which increased to the same levels as that of wild-type form upon PAR2 activation (Figure 4C and Supplementary Figure S2). Moreover, the binding of fVIIa-HRP to TF was unaffected by Ser241→Phe substitution (Table 2). Basal fXa-generation activity in cells expressing TFSer242-tGFP was comparable to that of TFWt-tGFP but did not increase following cellular activation (Figure 4D and Supplementary Figure S2). However, the fVIIa-HRP binding remained unaffected by Leu242→Ser substitution (Table 2). Finally, the requirement for a ﬂexible transmembrane domain, capable of accommodating different orientations of the protein, was explored by substituting Gly225 for a valine residue and therefore reducing the ﬂexibility of the transmembrane domain. The expression of TFVal225-tGFP supported basal levels of fXa- generation in resting cells, but again, there was no enhancement in fXa-generation following PAR2 activation (Figure 4E), which was concurrent with a reduction in fVIIa-HRP binding ability (Table 2). EW 12 of 21 Figure 4. Cont. Figure 4. Cont. Figure 4. 3.5. TF Activity Is Regulated by the Length and Orientation of the Transmembrane Domain Cont. Figure 4. Cont. 11 of 20 Cancers 2021, 13, 3837 Figure 4. The influence of the modifications of TF transmembrane domain on TF activity. HDBEC (5 × 104) were transfected to express (A) TFShortTM-tGFP, (B) TFLongTM-tGFP, (C) TFPhe241-tGFP, (D) TFSer242-tGFP or (E) TFVal225-tGFP, and fXa-generation was measured. (n = 4, * = p < 0.05 vs. the respective cells expressing TFWt-tGFP.) Figure 4. The inﬂuence of the modiﬁcations of TF transmembrane domain on TF activity. HDBEC (5 × 104) were transfected to express (A) TFShortTM-tGFP, (B) TFLongTM-tGFP, (C) TFPhe241-tGFP, (D) TFSer242-tGFP or (E) TFVal225-tGFP, and fXa-generation was measured. (n = 4, * = p < 0.05 vs. the respective cells expressing TFWt-tGFP.) Figure 4. The influence of the modifications of TF transmembrane domain on TF activity. HDBEC (5 × 104) were transfected to express (A) TFShortTM-tGFP, (B) TFLongTM-tGFP, (C) TFPhe241-tGFP, (D) TFSer242-tGFP or (E) TFVal225-tGFP, and fXa-generation was measured. (n = 4, * = p < 0.05 vs. the respective cells expressing TFWt-tGFP.) Figure 4. The inﬂuence of the modiﬁcations of TF transmembrane domain on TF activity. HDBEC (5 × 104) were transfected to express (A) TFShortTM-tGFP, (B) TFLongTM-tGFP, (C) TFPhe241-tGFP, (D) TFSer242-tGFP or (E) TFVal225-tGFP, and fXa-generation was measured. (n = 4, * = p < 0.05 vs. the respective cells expressing TFWt-tGFP.) 3.6. The Transmembrane Domain Regulates TF Phosphorylation at Ser253 and Incorporation into Microvesicles 3.6. The Transmembrane Domain Regulates TF Phosphorylation at Ser253 and Incorporation into Microvesicles De-palmitoylation of TF precedes its phosphorylation at Ser253 [11], which in turn is required for the incorporation of TF into microvesicles [13,15]. Therefore, an attempt was made to examine the outcome of TF de-palmitoylation and the change in the transmem- brane domain on the phosphorylation of TF. Cells were transfected with PPT-siRNA to- gether with TFWt-tGFP or the mutant forms of TF-tGFP described above. TF-tGFP was then immunoprecipitated and the phosphorylation state of Ser253 within TF-tGFP was as- sessed by western blot analysis. Because of immunoprecipitation, ratios were calculated against TF antigen, not a housekeeping gene. Moreover, due to the low yield of immuno- precipitation in HCAEC, only the data from HDBEC have been presented. The suppres- sion of PPT expression reduced the phosphorylation of TFWt-tGFP at Ser253 following the De-palmitoylation of TF precedes its phosphorylation at Ser253 [11], which in turn is required for the incorporation of TF into microvesicles [13,15]. 3.5. TF Activity Is Regulated by the Length and Orientation of the Transmembrane Domain Therefore, an attempt was made to examine the outcome of TF de-palmitoylation and the change in the trans- membrane domain on the phosphorylation of TF. Cells were transfected with PPT-siRNA together with TFWt-tGFP or the mutant forms of TF-tGFP described above. TF-tGFP was then immunoprecipitated and the phosphorylation state of Ser253 within TF-tGFP was assessed by western blot analysis. Because of immunoprecipitation, ratios were calculated against TF antigen, not a housekeeping gene. Moreover, due to the low yield of immuno- precipitation in HCAEC, only the data from HDBEC have been presented. The suppression of PPT expression reduced the phosphorylation of TFWt-tGFP at Ser253 following the acti- vation of PAR2 compared to cells transfected with control siRNA (Figure 5A). In addition, Ser253 was shown to be phosphorylated in non-activated cells expressing TFSer245-tGFP and further increased following PAR2 activation (Figure 5B,C). In contrast, phosphorylation of Ser253 was signiﬁcantly lower in cells expressing TFPhe245-tGFP, even following PAR2 ac- tivation. Similarly, Ser253 appeared to be phosphorylated in non-activated cells expressing TFPhe241-tGFP but not in cells expressing TFSer242-tGFP or TFShortTM-tGFP (Figure 5C,D). Ser253-phosphorylation also increased in TFPhe241-tGFP expressing cells following activa- tion but was lower in cells expressing TFSer242-tGFP and TFShortTM-tGFP. TFLongTM-tGFP was not tested in this study. The levels of TF-tGFP incorporated into released microvesi- cles also reﬂected the state of Ser253 phosphorylation but not in the same magnitudes (Figure 5E). Furthermore, the density of released microvesicles was unaltered by the type of TF mutation (Supplementary Figure S3). Finally, to discriminate between the contribu- tions of de-palmitoylation and phosphorylation to the fXa-generation associated with cell 12 of 20 t in the Cancers 2021, 13, 3837 12 of 20 t in the surface TF, HDBEC transfected to express TFWt-tGFP, or alternatively, MDA-MB-231 cells were pre-incubated with the PKCα inhibitor Gö6976 (100 nM). Inhibition of PKCα with Gö6976 did not alter fXa-generation in HDBEC expressing TFWt-tGFP (Figure 5F) or in MDA-MB-231 cells (Figure 5G). between the contributions of de-palmitoylation and phosphorylation to the fXa-genera- tion associated with cell surface TF, HDBEC transfected to express TFWt-tGFP, or alterna- tively, MDA-MB-231 cells were pre-incubated with the PKCα inhibitor Gö6976 (100 nM). Inhibition of PKCα with Gö6976 did not alter fXa-generation in HDBEC expressing TFWt- tGFP (Figure 5F) or in MDA-MB-231 cells (Figure 5G). MDA-MB-231 cells (Figure 5G). g p g tGFP (Figure 5F) or in MDA-MB-231 cells (Figure 5G). gure 5. 3.5. TF Activity Is Regulated by the Length and Orientation of the Transmembrane Domain Analysis of Ser253 phosphorylation in the TF variants. HDBEC (2 × 105) were transfected (A) to express TFWt-tGFP the presence and absence of PPT-siRNA or (B) to express TFSer245-tGFP or TFPhe245-tGFP. Cells were activated with PAR2- (20 µM) for 20 min, and TF-tGFP was then immunoprecipitated and analysed by western blot and (C) the phosphorylation Ser253 was quantiﬁed. (n = 3, * = p < 0.05 vs. the respective cells expressing TFWt-tGFP.) (D) HDBEC (2 × 105) were nsfected to express TFPhe241-tGFP, TFSer242-tGFP, TFShortTM-tGFP or TFVal225-tGFP, and the phosphorylation of Ser253 was alysed and (C) quantiﬁed. (E) HDBEC (5 × 104) were transfected with the TF variants as shown, and the concentration of released from cells was measured by TF-ELISA. (n = 4, * = p < 0.05 vs. the respective cells expressing TFWt-tGFP.) (F) DBEC (5 × 104) were transfected to express TFWt-tGFP and pre-incubated with Gö6976 (100 nM) prior to PAR2-activation d fXa-generation measured in resting and activated cells. (n = 4). (G) MDA-MB-231 (5 × 104) cells expressing endogenous were incubated with Gö6976 and fXa-generation measured (n = 3). e 5. Analysis of Ser253 phosphorylation in the TF variants. HDBEC (2 × 105) were transfected (A) to express TFWt-tG presence and absence of PPT-siRNA or (B) to express TFSer245-tGFP or TFPhe245-tGFP. Cells were activated with PA 0 µM) for 20 min, and TF-tGFP was then immunoprecipitated and analysed by western blot and (C) the phosphorylat 253 was quantiﬁed. (n = 3, * = p < 0.05 vs. the respective cells expressing TFWt-tGFP.) (D) HDBEC (2 × 105) w ected to express TFPhe241-tGFP, TFSer242-tGFP, TFShortTM-tGFP or TFVal225-tGFP, and the phosphorylation of Ser253 w sed and (C) quantiﬁed. (E) HDBEC (5 × 104) were transfected with the TF variants as shown, and the concentration eased from cells was measured by TF-ELISA. (n = 4, * = p < 0.05 vs. the respective cells expressing TFWt-tGFP.) C (5 × 104) were transfected to express TFWt-tGFP and pre-incubated with Gö6976 (100 nM) prior to PAR2-activat Xa-generation measured in resting and activated cells. (n = 4). (G) MDA-MB-231 (5 × 104) cells expressing endogen b d h G d f d ( ) Figure 5. Analysis of Ser253 phosphorylation in the TF variants. HDBEC (2 × 105) were transfected (A) to express TFWt-tGFP in the presence and absence of PPT-siRNA or (B) to express TFSer245-tGFP or TFPhe245-tGFP. 3.5. TF Activity Is Regulated by the Length and Orientation of the Transmembrane Domain Cells were activated with PAR2- AP (20 µM) for 20 min, and TF-tGFP was then immunoprecipitated and analysed by western blot and (C) the phosphorylation of Ser253 was quantiﬁed. (n = 3, * = p < 0.05 vs. the respective cells expressing TFWt-tGFP.) (D) HDBEC (2 × 105) were transfected to express TFPhe241-tGFP, TFSer242-tGFP, TFShortTM-tGFP or TFVal225-tGFP, and the phosphorylation of Ser253 was analysed and (C) quantiﬁed. (E) HDBEC (5 × 104) were transfected with the TF variants as shown, and the concentration of TF released from cells was measured by TF-ELISA. (n = 4, * = p < 0.05 vs. the respective cells expressing TFWt-tGFP.) (F) HDBEC (5 × 104) were transfected to express TFWt-tGFP and pre-incubated with Gö6976 (100 nM) prior to PAR2-activation and fXa-generation measured in resting and activated cells. (n = 4). (G) MDA-MB-231 (5 × 104) cells expressing endogenous TF were incubated with Gö6976 and fXa-generation measured (n = 3). Cancers 2021, 13, 3837 (F) HDBEC (5 × vation and fXa 13 of 20 cti- ng 13 of 20 cti- ng 3.7. Sequential De-Palmitoylation and Phosphorylation of TF Can Co-Ordinate Cell Proliferation and Apoptosis Following PAR2 Activation 3.7. Sequential De-Palmitoylation and Phosphorylation of TF Can Co-Ordinate Cell Proliferation and Apoptosis Following PAR2 Activation 3.7. Sequential De-Palmitoylation and Phosphorylation of TF Can Co-Ordinate Cell Proliferation and Apoptosis Following PAR2 Activation 3.7. Sequential De-Palmitoylation and Phosphorylation of TF Can Co-Ordinate Cell Proliferation and Apoptosis Following PAR2 Activation The activation of TF and its interactions with other proteins have been associated with changes in cell proliferation and the induction of apoptosis. To further elucidate the mechanisms by which TF mediates these cellular processes, we examined the change in cell numbers and the level of cellular apoptosis in HDBEC and MCF-7 cells expressing the TF-tGFP variants as above. For comparison, additional samples were transfected to express TFAla253-tGFP to prevent TF phosphorylation. Determination of cell numbers showed increases in cell numbers in both non-activated HDBEC (Figure 6A) and MCF- 7 cells (Figure 6C) on expression of TFSer245-tGFP and TFPhe245-tGFP compared to cells expressing TFWT-tGFP, but a signiﬁcant reduction in expression of TFShortTM-tGFP. Fur- thermore, PAR2 activation of the cells resulted in increased proliferation at 24 h in cells expressing TFWt-tGFP, TFPhe241-tGFP and TFLongTM-tGFP compared to their respective control cells expressing tGFP. 3.5. TF Activity Is Regulated by the Length and Orientation of the Transmembrane Domain However, a reduction in the number of both cell types was observed in PAR2-activated cells expressing TFAla253-tGFP and TFPhe245-tGFP and also in HDBEC expressing TFSer242-tGFP compared to their respective control cells expressing tGFP (Figure 6A,C). In agreement with the proliferation data, lower levels of endothelial but not MCF-7 cell apoptosis was detectable in cells expressing TFWt-tGFP, TFSer245-tGFP, TFPhe241-tGFP and TFLongTM-tGFP, while increased levels of apoptosis were detected in endothelial and MCF-7 cells expressing TFAla253-tGFP at 24 h post-activation (Figure 6B,D). The activation of TF and its interactions with other proteins have been associated with changes in cell proliferation and the induction of apoptosis. To further elucidate the mechanisms by which TF mediates these cellular processes, we examined the change in cell numbers and the level of cellular apoptosis in HDBEC and MCF-7 cells expressing the TF-tGFP variants as above. For comparison, additional samples were transfected to ex- press TFAla253-tGFP to prevent TF phosphorylation. Determination of cell numbers showed increases in cell numbers in both non-activated HDBEC (Figure 6A) and MCF-7 cells (Fig- ure 6C) on expression of TFSer245-tGFP and TFPhe245-tGFP compared to cells expressing TFWT-tGFP, but a significant reduction in expression of TFShortTM-tGFP. Furthermore, PAR2 activation of the cells resulted in increased proliferation at 24 h in cells expressing TFWt- tGFP, TFPhe241-tGFP and TFLongTM-tGFP compared to their respective control cells express- ing tGFP. However, a reduction in the number of both cell types was observed in PAR2- activated cells expressing TFAla253-tGFP and TFPhe245-tGFP and also in HDBEC expressing TFSer242-tGFP compared to their respective control cells expressing tGFP (Figure 6A,C). In agreement with the proliferation data, lower levels of endothelial but not MCF-7 cell apoptosis was detectable in cells expressing TFWt-tGFP, TFSer245-tGFP, TFPhe241-tGFP and TFLongTM-tGFP, while increased levels of apoptosis were detected in endothelial and MCF- 7 cells expressing TFAla253-tGFP at 24 h post-activation (Figure 6B,D). Figure 6. The inﬂuence of TF variants on cell proliferation and apoptosis. HDBEC and MCF-7 cells (2.5 × 104) were transfected to express the TF variants and were activated with PAR2-AP (20 µM). The rate of cell proliferation (A,C) and cell apoptosis (B,D) were determined using the crystal violet and a chromogenic apoptosis assay, respectively (n = 3, * = p < 0.05 vs. the respective tGFP-expressing cell sample). Figure 6. The inﬂuence of TF variants on cell proliferation and apoptosis. 3.5. TF Activity Is Regulated by the Length and Orientation of the Transmembrane Domain HDBEC and MCF-7 cells (2.5 × 104) were transfected to express the TF variants and were activated with PAR2-AP (20 µM). The rate of cell proliferation (A,C) and cell apoptosis (B,D) were determined using the crystal violet and a chromogenic apoptosis assay, respectively (n = 3, * = p < 0.05 vs. the respective tGFP-expressing cell sample). 4. Discussion The ability of fVIIa to activate coagulation is dependent on the formation of a complex with cell-surface TF. However, encrypted TF may form a complex with fVIIa without promoting signiﬁcant coagulation [3–8]. The kinetics of these interactions have previously Cancers 2021, 13, 3837 14 of 20 14 of 20 been studied [3,22,37]. In agreement with this concept, similar amounts of fVIIa-HRP were bound to the surface of HDBEC transfected to express TF before and after activation (Figure 1B and Table 2). Previously, we showed that PAR2 activation of MDA-MB-231 cells resulted in a 56% increase in cell-surface TF antigen at 30 min post-activation [14]. However, endothelial cells exhibit a relatively slower response rate to PAR2 activation compared to MDA-MB-231 cells [13], and also, the 20 min activation time used in this study was even shorter than that previously used for MDA-MB-231 cells [14]. Accordingly, neither the amount of TF-tGFP antigen expressed on the surface of HDBEC (not shown) nor the binding of fVIIa-HRP changed following PAR2 activation (Table 2). Therefore, the increases in fXa-generation observed did not arise from changes in cell-surface TF-tGFP antigen. g Reversible S-palmitoylation of TF was one of the earliest post-translational modiﬁca- tions reported, and it occurs on Cys245 of human TF [18,19]. Currently, palmitoyl-protein thioesterase (PPT) 1 and 2 (also known as acyl-protein thioesterase) are the identiﬁed enzymes responsible for the de-palmitoylation of cellular proteins, although controversies regarding the role, location and number of these enzymes do exist [32,38]. These enzymes have been implicated in the regulation of proteins as diverse as eNOS [39], p21RAS [40], PSD-95 [32] and growth-associated protein-43 (GAP-43) [41]. PPT enzymes may be initiated in response to intracellular Ca2+ ion release [39] and regulate proteins through reversible de-palmitoylation of cysteine residues [42]. The de-palmitoylation of proteins is a sequence- speciﬁc process [32] and determines the trafﬁcking and localisation of proteins within cells [43]. Due to the homology between PPT1 and PPT2, we employed a conﬁrmed siRNA that is suitable for silencing both genes concurrently. The suppression of PPT expression in cells expressing TFWt-tGFP reduced fXa-generation following the activation of the cells without affecting fVIIa-HRP binding (Figure 2 and Table 2). However, knock-down of PPT may have additional unknown effects on cells other than preventing de-palmitoylation of TF. Therefore, in further studies, palmitoylation of TF was prevented by Cys245→Ser sub- stitution. 4. Discussion This substitution increased fXa-generation in resting cells (Figure 2D), whereas replacing this residue with phenylalanine to mimic palmitoylation curtailed the increase in TF procoagulant activity following PAR2 activation (Figure 2E) and partially hindered fVIIa binding. Therefore, while the ability of TF to bind fVIIa is not altered by its palmitoylation state, the ability of the complex to generate fXa is signiﬁcantly ampliﬁed following de- palmitoylation of TF. A possible explanation for this enhanced fXa-generation may include the translocation of TF to lipid-raft domains that support TF activity through the exposure of phosphatidylserine on the surface of the cells. This hypothesis is supported by the suggestion that, following cellular activation, TF translocates across membrane domains of differing densities [37,44]. The purpose of the various mutational changes and possible explanations for the main observations have been summarised in Table 4. In fact, TF is released within low-density microvesicles [30], which must accordingly arise from mem- brane domains with low density [20,45,46]. Mutations in palmitoyl-protein thioesterase are known to lead to neurodegenerative disorders [47]. However, different mutations are thought to result in a broader spectrum of clinical presentations [48,49]. These include inﬂuences on the pro-inﬂammatory and immune responses [50,51], the apoptotic mech- anisms [52] and also tumour progression [53]. Therefore, currently any association with blood haemostasis remains tenuous, and we have refrained from speculation. However, the availability of PPT knock-out mice [48,54] may produce novel and interesting connections, particularly regarding the role of TF during the inﬂammatory responses and/or in cancer progression [55] 15 of 20 Cancers 2021, 13, 3837 Table 4. Summary of the purpose of TF modiﬁcations and the main observation. Plasmid Purpose Observation Possible Explanation pCMV-Ac-TFSer245-tGFP Prevent palmitoylation ↑TF activation TF can mobilise within the membrane ↑Proliferation (non-activated) pCMV-Ac-TFPhe245-tGFP Mimic palmitoylation ↓TF activation TF cannot mobilise or be released–Increased membrane thickness hampers fVIIa binding ↓Proliferation ↓fVIIa binding (activated) pCMV-Ac-TFPhe241-tGFP Increase hydrophobicity of TMD ↑TF activation Facilitates mobilisation to thicker membrane regions ↑Proliferation (activated) pCMV-Ac-TFSer242-tGFP Decrease hydrophobicity of TMD ↓TF activation Hampers mobilisation to thicker membrane regions ↓Proliferation (activated) pCMV-Ac-TFShortTM-tGFP Shorten TMD ↓TF activation Cannot mobilise to to thicker membrane regions ↓Proliferation ↓fVIIa binding pCMV-Ac-TFLongTM-tGFP Lengthen TMD ↑TF activation Facilitates mobilisation to thicker membrane regions ↑Proliferation (activated) pCMV-Ac-TFVal225-tGFP Reduce TMD ﬂexibility ↓TF activation Cannot accommodate correct structure for interaction with fVIIa ↓fVIIa binding Table 4. Summary of the purpose of TF modiﬁcations and the main observation. 4. Discussion Activation of cells expressing TFWt-tGFP resulted in the aggregation of TF on cells (Figure 3, Table 3). Moreover, prevention of palmitoylation induced spontaneous aggre- gation of TFSer245-tGFP even in resting cells. In contrast, activation of cells did not result in signiﬁcant aggregation either in cells expressing TFPhe245-tGFP or in cells following suppression of PPT1 and PPT2 expression. These ﬁndings are in line with those reporting a lower procoagulant activity associated with palmitoylated TF [11,12,19] arising from decreased association with cell-surface lipid rafts [5]. The translocation/aggregation of TF and the association with lipid rafts following de-palmitoylation also suggests alter- ations in the orientation of the transmembrane domain. Recently, alternative mechanisms by which palmitoylation can modify receptor protein function have emerged. These are mainly mediated through adjusting the orientation and length of the transmembrane do- main [33,36]. Modiﬁcations in the length and orientation of the transmembrane domain allow the incorporation of these membrane proteins into different membrane domains with varying membrane thickness. In fact, the transmembrane domain of TF consists of 21 amino acid residues, which is shorter than the average 23 residue length of the integral plasma membrane proteins [56]. Consequently, an increase in the span of the transmembrane domain may permit the transfer of TF into thicker lipid-raft domains [33,34,36] and could result in higher procoagulant activity. The presence of the Ser241-Leu242 di-peptide at the transmembrane-cytoplasmic interface within TF produces a motif of neutral hydrophobic- ity (Figure 7). This suggests that these two amino acids may remain within the cytoplasm or alternatively be buried within the membrane bilayer, depending on other inﬂuences such as palmitoylation of the proximal Cys245 residue. In support of this, increasing the length of the available membrane-spanning region resulted in higher activity in resting cells while shortening, and discouraging the incorporation of the di-peptide into the mem- Cancers 2021, 13, 3837 16 of 20 brane suppressed the increase in fXa-generation following cellular activation (Figure 4). Interestingly, the ability of the shortened TF variant to bind fVIIa was signiﬁcantly reduced but only following cellular activation, suggesting a robust cellular attempt to translocate TF to thicker membrane regions, which may in turn disrupt the TF-fVIIa complex due to steric hindrance. Interference with the TF-fVIIa complex was also observed following the expression of TFVal225-tGFP to reduce the ﬂexibility at the mid-section of the transmem- brane domain, also indicating that the TF–fVIIa interaction could not be accommodated. 4. Discussion In previous studies, the expression of truncated forms of TF in cells showed little variation in activity compared to full-length TF [9,10]. However, these studies measured the TF activity in activated/perturbed cells only. Our data explain the results obtained using cytoplasmic domain-deleted TF since the palmitoylation of the cytoplasmic domain appears to be essential for the encryption rather than the decryption of TF. yp yp De-palmitoylation of TF precedes and enhances the rate of TF phosphorylation [11,12]. The subsequent TF phosphorylation at Ser253 is concurrent with increased TF activity [14] and release within cell-derived microvesicles [13]. In agreement with a previous re- port [11], the lack of TF palmitoylation (TFSer245-tGFP) enhanced TF phosphorylation and release within microvesicles in resting cells (Figure 5). Moreover, encouraging membrane- incorporation of the transmembrane-cytoplasmic interphase domain (TFPhe241-tGFP) was equally effective, suggesting that TF phosphorylation at Ser253 is dependent on the length of the transmembrane domain. In contrast, prevention of de-palmitoylation using either PPT siRNA or by mimicking palmitoylation (TFPhe245-tGFP) reduced TF phosphorylation at Ser253 and its release within microvesicles. Furthermore, shortening of the transmem- brane region by expressing either TFSer242-tGFP or TFShortTM-tGFP equally decreased the level of TF phosphorylation at Ser253 and reduced TF release (Figure 5). It is possible that the lengthening of the transmembrane domain permits the approximation of PKCα to the TF cytoplasmic domain containing Ser253, prior to translocation of TF to lipid-rafts. However, PKCα has been suggested to be associated with lipid-rafts, which explains how the translocation of TF to lipid-rafts may promote TF phosphorylation by PKCα. Moreover, since inhibition of PKCα using Gö6976 did not alter the fXa-generation capacity, our data suggest that the fXa-generation may be independent of TF phosphorylation and release from cells. The expression of TFWt-tGFP induced cell proliferation in both endothelial cells and the MCF-7 cell line following PAR2-activation (Figure 6). This was concurrent with a reduction in cell apoptosis in endothelial cells but was not signiﬁcant in the MCF-7 cell line. Intriguingly, the expression of TFSer245-tGFP or TFPhe245-tGFP resulted in increased proliferation in resting cells but declined signiﬁcantly with TFPhe245-tGFP, following PAR2 activation. This suggests that de-palmitoylation of TF per se, is not a signal for induction of cell proliferation. Additionally, extending the transmembrane domain (TFPhe241-tGFP or TFLongTM-tGFP) was highly proliferative following PAR2 activation, while reduced rates of proliferation were associated with the expression of the shorter TFSer242-tGFP and TFShortTM- tGFP. 4. Discussion Therefore, the induction of cellular proliferation signalling appears to involve the translocation of TF within the cell membrane and is complemented with a signal arising from PAR2 activation [13]. The expression of TFWt-tGFP, TFSer245-tGFP, TFPhe241-tGFP or TFLongTM-tGFP also reduced the rate of cell apoptosis in HDBEC but not MCF-7 cells (Figure 6). However, the rate of apoptosis did not match the reduction in cell proliferation observed in cells expressing TFPhe245-tGFP, TFSer242-tGFP and TFShortTM-tGFP. Together, these data suggest that the induction of apoptosis is likely to be associated with the lack of phosphorylation of Ser253, and therefore, the impairment in the release of excess TF from cells and may hence be distinct from the processes that induce proliferation [28,57,58]. 17 of 20 18 of 21 Cancers 2021, 13, 3837 Cancers 2021 13 x FOR . Schematic representation of the outcome of TF de-palmitoylation on the transmembrane and cytoplasmic A) Palmitoylation of Cys245 retains the transmembrane-cytoplasmic interface-region within the cytoplasm mitoylation of TF permits Ser241-Leu242 to be incorporated into the cell membrane elongating the transmemb from 21 to 23 amino acids. 7. Schematic representation of the outcome of TF de-palmitoylation on the transmembrane and cytopla s. (A) Palmitoylation of Cys245 retains the transmembrane-cytoplasmic interface-region within the cytoplasm mitoylation of TF permits Ser241-Leu242 to be incorporated into the cell membrane elongating the transmemb from 21 to 23 amino acids. Figure 7. Schematic representation of the outcome of TF de-palmitoylation on the transmembrane and cytoplasmic do- mains. (A) Palmitoylation of Cys245 retains the transmembrane-cytoplasmic interface-region within the cytoplasm. (B) De-palmitoylation of TF permits Ser241-Leu242 to be incorporated into the cell membrane elongating the transmembrane domain from 21 to 23 amino acids. Figure 7. Schematic representation of the outcome of TF de-palmitoylation on the transmembrane and cytoplasmic domains. (A) Palmitoylation of Cys245 retains the transmembrane-cytoplasmic interface-region within the cytoplasm. (B) De-palmitoylation of TF permits Ser241-Leu242 to be incorporated into the cell membrane elongating the transmembrane domain from 21 to 23 amino acids. Cancers 2021, 13, 3837 18 of 20 18 of 20 5. Conclusions In conclusion, this study indicates that the procoagulant activity of TF may be en- hanced by the palmitoylation-dependent modiﬁcations in the orientation of the trans- membrane domain of TF and is likely to mobilise TF to particular regions within the cell membrane. Furthermore, the increase in the procoagulant activity is initiated by de- palmitoylation and appears to be independent of TF phosphorylation. Finally, following cellular activation, separate proliferative and pro-apoptotic TF signals are initiated through phosphorylation-independent and -dependent mechanisms, respectively. At least some of these mechanisms appear to be independent of the procoagulant function of TF and may therefore determine the level of damage and the fate of the cells following injury or trauma. Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/cancers13153837/s1, Figure S1: Conﬁrmation of fVIIa-HRP activity and binding to TF, Figure S2: Analysis of the association of TF and PAR2 in the presence and absence of fVIIa, examined in HCAEC, Figure S3: Quantiﬁcation of HDBEC-derived microvesicles using the Zymuphen Assay l. Author Contributions: The study was designed by C.E. and the experimental work carried out by C.E. and S.F. The data were evaluated by C.E., and the manuscript was prepared by C.E., A.M.R.R., J.G., H.H.V. and A.M. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Funding: This research received no external funding. Funding: This research received no external funding. 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: Not applicable. Data Availability Statement: Not applicable. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. References 1. Grover, S.P.; Mackman, N. Tissue Factor: An Essential Mediator of Hemostasis and Trigger of Thromb Vasc. Biol. 2018, 38, 709–725. [CrossRef] Vasc. Biol. 2018, 38, 709–725. [CrossRef] 2. Branchford, B.R.; Carpenter, S.L. The Role of Inﬂammation in Venous Thromboembolism. Front. Pediatr. 2018, 6, 142. [CrossRef] [PubMed] 2. Branchford, B.R.; Carpenter, S.L. The Role of Inﬂammation in Venous Thromboembolism. Front. Pediatr. 2018, 6, 142. [CrossRef] [PubMed] [ ] 3. Kothari, H.; Pendurthi, U.R.; Rao, L.V. 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https://openalex.org/W2985276039	https://www.frontiersin.org/articles/10.3389/fnagi.2019.00299/pdf	English	null	Age and Sex Influence the Neuro-inflammatory Response to a Peripheral Acute LPS Challenge	Frontiers in aging neuroscience	2,019	cc-by	9,915	ORIGINAL RESEARCH published: 05 November 2019 doi: 10.3389/fnagi.2019.00299 Citation: Murtaj V, Belloli S, Di Grigoli G, Pannese M, Ballarini E, Rodriguez-Menendez V, Marmiroli P, Cappelli A, Masiello V, Monterisi C, Bellelli G, Panina-Bordignon P and Moresco RM (2019) Age and Sex Inﬂuence the Neuro-inﬂammatory Response to a Peripheral Acute LPS Challenge. Front. Aging Neurosci. 11:299. doi: 10.3389/fnagi.2019.00299 Keywords: aging, neuroinﬂammation, microglia, astrocytes, 18 kDa translocator protein, triggering receptor expressed on myeloid cells 2 Age and Sex Inﬂuence the Neuro-inﬂammatory Response to a Peripheral Acute LPS Challenge Valentina Murtaj 1,2, Sara Belloli 2,3, Giuseppe Di Grigoli 2,3, Maria Pannese 4, Elisa Ballarini 5,6, Virginia Rodriguez-Menendez 5,6, Paola Marmiroli 5,6, Andrea Cappelli 7, Valeria Masiello 2, Cristina Monterisi 6, Giuseppe Bellelli 8, Paola Panina-Bordignon 4,9† and Rosa Maria Moresco 2,6† 1PhD Program in Neuroscience, School of Medicine and Surgery, University of Milano-Bicocca, Monza, Italy, 2PET and Nuclear Medicine Unit, San Raffaele Scientiﬁc Institute, Milan, Italy, 3Institute of Molecular Bioimaging and Physiology of National Reasearch Council, Segrate, Italy, 4Neuroimmunology Unit, Division of Neuroscience, IRCCS San Raffaele Scientiﬁc Institute, Milan, Italy, 5Milan Center for Neuroscience, School of Medicine and Surgery, University of Milano-Bicocca, Monza, Italy, 6Department of Medicine and Surgery, Tecnomed Foundation, University of Milano-Bicocca, Monza, Italy, 7Department of Biotechnology, Chemistry and Pharmacy, University of Siena, Siena, Italy, 8Acute Geriatric Unit, School of Medicine and Surgery, San Gerardo Hospital, University of Milano-Bicocca, Monza, Italy, 9School of Medicine and Surgery, San Raffaele Vita-Salute University, Milan, Italy Reviewed by: Tahir Ali, Tahir Ali, University of Calgary, Canada Chan Young Shin, Konkuk University, South Korea Bernd L. Fiebich, University of Freiburg, Germany , University of Calgary, Canada Chan Young Shin, Konkuk University, South Korea Bernd L. Fiebich, University of Freiburg, Germany Correspondence: Rosa Maria Moresco moresco.rosamaria@hsr.it Paola Panina-Bordignon panina.paola@hsr.it Correspondence: Rosa Maria Moresco moresco.rosamaria@hsr.it Paola Panina-Bordignon panina.paola@hsr.it †These authors have contributed equally to this work as co-last authors Received: 24 June 2019 Accepted: 17 October 2019 Published: 05 November 2019 Aging is associated with an exaggerated response to peripheral inﬂammatory challenges together with behavioral and cognitive deﬁcits. Studies considering both age and sex remain limited, despite sex dimorphism of astrocytes and microglial cells is largely recognized. To ﬁll this knowledge gap, we investigated the effect of a single intraperitoneal lipopolysaccharide (LPS) administration in adult and aged mice. We assessed the expression of different inﬂammatory mediators, and the microglial response through binding of [18F]-VC701 tracer to translocator protein (TSPO) receptors in the male and female brain. Aged female brain showed a higher pro-inﬂammatory response to LPS compared to adult female and to aged male, as revealed by ex vivo binding to TSPO receptors and pro-inﬂammatory mediator transcript levels. The highest astroglial reaction was observed in the brain of aged females. Differently to the other groups of animals, in aged males LPS challenge did not affect transcription of triggering receptor expressed on myeloid cells 2 (TREM2). In conclusion, our study shows that in the mouse’s brain the neuro-inﬂammatory response to an acute peripheral insult is sex- and age-dependent. Moreover, our results might set the basis for further studies aimed at identifying sex-related targets involved in the modulation of the aberrant neuro- inﬂammatory response that characterizes aging. This knowledge could be relevant for the treatment of conditions such as delirium and dementia. Edited by: Diego Ruano, University of Seville, Spain Reviewed by: Tahir Ali, University of Calgary, Canada Chan Young Shin, Konkuk University, South Korea Bernd L. Fiebich, University of Freiburg, Germany Edited by: Diego Ruano, University of Seville, Spain INTRODUCTION (2017) demonstrated the presence of AD endo-phenotypes in the brain of asymptomatic peri-menopausal or menopausal women when compared to age-matched men. Sex dimorphism of astrocytes and microglial cells is largely recognized and has been recently demonstrated by Villa et al. (2018). Adult female microglial cells carry a neuroprotective phenotype even when transplanted into male brain (Amateau and McCarthy, 2002; Hanamsagar et al., 2017; Villa et al., 2018). Interestingly, this protective phenotype seems to be in contrast with what has been observed in aged subjects, as suggested by a whole genome profile showing that old female brains exhibit higher transcription of genes of the complement system when compared to old males. The different neuro-inflammatory signatures may explain the sex-specific susceptibility to cognitive disorders (Mangold et al., 2017). A neuro-inflammatory response induced by peripheral challenge with lipopolysaccharide (LPS) has been recently demonstrated in the brain of adult healthy volunteers using Positron Emission Tomography (PET) and a radio ligand for in vivo imaging of brain inflammation (Sandiego et al., 2015). However, most evidence on molecular and pathological CNS effects induced by peripheral inflammatory challenges, particularly during aging, derive from pre-clinical studies on rodents (Cunningham and Maclullich, 2013). As shown in the literature, aging is associated with an exaggerated response to peripheral inflammatory challenges together with behavioral and cognitive deficits (Hoogland et al., 2015; Schreuder et al., 2017). Indeed, in neurodegenerative disorders as well as in normal aging, microglia cells lose their supportive role in neuroplasticity and undertake a primed over-reactive phenotype promoting cognitive decline and synaptic dysfunction (Godbout and Johnson, 2006; Maclullich et al., 2008; Teeling and Perry, 2009). A recent gene expression profiling of microglia showed that aging is associated with over-expression of immune-related genes with an intermediate signature between acute and primed microglial genes (Holtman et al., 2015). Translocator protein (TSPO) has been shown to be a reliable biomarker of microglia activation in a large number of studies of neuro-inflammation by pre-clinical imaging (Liu et al., 2014). During brain injury or inflammatory insults, TSPO is overexpressed in activated microglia cells. For this reason, TSPO ligands for PET imaging have been extensively used for the in vivo monitoring of microglia activation/macrophage infiltration in different neuropsychiatric conditions as well as in neurodegenerative and neuro-inflammatory animal models (Liu et al., 2014). INTRODUCTION Currently, not only is there a lack of studies on the inflammatory responses in males and females but also studies considering both sex and age remain limited. For this reason, the main goal of this study was to test whether sex and age influence the early brain response to an acute peripheral inflammatory challenge. This experimental setting could reproduce the delirium syndrome, a transient and serious neurocognitive disorder characterized by an acute onset and fluctuating course (Inouye et al., 2014) To this aim, we investigated the effect of a single intraperitoneal LPS administration in male and female, adult and aged mice. The association between genes regulating monocytes or microglial response with neurodegenerative disorders also supports the major role that neuroinflammation exerts in cognitive dysfunction. An example of this is the Triggering Receptor Expressed on Myeloid (TREM), a key component of innate and adaptive immunity, which is expressed by a variety of innate cells of the myeloid lineage including neutrophils, monocytes, osteoclasts, macrophages, dendritic cells and microglia. In particular, TREM2 has been shown to bind to poly-anionic ligands such as bacterial LPS and phospholipids (Wang et al., 2015). Upon ligand binding, TREM2 signals intracellularly through the adaptor protein DAP12, eventually regulating different cellular functions like phagocytosis, cytokine production, proliferation and survival (Thankam et al., 2016). Genetic studies have identified TREM2 variants that are associated with an increased risk of Alzheimer’s disease (AD; Zheng et al., 2018). Another protein of potential interest is the TREM cells Like 2 (TREML2 also named TLT2). TREML2 is upregulated on B cells, neutrophils and macrophages during inflammation, and recent data suggest a potential modulatory role in pro-inflammatory responses (Thomas et al., 2016). Indeed, a missense variant of TREML2 (rs3747742) has been associated with a reduced susceptibility to develop INTRODUCTION The activation of the peripheral immune system is reflected by a pro-inflammatory milieu in the central nervous system (CNS; Norden and Godbout, 2013). The mechanisms of neuroinflammation are the subject of an intense pre-clinical research effort. However, most studies focus only on male animals, as preferred sex. A recent systematic review showed that only The activation of the peripheral immune system is reflected by a pro-inflammatory milieu in the central nervous system (CNS; Norden and Godbout, 2013). The mechanisms of neuroinflammation are the subject of an intense pre-clinical research effort. However, most studies focus only on male animals, as preferred sex. A recent systematic review showed that only November 2019 \| Volume 11 \| Article 299 Frontiers in Aging Neuroscience \| www.frontiersin.org Age and Sex Inﬂuence Neuroinﬂammation Murtaj et al. 3 out of 51 publications included both male and female mice or rats, and only a few of them focused on aged animals (Hoogland et al., 2015). This issue is relevant since ample evidence support sex-related differences in age-dependent neurodegenerative disorders (Bouman et al., 2005; Marriott and Huet-Hudson, 2006; Azad et al., 2007; Ycaza Herrera and Mather, 2015; Vom Steeg et al., 2016). Precise regulation of the immune responses maintains brain tissue homeostasis, while a chronic inflammatory state may influence the loss of neuronal function and plasticity (Besedovsky and Rey, 2008). In the presence of immune senescence, systemic inflammation represents a major precipitating factor for cognitive disorders (Perry, 2004; Costantini et al., 2018). 3 out of 51 publications included both male and female mice or rats, and only a few of them focused on aged animals (Hoogland et al., 2015). This issue is relevant since ample evidence support sex-related differences in age-dependent neurodegenerative disorders (Bouman et al., 2005; Marriott and Huet-Hudson, 2006; Azad et al., 2007; Ycaza Herrera and Mather, 2015; Vom Steeg et al., 2016). Precise regulation of the immune responses maintains brain tissue homeostasis, while a chronic inflammatory state may influence the loss of neuronal function and plasticity (Besedovsky and Rey, 2008). In the presence of immune senescence, systemic inflammation represents a major precipitating factor for cognitive disorders (Perry, 2004; Costantini et al., 2018). AD (Benitez et al., 2014; Bhattacharjee et al., 2014; Zhao and Lukiw, 2015). Females have a higher prevalence of AD compared to males, thus sex is included among the risk factors for dementia (McCarthy et al., 2012). Using in vivo imaging, Mosconi et al. Animals and LPS Challenge The 2−∆∆CT method was used to calculate the relative changes in gene expression in LPS-treated relative to vehicle-treated mice. The two-way ANOVA following by Tukey post hoc test was used to evaluate statistical significance between LPS-treated groups while parametric unpaired Student’s t-test were used to compare LPS-treated to control group. Genes and relative primer sets used are listed in Table 2. homogenized in the supplied homogenization buffer and stored at −80◦C. Total RNA was extracted with the Promega Maxwell® 16 LEV simplyRNA kit using the Maxwell® 16 Instrument (Promega). cDNA synthesis was performed using 250 ng of total RNA with the ThermoScript RT-PCR System (Invitrogen) and Random Primers (Promega) in a final volume of 20 µl according to the manufacturer’s instructions. The cDNA from each sample was used to perform RT-qPCR using LightCycler 480 SYBR Green I Master Mix (Roche) on the LightCycler 480 Instrument (Roche). β-actin was used as housekeeping gene for sample normalization. All analyses were performed in triplicate. The 2−∆∆CT method was used to calculate the relative changes in gene expression in LPS-treated relative to vehicle-treated mice. The two-way ANOVA following by Tukey post hoc test was used to evaluate statistical significance between LPS-treated groups while parametric unpaired Student’s t-test were used to compare LPS-treated to control group. Genes and relative primer sets used are listed in Table 2. maintained until reaching the age of 17–18 months (aged) and were further processed. A total number of 76 mice were included in the study. For each experimental task, adult and aged mice of both sexes were randomly assigned to LPS or vehicle (saline) treatment as reported in Table 1 and in the diagram below (Figure 1). Animals were intraperitoneally injected with 0.63 mg/kg of LPS (Lipopolysaccharides of Escherichia coli, serotype 055:B5; Sigma-Aldrich) freshly dissolved in sterile saline prior to injection, or vehicle (2 mg/5 ml saline), and neuro-inflammatory responses were evaluated 6 h later (Biesmans et al., 2013). Ex vivo Binding of [18F]-VC701 to TSPO Protein There were eight mice per group and treatment was dedicated to ex vivo imaging studies. Microglia activation was evaluated using the [18F]-labeled VC701, a TSPO radio ligand developed in our facility (Di Grigoli et al., 2015; Belloli et al., 2018). This radiopharmaceutical binds to 18 kDa TSPO. In this study, we used the novel TSPO tracer [18F]-VC701 that allows to obtain adequate signal to noise ratios for the measurements of TSPO binding in mouse brain (Belloli et al., 2018). Binding of [18F]- VC701 was evaluated ex vivo at 6 h after the intraperitoneal administration of LPS or saline. Animals were injected with 4.55 ± 1.85 MBq (specific activity: 3.17 ± 1.4 Ci/µMole) of [18F]-VC701 through the tail vein and sacrificed under light anesthesia 120 min later. Blood samples were collected and counted in a gamma counter (LKB Compugamma CS1282, Wallac). Brains were rapidly removed and divided into two hemispheres. Cortex, hippocampus and cerebellum were collected and placed in pre-weighed tubes. Radioactivity concentration was counted while the tissue used for RT-PCR was stored at −80◦C for transcript measurement. After counting, tissue and blood radioactivity concentration were corrected for the half-life of [18F] (108.9 min) and expressed as percentage of the injected dose per gram of tissue (%I.D./g). To avoid a potential confounding effect due to differences in blood circulating levels, binding data were also normalized to blood uptake value. Animals and LPS Challenge Animals and LPS Challenge Adult (2 months) and aged (17–18 months) non-breeding male and female C57Bl/6J mice were purchased from Charles River. Animals were maintained and handled in compliance with our institutional guidelines for the care and use of experimental animals (IACUC) and the national law for animals used in research (Prot. N. SK552/2012 D.lsg. 116/1992 and N. 722/2016-PR D.lsg. 26/2016). Mice were housed in the San Raffaele Hospital animal facility, maintained in 12/12 h light/dark cycle with access ad libitum to food and water. Following 1 week of acclimation, one group of 2 months old mice (adult) were processed according to the experimental tasks described below, whereas another group of mice was Frontiers in Aging Neuroscience \| www.frontiersin.org November 2019 \| Volume 11 \| Article 299 Frontiers in Aging Neuroscience \| www.frontiersin.org 2 Age and Sex Inﬂuence Neuroinﬂammation Murtaj et al. TABLE 1 \| Age, body weight, sample size and treatment dosage of each experimental group. Experimental group Age Body weight (g) [18F]-VC701 binding (n) RT-PCR (n) IHC (n) Dosage (LPS/vehicle) Adult males + LPS 2 months 26.9 ± 2.7 8 5/8 3 0.63 mg/kg Adult males vehicle 2 months 26.1 ± 1.6 8 5/8 3 50 µl saline Adult females + LPS 2 months 18.3 ± 3.6 8 4/8 3 0.63 mg/kg Adult females vehicle 2 months 19.6 ± 1.1 8 5/8 3 30 µl saline Aged males + LPS 17/18 months 35.5 ± 2.5 8 5/8 3 0.63 mg/kg Aged male vehicle 17/18 months 36.4 ± 3.3 8 5/8 3 50 µl saline Aged females + LPS 17/18 months 31.1 ± 4.2 8 5/8 3 0.63 mg/kg Aged females vehicle 17/18 months 34.2 ± 6.6 8 5/8 3 50 µl saline homogenized in the supplied homogenization buffer and stored at −80◦C. Total RNA was extracted with the Promega Maxwell® 16 LEV simplyRNA kit using the Maxwell® 16 Instrument (Promega). cDNA synthesis was performed using 250 ng of total RNA with the ThermoScript RT-PCR System (Invitrogen) and Random Primers (Promega) in a final volume of 20 µl according to the manufacturer’s instructions. The cDNA from each sample was used to perform RT-qPCR using LightCycler 480 SYBR Green I Master Mix (Roche) on the LightCycler 480 Instrument (Roche). β-actin was used as housekeeping gene for sample normalization. All analyses were performed in triplicate. November 2019 \| Volume 11 \| Article 299 Immunohistochemistry (IHC) Immunohistochemistry (IHC) and histopathologic analysis were performed in distinct adult and aged female mice (n = 3 per group and treatment). Mice were sacrificed 6 h after LPS or vehicle challenge and perfused with PBS followed by 4% paraformaldehyde (PFA). Brains were removed, post-fixed in PFA 4% for 24 h, cryo-protected overnight in 20% sucrose and 10 µm sections were cut on a cryostat for histological TABLE 2 \| Genes and relative primer list used in gene expression analysis. Name Primer sequence (5′-3′) β-actin F 5′-gactcctatgtgggtgacgagg-3′ β-actin R 5′ catggctggggtgttgaaggtc-3′ Trem2 F 5′-gcacctccaggaatcaagag-3′ Trem2 R 5′-gggtccagtgaggatctgaa-3′ Trem2L F 5′-tggtggtggtgttgacatttcttcc-3′ Trem2L R 5′atccagggtttagcatagttgctgc-3′ Tspo F 5′-tcagcggctaccaacct-3′ Tspo R 5′-caggattcaggcatggtgat-3′ Il-1β F 5′-gcccatcctctgtgactcat-3′ Il-1β R 5′-aggccacaggtattttgtcg-3′ Tnf-α F 5′-cctgtagcccacgtcgtag-3′ Tnf-α R 5′-gggagtagacaaggtacaaccc-3′ Il4 F 5′-tcaacccccagctagttgtc-3′ Il4 R 5′-tgttcttcgttgctgtgagg-3′ Arg1 F 5′-ttgggtggatgctcacactg-3′ Arg1 R 5′-gtacacgatgtctttggcaga-3′ IL-6 F 5′-agttgccttcttgggactga-3′ IL-6 R 5′-tccacgatttcccagagaac-3′ Statistical Analysis y Statistical evaluation of [18F]-VC701 tracer uptake was carried out using two-way ANOVA for Multiple Comparisons with a Tukey post hoc correction with gender, aging and treatment as covariates. Data representing radio ligand uptake in each LPS-treated and vehicle group were analyzed through parametric unpaired Student’s t-test. For gene expression studies, data were normalized to housekeeping gene followed by a fold change evaluation comparing data to internal control group. Data derived from comparison of LPS-treated groups were statistically analyzed using two-way ANOVA for Multiple Comparisons with a Tukey post hoc correction, while for those reported on Supplementary Figures, data were statistically evaluated using parametric unpaired Student’s Peripheral Exposure to LPS Inﬂuences Sex- and Age-Dependent Microglia Activation TSPO expression in different brain areas of adult and aged untreated and LPS-treated male and female mice was evaluated by ex vivo binding of [18F]-VC701 to TSPO, 6 h after LPS/vehicle injection. LPS treatment induced a statistically significant increase (p < 0.05) of tracer’s uptake in the cortex and cerebellum of aged males, and in cortex and hippocampus of aged females (Supplementary Figure S1). Higher uptake of [18F]-VC701 was observed in the cortex of aged vs. adult LPS-treated females (Figure 2). This age-dependent effect was not observed in males. The percentage of tracer’s uptake in the cortex and hippocampal areas was significantly higher in LPS-treated aged females compared to age-matched vehicle (cortex 51.6%, p ≤0.017; hippocampus 86.4% p ≤0.010; cerebellum ns, 36.1%, data not shown). Tissue to blood ratio of tracer’s uptake in LPS-treated females, expressed as % of injected dose per gram of tissue (%I.D./g), is shown in Table 3. RNA Extraction and RT-qPCR Analysis RNA Extraction and RT-qPCR Analysis RT-PCR analysis was performed on five out of the eight animals/group that was used for the [18F]-VC701 experiments. One mouse in the adult female LPS-treated group was excluded due to insufficient mRNA availability. The brain areas (cortex, striatum, hippocampus and cerebellum) were dissected, November 2019 \| Volume 11 \| Article 299 Frontiers in Aging Neuroscience \| www.frontiersin.org 3 Murtaj et al. Age and Sex Inﬂuence Neuroinﬂammation FIGURE 1 \| Schematic diagram of the experimental design and readouts. \| Schematic diagram of the experimental design and readouts. t-test. Immunohistochemical analyses were performed using two-way ANOVA for Multiple Comparison with Tukey post hoc correction. analysis in bright-field microscopy, slices were stained using standard protocols for hematoxylin and eosin (using Mayer’s Hematoxylin, BioOptica #05-06002/L and Eosin, BioOptica #05-10002/L). Analyses were performed using the Prism V6.0 software (GraphPad Prism, San Diego, CA, USA). Statistical significance was accepted when ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. For IHC, slices were immuno-stained with polyclonal rabbit anti-Iba-1 antibody (Wako, #019-19741) and polyclonal rabbit anti-GFAP antibody (Novus Biologicals, #NB300-141). Any endogenous peroxidase activity was quenched by incubating the slices with 0.3% H2O2 in methanol for 10 min at RT. Both antibodies were used at a dilution of 1:1,000, incubated for 1 h at RT with EXPOSE rabbit specific HRP/DAB detection IHC kit (Abcam, #ab80437). DAB chromogen was than applied to each section for 5 min at RT and counterstained with Mayer’s hematoxylin, dehydrated and mounted with Eukitt (BioOptica, #09-00100). Positive and negative controls were run simultaneously. Slides were acquired with Aperio AT2 digital scanner at a magnification of 40× (Leica Biosystems). Region of interest (ROI) were drowned manually on half hemisphere for cortex, on the entire hippocampus, and on a selected region of the cerebellum. Percentage of positive cells were calculated using Aperio eSlide Manager (Leica Biosystems). From the data obtained (weak, medium and strong positive intensity), weak positive were not included in the analysis as considered as background. Sex and Age Effect on Transcript Levels of Markers of Microglia Activation Interestingly, we found an age- and sex-dependent effect on the expression of the neuro-inflammatory markers TREM2 and TREML2 after LPS administration. Our results showed that peripheral LPS injection induced a significant decrease of TREM2 transcript levels in all brain regions of adult males and females (Supplementary Figure S2D). Yet in aged males, LPS injection did not decrease TREM2 transcript levels, which were instead significantly decreased in all brain regions of aged females (Supplementary Figure S3D). When we specifically compared TREM2 transcript levels among LPS-treated mice, we found that they were significantly higher in aged males compared to aged females (p < 0.0002), while no differences were detected in adult mice of both sexes (Figure 4A). TREML2 transcript levels were significantly increased in the hippocampus of aged males compared to aged females (p < 0.01), and also in the cortex, although not reaching statistical significance (Figure 4B, Supplementary Figures S2E, S3E). Female Brain Shows a Higher Pro-inﬂammatory Response to LPS During Aging In order to confirm that the higher [18F]-VC701 uptake was associated with a pro-inflammatory response of microglia, we measured the levels of IL-1β, TNF-α, IL-6, IL-4 and Arg-1 transcripts in LPS-treated and untreated mice. In all brain regions, peripheral LPS injection induced neuroinflammation irrespective of sex and age, as shown by significantly increased November 2019 \| Volume 11 \| Article 299 Frontiers in Aging Neuroscience \| www.frontiersin.org 4 Murtaj et al. Age and Sex Inﬂuence Neuroinﬂammation TABLE 3 \| Tissue to blood [18F]-VC701 uptake ratio. Brain Region Tissue/blood ratio (%I.D./g; mean ± SD) LPS-treated adult females LPS-treated aged females Cortex 1.6 ± 0.6 2.52 ± 0.7 Hippocampus 2.31 ± 1.4 2.46 ± 0.9 Cerebellum 2.75 ± 1.3 3.75 ± 1.1 TABLE 3 \| Tissue to blood [18F]-VC701 uptake ratio. FIGURE 2 \| Translocator protein (TSPO) tracer uptake reveal microglial activation increased in aged female lipopolysaccharide (LPS)-treated animals. [18F]-VC701 brain tracer uptake in cortex (A); hippocampus (B) and cerebellum (C) on LPS-treated adult and aged male and female mice. Data were analyzed using two-way ANOVA analysis with Tukey post hoc and are expressed as percentage (%) of injected dose per gram of tissue to blood ratio, mean ± SD, eight mice per group; Mouse brain stained slices (cortex, hippocampus and cerebellum) derived from immunohistochemistry (IHC) studies. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. IL-6 in the cerebellum were detected in aged females compared to aged males (Figures 3A,C). TNF-α transcript levels were increased in the cortex and hippocampus of aged compared to adult females, although the differences were not statistically significant (Figure 3B). Microglia Activation and Astrogliosis in Aged Female Animals FIGURE 2 \| Translocator protein (TSPO) tracer uptake reveal microglial activation increased in aged female lipopolysaccharide (LPS)-treated animals. [18F]-VC701 brain tracer uptake in cortex (A); hippocampus (B) and cerebellum (C) on LPS-treated adult and aged male and female mice. Data were analyzed using two-way ANOVA analysis with Tukey post hoc and are expressed as percentage (%) of injected dose per gram of tissue to blood ratio, mean ± SD, eight mice per group; Mouse brain stained slices (cortex, hippocampus and cerebellum) derived from immunohistochemistry (IHC) studies. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. As major differences in the neuro-inflammatory response to LPS were observed in females, we further evaluated glia activation in females by IHC. In all LPS-treated females, H&E staining showed no sign of neuronal loss or morphological changes in the three brain areas examined (cortex, cerebellum and hippocampus, data not shown). In LPS-treated aged females an increased percentage of Iba-1 positive cells were detected when compared to LPS-treated adult females, and vehicle- treated adult and aged females, which is indicative of a higher inflammatory status (Figure 5A, cortex). Microglia activation was suggested by cell morphology in the cerebral and cerebellar cortex of LPS-treated aged vs. LPS-treated adult females and vehicle-treated females (Figures 5A–C, cortex and cerebellum). In the hippocampus both LPS and vehicle-treated aged females showed a higher degree of Iba-1 immunoreactivity in comparison with adult females reaching statistical significance in vehicle-treated aged vs. vehicle-treated adult mice (Figure 5C, hippocampus). transcript levels of IL-1β, IL-6 and TNF-α (Supplementary Figures S2A–C, S3A–C). Arg-1 and IL-4 transcripts were negligibly expressed in both LPS-treated and untreated mice (data not shown). transcript levels of IL-1β, IL-6 and TNF-α (Supplementary Figures S2A–C, S3A–C). Arg-1 and IL-4 transcripts were negligibly expressed in both LPS-treated and untreated mice (data not shown). In agreement with [18F]-VC701 uptake results, in female but not in male brain, age affected levels of the pro-inflammatory cytokines IL-1β, TNF-α and IL-6 in response to the LPS challenge. Aged females compared to adult females showed increased levels of IL-1β and IL-6 in all areas, which was statistically significant in the cortex for IL-1β, and in both cortex and cerebellum for IL-6 (Figures 3A,C). No age-dependent effects on IL-1β, TNF-α and IL-6 transcript levels were detected in males. Microglia Activation and Astrogliosis in Aged Female Animals Increased IL-1β transcript levels in the cortex and Astroglial cells showed an increase in GFAP immunoreactivity in aged compared to adult females (Figures 6A,B). This observation was consistent in the cortex, Astroglial cells showed an increase in GFAP immunoreactivity in aged compared to adult females (Figures 6A,B). This observation was consistent in the cortex, Frontiers in Aging Neuroscience \| www.frontiersin.org November 2019 \| Volume 11 \| Article 299 5 Murtaj et al. Age and Sex Inﬂuence Neuroinﬂammation FIGURE 3 \| Increased expression levels of pro-inﬂammatory cytokines in aged LPS treated mice. Gene expression analysis of IL-1β (A), TNF-α (B) and IL-6 (C) by RT-qPCR in cortex, hippocampus and cerebellum of all LPS-treated animals. All the values were normalized to mouse β-actin and expressed as fold change referred to internal control group. Bars represent the average of triplicate measurements and error bars represent ± SEM, ﬁve animals per group. Statistical analysis was performed using two-way ANOVA following by Tukey post hoc test of the relative mRNA expression of LPS-treated mice previously normalized to internal control group. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. FIGURE 3 \| Increased expression levels of pro-inﬂammatory cytokines in aged LPS treated mice. Gene expression analysis of IL-1β (A), TNF-α (B) and IL-6 (C) by RT-qPCR in cortex, hippocampus and cerebellum of all LPS-treated animals. All the values were normalized to mouse β-actin and expressed as fold change referred to internal control group. Bars represent the average of triplicate measurements and error bars represent ± SEM, ﬁve animals per group. Statistical analysis was performed using two-way ANOVA following by Tukey post hoc test of the relative mRNA expression of LPS-treated mice previously normalized to internal control group. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. FIGURE 3 \| Increased expression levels of pro-inﬂammatory cytokines in aged LPS treated mice. Gene expression analysis of IL-1β (A), TNF-α (B) and IL-6 (C) by RT-qPCR in cortex, hippocampus and cerebellum of all LPS-treated animals. All the values were normalized to mouse β-actin and expressed as fold change referred to internal control group. Bars represent the average of triplicate measurements and error bars represent ± SEM, ﬁve animals per group. Statistical analysis was performed using two-way ANOVA following by Tukey post hoc test of the relative mRNA expression of LPS-treated mice previously normalized to internal control group. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. Microglia Activation and Astrogliosis in Aged Female Animals in LPS-treated aged females for Iba-1 and in vehicle and LPS-treated aged females for GFAP (data not shown). cerebellum and hippocampus of aged females where astrocytes also showed hyperplasia of cytoplasm and of cellular processes resembling an activated phenotype. Quantification analysis showed a significant higher percentage of GFAP positive cells in cortex and hippocampus of aged compared to adult females. A similar trend was observed also in the cerebellar region (Figure 6C). Furthermore, in the sections where olfactory bulb was present, a general increase in immunoreactivity for both Iba-1 and GFAP was observed, again more evident DISCUSSION In this study, we demonstrate for the first time that both age and sex influence an early neuro-inflammatory response after an acute mild peripheral LPS challenge in mice. In comparison to control conditions, LPS-treated aged male and female mice November 2019 \| Volume 11 \| Article 299 Frontiers in Aging Neuroscience \| www.frontiersin.org 6 Murtaj et al. Age and Sex Inﬂuence Neuroinﬂammation FIGURE 4 \| Triggering receptor expressed on myeloid cells like 2 (TREM2) is differentially expressed in aged LPS treated mice. Gene expression analysis of TREM2 (A), TREML2 (B), two immunomodulatory markers by RT-qPCR in cortex, hippocampus and cerebellum of all LPS-treated animals. All the values were normalized to mouse β-actin and expressed as fold change referred to internal control group. Bars represent the average of triplicate measurements and error bars represent ± SEM, ﬁve animals per group. Statistical analysis was performed using two-way ANOVA following by Tukey post hoc test of the relative mRNA expression of LPS-treated mice previously normalized to internal control group. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. showed an increase [18F]-VC701 binding indicative of higher expression of TSPO. A prominent effect was observed in LPS-treated aged compared to adult females. [18F]-VC701 is l l f h h k (Di Grigoli et al., 2015). TSPO’s main role is to transport cholesterol across the outer mitochondrial membrane. While TSPO is negligibly expressed in the normal brain parenchyma, its l d h FIGURE 4 \| Triggering receptor expressed on myeloid cells like 2 (TREM2) is differentially expressed in aged LPS treated mice. Gene expression analysis of TREM2 (A), TREML2 (B), two immunomodulatory markers by RT-qPCR in cortex, hippocampus and cerebellum of all LPS-treated animals. All the values were normalized to mouse β-actin and expressed as fold change referred to internal control group. Bars represent the average of triplicate measurements and error bars represent ± SEM, ﬁve animals per group. Statistical analysis was performed using two-way ANOVA following by Tukey post hoc test of the relative mRNA expression of LPS-treated mice previously normalized to internal control group. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. showed an increase [18F]-VC701 binding indicative of higher expression of TSPO. A prominent effect was observed in LPS-treated aged compared to adult females. [18F]-VC701 is a structural analog of PK11195, which targets 18 kDa TSPO (Di Grigoli et al., 2015). DISCUSSION TSPO’s main role is to transport cholesterol across the outer mitochondrial membrane. While TSPO is negligibly expressed in the normal brain parenchyma, its expression increases upon microglia activation and macrophage November 2019 \| Volume 11 \| Article 299 Frontiers in Aging Neuroscience \| www.frontiersin.org Frontiers in Aging Neuroscience \| www.frontiersin.org 7 Age and Sex Inﬂuence Neuroinﬂammation Murtaj et al. were sexually differentiated in the adult brain, as male mice carried a phenotype more poised to inflammatory reactions than female microglia. Moreover, in females, microglia displayed neuroprotective features in an acute pre-clinical model of ischemic insult (Villa et al., 2018). Gene sexual dimorphism has also been reported in humans by Berchtold et al. (2008). This study shows that males display a greater number of gene expression changes (mainly downregulation) during the transition to the sixth and seventh decade of life while females show more prominent gene expression changes later in life, during the eighth and ninth decade of life. Moreover, increased immune activation was greater in the female brain, as shown by a higher percentage of up-regulated genes involved in the immune response and inflammation (34% in males and 50% in females; Berchtold et al., 2008). FIGURE 5 \| Increased IBA1 immunoreactivity and changes in glia morphology in brain areas of LPS-treated aged vs. LPS-treated adult females. (A) Immunostaining of Iba-1+ cells in cerebral cortex, hippocampus and cerebellum from one representative vehicle and one representative LPS-treated adult female mouse. (B) Immunostaining of Iba-1+ cells in cerebral cortex, hippocampus and cerebellum from one representative vehicle and one representative LPS-treated aged female mouse. (C) Quantiﬁcation of the percentage of Iba-1+ cells in cortex, hippocampus and cerebellum of all female groups used in the study. Data are expressed as mean ± SEM of selected region of interest (ROI) performed on three animals per group, scale bar = 100 µm. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. To better understand the nature of the microglia response, we evaluated the effect of the peripheral LPS challenge on transcription of a few inflammatory mediators: IL-1β, TNF-α, IL-6, Arg1 and IL-4. We showed that LPS induced a pro-inflammatory reaction in the brain of male and female mice as indicated by the up-regulation of IL-1β, TNF-α and IL-6 gene expression. DISCUSSION However, in agreement with [18F]-VC701 studies, the effect of LPS on pro-inflammatory cytokine transcript levels was significantly increased in aged compared to adult females, as well as in aged females compared to aged males. As a major age effect was observed in females, we further analyzed microglia and astrocyte status by IHC in all brain regions of adult and aged females. In agreement with TSPO expression and pro-inflammatory cytokine transcript levels, we found an increased expression of microglia and astrocyte markers in LPS-treated and vehicle-treated aged compared to adult females, with some regional differences. Increased Iba-1 expression was present particularly in the cerebral cortex of LPS-treated aged animals, whereas GFAP immunoreactivity was higher in aged females in all the cerebral areas examined, regardless of LPS administration. Finally, the higher Iba-1 and GFAP signals detected in aged females was associated with a different cellular morphology. Altogether, our results highlight the acute onset of a higher neuro-inflammatory response in aged females. A similar response was also reported in female 3xTg-AD mice, where neuroinflammation was associated with progressive cognitive decline that increased as a function of age (Belfiore et al., 2019). FIGURE 5 \| Increased IBA1 immunoreactivity and changes in glia morphology in brain areas of LPS-treated aged vs. LPS-treated adult FIGURE 5 \| Increased IBA1 immunoreactivity and changes in glia morphology in brain areas of LPS-treated aged vs. LPS-treated adult females. (A) Immunostaining of Iba-1+ cells in cerebral cortex, hippocampus and cerebellum from one representative vehicle and one representative LPS-treated adult female mouse. (B) Immunostaining of Iba-1+ cells in cerebral cortex, hippocampus and cerebellum from one representative vehicle and one representative LPS-treated aged female mouse. (C) Quantiﬁcation of the percentage of Iba-1+ cells in cortex, hippocampus and cerebellum of all female groups used in the study. Data are expressed as mean ± SEM of selected region of interest (ROI) performed on three animals per group, scale bar = 100 µm. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. GURE 5 \| Increased IBA1 immunoreactivity and changes in glia FIGURE 5 \| Increased IBA1 immunoreactivity and changes in glia morphology in brain areas of LPS-treated aged vs. LPS-treated adult females. (A) Immunostaining of Iba-1+ cells in cerebral cortex, hippocampus and cerebellum from one representative vehicle and one representative LPS-treated adult female mouse. DISCUSSION (B) Immunostaining of Iba-1+ cells in cerebral cortex, hippocampus and cerebellum from one representative vehicle and one representative LPS-treated aged female mouse. (C) Quantiﬁcation of the percentage of Iba-1+ cells in cortex, hippocampus and cerebellum of all female groups used in the study. Data are expressed as mean ± SEM of selected region of interest (ROI) performed on three animals per group, scale bar = 100 µm. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. Microglia, as the primary source of pro-inflammatory cytokines, are pivotal mediators of neuroinflammation (Colonna and Butovsky, 2017). The amplitude and duration of the context-dependent activation of microglia are regulated by many pattern-recognition receptors, and immune receptors that deliver either activating or inhibitory signals. TREM2 and TREML2 are two microglia/monocyte regulators with potential and opposite roles in neurodegenerative disorders such as AD (Zsido et al., 2017). We show that acute peripheral administration of LPS induced an opposite modulation of TREM2 and TREML2 transcripts. In all groups analyzed, except aged male mice, we showed that LPS reduced TREM2 while increasing TREML2 transcript levels. TREM2 regulates critical functions of microglia including inhibition of pro-inflammatory responses infiltration (Betlazar et al., 2018). Thus TSPO up-regulation has been mainly associated with a pro-inflammatory status of microglia (Bonsack et al., 2016). PET-TSPO has been successfully applied to image the neuro-inflammatory reaction to LPS administration in the brain of human subjects, showing an increased binding of the TSPO radiotracer [11C]-PBR28 in all brain regions (Sandiego et al., 2015). Co-localization of TSPO with CD206-positive alternatively-activated microglia has also been reported (Liu et al., 2015). Our results, although obtained from analysis of whole tissue are in agreement with transcriptomic data of purified microglia (Villa et al., 2018). Villa et al. (2018) demonstrated that microglia cells Frontiers in Aging Neuroscience \| www.frontiersin.org Frontiers in Aging Neuroscience \| www.frontiersin.org November 2019 \| Volume 11 \| Article 299 8 Age and Sex Inﬂuence Neuroinﬂammation Murtaj et al. FIGURE 6 \| Increased astrocytosis and changes in glia morphology in brain areas of LPS-treated and vehicle-treated aged females. (A) Immunostaining of GFAP+ cell in cerebral cortex, hippocampus and cerebellum from one representative vehicle and one representative LPS-treated adult female mouse. (B) Immunostaining of GFAP+ astrocyte cells in cerebral cortex, hippocampus and cerebellum from one representative vehicle and one representative LPS-treated aged female mouse. DISCUSSION (C) Quantiﬁcation of percentage of GFAP+ cells in cortex, hippocampus and cerebellum of all female groups used in the study. Data are expressed as mean ± SEM of selected ROI performed on three animals per group, scale bar = 100 µm. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. 2017). In particular, reduction of TREM2 was associated with a decreased microglia proliferation, an effect that was absent when TREML2 was knocked down. During aging, microglia undergo cellular senescence losing regenerative and trophic functions (Mecca et al., 2018). According to Colonna and Wang (2016) TREM2 protects the aged brain from insults. Indeed, carrying a loss of function mutations in the TREM2 gene represents an important risk factor for dementia (Gao et al., 2017). In vitro experiments demonstrated that, silencing of TREM2 exacerbated LPS-induced pro-inflammatory response in BV2 microglia (Zhong et al., 2015), In contrast, overexpression of TREM2 lowered the pro-inflammatory response, thus supporting the hypothesis that TREM2 expression modulates the acute and transient pro-inflammatory responses induced by LPS (Zhong et al., 2015). Thus, the higher TREM2 expression observed in aged male mice might represent a strategy to counterbalance the pro-inflammatory milieu of aging, partially sustained by activation of signaling pathways mediated by TREML2, which likely activates apoptosis and modulates microglial functions (de Freitas et al., 2012; Zheng et al., 2016). In aged female brains, we observed up-regulation of reactive GFAP-positive astroglia in all the regions examined independently from LPS administration. Although GFAP expression is limited to only a fraction of astrocytes with a substantial regional heterogeneity, its expression has been associated with reactive astrogliosis (Verkhratsky and Nedergaard, 2018). During aging, high levels of GFAP expression were identified as ‘‘mild to moderate astrogliosis’’ while the presence of cellular hypertrophy in addition to GFAP increase appears to be associated to ‘‘severe diffuse astrogliosis’’ (Cohen and Torres, 2019). Matias et al. (2019) reported that the astrocyte response to brain aging showed intra-regional heterogeneity (Matias et al., 2019): astrocytes in the hippocampal region presented an age-dependent hypertrophy, similar to the astrocyte morphology detected in our study, which may contribute to synapse loss and neuroinflammation. FIGURE 6 \| Increased astrocytosis and changes in glia morphology in brain areas of LPS-treated and vehicle-treated aged females. (A) Immunostaining of GFAP+ cell in cerebral cortex, hippocampus and cerebellum from one representative vehicle and one representative LPS-treated adult female mouse. ACKNOWLEDGMENTS We thank Pasquale Simonelli for technical support with animal preparation and ex vivo binding experiments. We thank Pasquale Simonelli for technical support with animal preparation and ex vivo binding experiments. CONCLUSION This work was supported by grants from: The Italian Ministry of Education, University and Research (Ministero dell’Istruzione, dell’Universitá e della Ricerca; MIUR) Project ‘‘Identification, validation and commercial development of new diagnostic and prognostic biomarkers for complex trait diseases (IVASCOMAR, Prot. CTN01 00177 165430)’’. In conclusion, the results of this study show that the neuro- inflammatory response of a mouse’s brain to an acute peripheral LPS challenge is sex- and age-dependent, and is likely to involve multiple cellular and molecular regulators of neuronal functions. In conclusion, the results of this study show that the neuro- inflammatory response of a mouse’s brain to an acute peripheral LPS challenge is sex- and age-dependent, and is likely to involve multiple cellular and molecular regulators of neuronal functions. Our results might set the basis for further studies aimed at characterizing sex-related targets involved in the modulation of the neuro-inflammatory response in aging. This knowledge could be relevant for the treatment of conditions such as delirium and dementia. Our results might set the basis for further studies aimed at characterizing sex-related targets involved in the modulation of the neuro-inflammatory response in aging. This knowledge could be relevant for the treatment of conditions such as delirium and dementia. DATA AVAILABILITY STATEMENT The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fnagi.2019.002 99/full#supplementary-material. The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher. DISCUSSION (B) Immunostaining of GFAP+ astrocyte cells in cerebral cortex, hippocampus and cerebellum from one representative vehicle and one representative LPS-treated aged female mouse. (C) Quantiﬁcation of percentage of GFAP+ cells in cortex, hippocampus and cerebellum of all female groups used in the study. Data are expressed as mean ± SEM of selected ROI performed on three animals per group, scale bar = 100 µm. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. Interestingly, increased levels of neuro-inflammatory markers, including GFAP, are present in the brain of AD patients. Recently Barroeta-Espar et al. (2019) demonstrated reduced levels of GFAP and a different pattern of cytokines expression in subjects resilient to AD pathology. This selected population of aged subjects harbor plaque and tangle loads that in some cases are equivalent to those found in AD, although in absence of the typical patterns of neuronal/synaptic loss seen in the pathology. These findings suggest that down-regulation of neuroinflammation is one of the differential traits of human brain resilience to AD pathology, a mechanism that might be less efficient in female brain. Implications of our findings may impact the development of clinical phenotypes such as delirium and dementia. Delirium is one of the most common neuropsychiatric conditions among older people, which is known to develop as an acute consequence of peripheral infection or inflammation originating outside the brain (Cerejeira et al., 2010; Franceschi and Campisi, 2014; Bu et al., 2015; Morandi et al., 2018). and stimulation of phagocytosis of apoptotic neurons (Piccio et al., 2008). TREM2-transduced myeloid precursors mediate nervous tissue debris clearance and facilitate recovery in an animal model of multiple sclerosis (Takahashi et al., 2005, 2007). Moreover, TREM2 binds poly-anionic ligands including LPS (Wang et al., 2015). Previous findings showed that LPS stimulation suppressed TREM2 levels in microglia, at the same time inducing pro-inflammatory cytokines secretion (Zheng et al., 2016). Peripheral LPS challenge (at a similar dose to that used in our study) induced up-regulation of TREML2 and down-regulation of TREM2 levels in microglia, and increase of pro-inflammatory cytokines (Zheng et al., 2016; Zhang et al., Frontiers in Aging Neuroscience \| www.frontiersin.org November 2019 \| Volume 11 \| Article 299 Frontiers in Aging Neuroscience \| www.frontiersin.org 9 Age and Sex Inﬂuence Neuroinﬂammation Murtaj et al. The higher the level of frailty, the higher the likelihood of developing delirium in reaction to an inflammatory stimulus (Bellelli et al., 2017; Persico et al., 2018). ETHICS STATEMENT The animal study was approved by our Institutional Guidelines for the Care and Use of Experimental Animals (IACUC) and the National Law for animals used in research (Prot. N. SK552/2012 D.lsg. 116/1992 and N. 722/2016-PR D.lsg. 26/2016). Future studies should explore chronic neuroinflammation and morphological-functional changes in glial cell upon repetitive systemic insult or an acute severe neural event. As the cortex represents in our study the most important region influenced by sex and age, we will further explore gene modulation of specific pathways trough transcriptomic analysis of cortical glia in adult and aged female and male mice. Furthermore, specific behavioral phenotypes such as attention and active inhibitory processes associated with cognitive frailty will be investigated. AUTHOR CONTRIBUTIONS VMu: data generation, collection, analysis and interpretation, and manuscript drafting. SB and GG: data generation and collection. MP: support in qRT-PCR data generation and analysis. EB, VR-M, and PM: support in IHC data analysis and interpretation. AC, VMa, and CM: production and validation of the tracer [18F]-VC701. GB: critical review of the manuscript. 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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. Wang, Y., Cella, M., Mallinson, K., Ulrich, J. D., Young, K. L., Robinette, M. L., et al. (2015). TREM2 lipid sensing sustains the microglial response in an Alzheimer’s disease model. Cell 160, 1061–1071. doi: 10.1016/j.cell.2015.01.049 Ycaza Herrera, A., and Mather, M. (2015). Actions and interactions of estradiol and glucocorticoids in cognition and the brain: implications for aging women. Neurosci. Biobehav. Rev. 55, 36–52. doi: 10.1016/j.neubiorev.2015.04.005 Wang, Y., Cella, M., Mallinson, K., Ulrich, J. D., Young, K. L., Robinette, M. L., et al. (2015). TREM2 lipid sensing sustains the microglial response in an Alzheimer’s disease model. 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https://openalex.org/W4372353101	https://periodicos.institutoiesa.com/index.php/avancoseolhares/article/download/33/22	Portuguese	null	UM ESTUDO SOBRE A GESTÃO DE PESSOAS NA PANDEMIA DO COVID-19	Avanços & Olhares	2,023	cc-by	6,608	1 Doutoranda em Administração de Empresas pela Falcultad Interamerica de Ciencias Sociales; Mestre em Gestão Empresarial pela Universidade de Lusófona. Graduada em Ciências Econômicas pela Universidade Federal de Viçosa. Pós-graduação em Gestão e Marketing do Turismo pela Universidade de Brasília e Direito Público pela Faculdade Projeção. Empresária na área de eventos com vasta experiência no planejamento execução de eventos nos eixos de Brasília, Goiânia e Anápolis e Professora Universitária - Centro Universitário Planalto do Distrito Federal (UNIPLAN). Resumo A gestão de pessoas precisa elaborar estratégias para alinhar os seus processos e competências, as pessoas são o principal ativo, sendo necessário, portanto, que as organizações sejam mais conscientes e atentas aos seus funcionários. Diante da pandemia, os gestores tiveram que criar táticas para lidar com esse cenário adverso, o processo tornou-se mais competitivo e extremamente agressivo, apresentou uma nova realidade para as empresas, decidindo a sua continuação no mercado. A metodologia da pesquisa bibliográfica consiste na utilização de plataformas científicas, tais como o Scielo, Google Scholar, entre outras. A fim de ampliar as fontes de pesquisa e enriquecer a análise das informações obtidas sobre a temática escolhida. Pode-se perceber a mudança que ocorreu em relação ao trabalho remoto, na realocação dinâmica de recursos, na aceleração da digitalização e na automação para atender às necessidades individuais e organizacionais em constante evolução. Para iniciar a organização do futuro, a contratação de novos colaboradores e da liderança devem ser dentro de uma nova proposta criativa e flexível. O novo conceito empresarial precisa ser ousado, buscar talentos, ter uma rede de equipe consolidada, além disso deve criar um ecossistema na organização favorável a todos os envolvidos. Palavras-chave: Gestão de pessoas. Recurso Humano. Pandemia Palavras-chave: Gestão de pessoas. Recurso Humano. Pandemia UM ESTUDO SOBRE A GESTÃO DE PESSOAS NA PANDEMIA DO COVID-19 Mirizete de Jesus dos Santos1 Katia Mary Casara Pellin2 2 Doutoranda em Administração de Empresas Faculdade de Ciências Sociais Interamericana. Mestre em Gestão Empresarial pela Universidade Lusófona. Graduada em Administração pela Faculdades Integradas – FIRB. Pós- graduação em Administração Pública Instituição União Educacional do Norte – UNINORTE e Licenciatura Pedagogia Faculdade de Teologia. Avanços & Olhares, ISSN: 2595-2579, Nº 9, Barra do Garças - MT Avanços & Olhares, ISSN: 2595-2579, Nº 9, Barra do Garças - MT 1 Palavras-chave: Gestão de pessoas. Recurso Humano. Pandemia Resumen La gestión de personas necesita diseñar estrategias para alinear sus procesos y habilidades, las personas son el principal activo, por lo tanto, las organizaciones necesitan estar más conscientes y atentas a sus empleados. Ante la pandemia, los directivos debieron crear tácticas para enfrentar este escenario adverso, el proceso se tornó más competitivo y sumamente agresivo, presentando una nueva realidad para las empresas, decidiendo continuar en el mercado. La metodología de investigación bibliográfica consiste en el uso de plataformas científicas, como Scielo, Google Scholar, entre otras. Con el fin de ampliar las fuentes de investigación y enriquecer el análisis de la información obtenida sobre el tema elegido. Puede ver el cambio que ha tenido lugar desde el trabajo remoto, la reasignación dinámica de recursos, la aceleración de la digitalización y la automatización para satisfacer las necesidades individuales y organizacionales en evolución. Para iniciar la organización del futuro, la contratación de nuevos empleados y el liderazgo deben estar dentro de una nueva propuesta creativa y flexible. El nuevo concepto de negocio debe ser audaz, buscar talento, tener una red de equipos consolidada y también debe crear un ecosistema en la organización favorable para todos los involucrados. Palabras clave: Gestión de personas. Recursos humanos. Pandemia Abstract People management needs to devise strategies to align its processes and skills, people are the main asset, therefore, organizations need to be more aware and attentive to their employees. Faced with the pandemic, managers had to create tactics to deal with this adverse scenario, the process became more competitive and extremely aggressive, presenting a new reality for companies, deciding to continue in the market. The bibliographic research methodology consists of the use of scientific platforms, such as Scielo, Google Scholar, among others. In order to expand the research sources and enrich the analysis of the information obtained on the chosen theme. You can see the shift that has taken place from remote working, the dynamic reallocation of resources, the acceleration of digitization and automation to meet evolving individual and organizational needs. To start the organization of the future, hiring new employees and leadership must be within a new creative and flexible proposal. The new business Avanços & Olhares, ISSN: 2595-2579, Nº 9, Barra do Garças - MT 2 concept needs to be bold, seek talent, have a consolidated team network, and it must also create an ecosystem in the organization favorable to all those involved. Keywords: People management. Human resource. Pandemic Keywords: People management. Human resource. Pandemic INTRODUÇÃO De acordo com Barnard (1971), as pessoas não atuam isoladamente, mas com interações para alcançar os seus objetivos. Nas relações humanas, os indivíduos sofrem influências umas das outras, devido às diferenças individuais, pois cada uma têm suas características, capacidades e limitações próprias. Os indivíduos precisam se relacionar para alcançarem os seus objetivos de forma mais eficiente, sendo assim, através da participação pessoal e do cooperativismo, surgem as organizações. Dessa forma, as instituições são sistemas cooperativos, que têm como fundamento a racionalidade, ou seja, as organizações são sistemas sociais que se baseiam na cooperação entre as pessoas. Conforme Chiavenato (1993, p. 565), "uma organização somente existe quando ocorrem três condições: a interação entre duas ou mais pessoas, desejo e disposição para a cooperação e finalidade de alcançar objetivo comum." Dentro das instituições existe a necessidade de ter uma área voltada para as pessoas, que se atente para além da documentação Avanços & Olhares, ISSN: 2595-2579, Nº 9, Barra do Garças - MT 3 3 e dos processos burocráticos, sendo indispensável uma ação que crie e organize uma forma de agir sobre o comportamento dos funcionários (FISCHER, 2002, p.11). Isso comprova que além da força de trabalho, é preciso uma equipe que se organize e dê ênfase nos objetivos organizacionais e nas relações pessoais dentro da instituição, sendo esse o setor de Recursos Humanos (RH). Fischer (2002) salienta que o setor de recursos humanos nas organizações desenvolveu-se à medida que os funcionários se tornaram fatores de produção. O conceito desse setor seria, concentrar-se no controle das pessoas que prestam serviço e não na sua satisfação e motivação como é discutido atualmente. Em relação aos Recursos Humanos, Springer e Springer (1990) afirmavam que o objetivo do setor era identificar entre os candidatos, os sujeitos que poderão ser colaboradores mais produtivos com menor custo, o demonstrando que o controle era crucial, uma vez que o foco principal era de avaliar a capacidade dos trabalhadores. A gestão de pessoas precisa elaborar estratégias para alinhar os seus processos e competências, ao cenário interno e externo da organização, proporcionando vantagens competitivas à empresa. (CHIAVENATO, 2009) Conforme Chiavenato (2014), as pessoas são o principal ativo da organização, sendo necessário, portanto, que as organizações sejam mais conscientes e atentas aos seus funcionários. As organizações com sucesso percebem que só crescem se maximizarem os retornos dos investimentos de todos os parceiros, sobretudo os funcionários. INTRODUÇÃO O mundo está se modificando com uma rapidez extraordinária, com intensidade cada vez maior. Sempre houve mudanças na história humana, mas não com a pressa, profundidade e efeitos que ocorrem hoje. Para Chiavenato (2014, p.1), ainda, diversos elementos ajudam tais como nesta mudança: "econômicas, tecnológicas, sociais, culturais, legais, políticas, demográficas e ecológicas que atuam de maneira conjugada e sistêmica [...] trazendo transformações, imprevisibilidade e incerteza para as organizações”. O que ocorre fora tem um forte impacto sobre as organizações. É crucial analisar o ambiente externo para ajustar o procedimento de cada organização e o seu propósito para o futuro, uma vez que ele será completamente diferente do cenário atual. A maioria das pessoas que trabalham no setor de recursos humanos (RH) são as que mais sofrem, pois as alterações são tantas que até a denominação da área está sendo alterada. Segundo Chiavenato (2014, p.1) em diversas instituições, Avanços & Olhares, ISSN: 2595-2579, Nº 9, Barra do Garças - MT [...] a denominação administração de recursos humanos (ARH) está sendo substituída por expressões como gestão de pessoas (GP), gestão com pessoas, gestão do talento humano, gestão de parceiros ou de colaboradores, gestão de competências, gestão do capital humano ou administração do capital intelectual. Ao longo dos anos de 2019, a pandemia causada pela covid-193, alterou a interação como conhecemos. Fechando, no primeiro semestre de 2020, as fronteiras entre os países, ainda hoje, o mundo sofre os impactos causados nas organizações e na sociedade em geral. Conforme Felipe et al. (2021, p.221), ficou evidente a dificuldade que as organizações tiveram na tomada de ações para eficácia do combate da situação. A tensão, tanto mental quanto emocional, dificultam o equilíbrio entre as demandas do trabalho e outros relevantes para estabilidade emocional, como as questões relacionadas família. Mudança da rotina e da jornada de trabalho, novas demandas, home office, entre outras situações dificultaram e limitaram o trabalho dos colaboradores. Segundo Panda (2020), estávamos diante de uma situação inédita na história mundial. Foi necessário criar ferramentas a curto prazo para proteger as companhias e a economia, uma vez que o setor privado é dependente do setor público. Os empresários acreditavam que ainda existiam atividades econômicas que poderiam sobreviver à crise da Covid-19, embora fossem poucas. Carvalho et al. (2020) complementa que o caos econômico anunciado pela pandemia atingiu as atividades econômicas das instituições, exigindo propostas governamentais para o menor impacto possível na economia do país. g ç 4 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior ç g p (SARS-CoV-2), conforme a Organização Mundial da Saúde (OMS). g ç 4 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior ID-19 é uma doença infectocontagiosa causada pelo coronavírus da síndrome respiratória aguda grave 2 ç g p p (SARS-CoV-2), conforme a Organização Mundial da Saúde (OMS). 3 A COVID-19 é uma doença infectocontagiosa causada pelo coronavírus da síndrome respiratória aguda grave 2 (SARS-CoV-2), conforme a Organização Mundial da Saúde (OMS). 4 C d d A f i d P l d Ní l S i 3 A COVID-19 é uma doença infectocontagiosa causada pelo coronavírus da síndrome respiratória aguda grave 2 (SARS-CoV-2), conforme a Organização Mundial da Saúde (OMS). 4 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior 5 O lockdown pode ser classificado em confinamento total ou parcial (BRASIL). A Recomendação nº 036, de 11 de maio de 2020, recomenda a implementação de medidas de distanciamento social mais restritivo (lockdown), nos municípios com ocorrência acelerada de novos casos de COVID-19 e com taxa de ocupação dos serviços atingido níveis críticos [Internet]. Brasília: Conselho Nacional de Saúde; 2020. Disponível em: http://conselho.saude.gov.br/recomendacoescns/1163-recomendac-a-o-n-036-de-11-de-maio-de-2020 Metodologia A metodologia da pesquisa bibliográfica consiste na utilização de plataformas científicas, tais como o Scielo, Google Scholar, entre outras, para buscar referências relevantes e atualizadas sobre o tema proposto. Os descritores "Gestão" e "Pessoas" foram utilizados para a fundamentação e discussão dos estudos encontrados. Além disso, realizou-se um levantamento no banco de teses da CAPES4, utilizando os descritores "Gestão de Pessoas", "Pandemia" e "Recursos Humanos", a fim de ampliar as fontes de pesquisa e enriquecer a análise das informações obtidas. Foi feita uma leitura crítica dos artigos e teses Avanços & Olhares, ISSN: 2595-2579, Nº 9, Barra do Garças - MT 5 5 encontrados, avaliando a qualidade das evidências apresentadas e verificando compatibilidade com a proposta da temática escolhida. Gestão empresarial diante do contexto da Covid19 Diante da pandemia, os gestores tiveram que criar táticas para lidar com esse cenário adverso. O processo competitivo extremamente agressivo, a utilização de decisões estratégicas em situações contrárias ou favoráveis apresentou uma nova realidade para as empresas, decidindo a sua continuação no mercado (PFANNEMULLER, 2006). Becker et al. (2020) sugere que devemos tentar contribuir analisando os impactos e adversidades da pandemia de Covid-19 nas instituições, bem como as boas práticas para o futuro. A pandemia demandou que os órgãos público e privado adaptassem ao novo contexto, fazendo com que a maioria dos funcionários ficasse em home office. O principal obstáculo foi como permitir que esta nova infraestrutura de Tecnologia da Informação (TI) fosse implementada em pouco tempo para uma quantidade enorme de usuários remotos (OKANO et al.,2020). Dessa maneira, foi um grande desafio para as companhias se ajustarem a esse novo cenário, pois, afetou diretamente a arrecadação, causando diversos problemas. Em pouco tempo, as organizações tiveram que elaborar processos e instrumento para se adaptarem ao novo contexto. Em geral, as companhias passaram a fazer os seus atendimentos de forma remota. Optar pelo ambiente virtual foi a forma de manter as normas de preservação e de proteção à saúde coletiva. Algumas organizações enfrentam dificuldades por não terem profissionais capacitados para lidar com tecnologias, ou por não ter condições de incentivar o trabalho à distância. O isolamento social que se instalou de forma repentina em diversos lugares do mundo, deixou as instituições sem um plano de ação para continuar o serviço (BECKER et al.,2020) Segundo Nogueira et al. (2020), a pandemia foi similar às principais crises econômicas que o mundo contemporâneo já sofreu. As atividades econômicas foram afetadas pelo isolamento social e falta de trabalhadores, devido ao medo do contágio, pois houve o lockdown 5imposto pelos governantes e o endógeno adotado pelos cidadãos. Avanços & Olhares, ISSN: 2595-2579, Nº 9, Barra do Garças - MT 6 A pandemia do Coronavírus sucedeu na acelerada e grave difusão da virose econômica, destruindo várias redes de produção no planeta (NOGUEIRA et al. 2020). Nesse cenário, de pandemia, um dos pontos que merecem ser considerados, é a repercussão das intenções de precaução e conduta diante ao vírus nas organizações e no ambiente de negócios. 6 FIA Business School. Coronavírus e o impacto nos negócios: como preparar sua empresa?. Disponível em: < https://fia.com.br/blog/coronavirus-e-o-impacto-nos-negocios/>. Acesso 23 mar 2023 FIA Business School. Coronavírus e o impacto nos negócios: como preparar sua empresa?. Disponív https://fia.com.br/blog/coronavirus-e-o-impacto-nos-negocios/>. Acesso 23 mar 2023 6 FIA Business School. Coronavírus e o impacto nos negócios: como preparar sua empresa?. Disponív Gestão empresarial diante do contexto da Covid19 Santos e Oliveira (2021) destacaram que a pandemia afetou várias áreas do setor organizacional, dados da FIA (2020)6 mostrou que 88% dos empreendedores não conseguiram se manter no período da pandemia, sendo esses os pequenos e microempreendedores. Ainda, conforme o Instituto Brasileiro de Geografia e Estatística - IBGE (2020), 70% das companhias em funcionamento no início da pandemia sofreram danos diretos ou indiretos. As pequenas companhias são importantes para a economia nacional, pois representam 99% das empresas com diversos empregos formais e 30% do PIB (PERREIRA, 2021). Apesar da relevância incontestável desse setor para o país, o mesmo enfrenta constantemente dificuldades para obter empréstimos para o financiamento de suas atividades, devido à burocracia excessiva, garantias enviáveis e custos de crédito exorbitantes (BEVILAQUA et al.,2018), dificultando o crescimento e a estabilidade dessas companhias, especialmente em tempos de recessão. Como medida preventiva contra a pandemia, o Governo Federal implementou medidas provisórias para conter a queda na economia e o aumento do desemprego (SEBRAE, 2022). O Governo concedeu diversos incentivos para dar um auxílio social, como o Programa Nacional de Apoio às Microempresas e Empresas de Pequeno Porte (PRONAMPE), que passou a ser um apoio às pequenas empresas diferenciado, proporcionando maior crédito para as empresas que estivesse operando há mais de um ano. E o Programa Emergencial de Suporte a Empregos – (PESE), executado pelo Banco Nacional de Desenvolvimento Econômico e Social (BNDES), elaborado com a Medida Provisória n. º 944/2020, e reescrito mais tarde na forma da Lei 14.043/2020 em agosto de 2020, que beneficiou aproximadamente 1,9 milhão de colaboradores no país (SANTOS; OLIVEIRA, 2021). O PESE pagou apenas folha salarial e quitação de verbas trabalhistas. O objetivo deste programa, segundo o BNDES (2021), foi ser um crédito emergencial para companhias com faturamento entre R$ 360 mil, exclusive, e R$ 10 milhões, com base no ano de 2019, em sua primeira fase, conforme a MP 944/2020. Numa segunda etapa, que foi reformulada pela lei Avanços & Olhares, ISSN: 2595-2579, Nº 9, Barra do Garças - MT 7 anterior, o crédito foi estendido para instituições com faturamento até R$ 50 milhões (PARMAIS, 2021). O financiamento foi usado para pagar a folha de salários de funcionários e, na segunda fase, para quitar verbas trabalhistas. Gestão empresarial diante do contexto da Covid19 A taxa de juros é 3,75% ao ano, sem remuneração ao BNDES e aos agentes financeiros, uma vez que o empréstimo foi definido em 36 meses, sendo os seis primeiros considerados carência, fazendo parte do período total disponibilizado para quitação do financiamento. As companhias também tiveram a obrigação de não dispensar os funcionários, com exceção dos casos de justa causa, até dois meses (60 dias) após terem recebido o total do crédito, o que garantiu 100% dos empregos na primeira fase. Na segunda fase, a exigência foi a mesma proporção da folha de pagamento paga com os recursos do programa (BNDES, 2021). O PESE foi criado para suprir a lacuna causada pela pandemia nos empregos formais e as dificuldades em preservá-los pelas organizações diante de tantas dificuldades nessa crise. Gestão de Pessoas As novas estruturas tecnológicas, econômicas, sociais e comerciais, atualmente, modificam as forças produtivas, estabelecendo altos níveis de competição entre empresas. A organização, de acordo com Chiavenato (2014), é um organismo vivo, em constante ação e crescimento. Quando bem-sucedidas, elas podem alavancar ou, pelo menos, manter a estabilidade. O crescimento exige mais complexidade, investimento, infraestrutura, tecnologia, ações de apoio, etc. Isso aumenta o contingente de indivíduos e a exigência da utilização de informações, habilidades e competências necessários ao aperfeiçoamento e a competitividade do negócio. Para aumentar os parâmetros competitivos mercantis, é necessário adotar estratégias empresariais modernas para reduzir custos, melhorar a qualidade e buscar vantagens competitivas e inovações sustentáveis. (MAESTRO et al., 2013) As pressões competitivas tornaram a gestão de pessoas nas empresas um importante elemento estratégico, enquanto, de forma progressiva, tem permitido funções e ações mais próximas ao campo estratégico das organizações (MASCARENHAS, 2008) As inovações tecnológicas nas instituições e os novos conceitos de gestão de pessoas provocam debate teórico que permitem novas perspectivas e o entendimento de como tornar a Avanços & Olhares, ISSN: 2595-2579, Nº 9, Barra do Garças - MT 8 atuação mais eficiente, em termos de suas capacidades, para conceber resultados coeso com as necessidades e estratégias (BOSELIE, 2010). Ainda afirma Chiavenato: Ainda afirma Chiavenato: Ainda afirma Chiavenato: A turbulência das mudanças sociais, tecnológicas, econômicas, culturais e políticas e suas tendências estão trazendo novas abordagens, mais flexíveis e ágeis, para garantir a competitividade e a sobrevivência das organizações. E os profissionais de GP devem saber como lidar com mudanças, se realmente querem contribuir para o sucesso da sua organização. São mudanças que se multiplicam exponencialmente e cujas soluções impõem novas estratégias, filosofias, programas, procedimentos e soluções. A GP deve estar comprometida com as mudanças. (CHIAVENATO, 2014, p.12). Dessa forma, a gestão de pessoas necessita estar preparada para propor inovações, procedimentos e abordagens para destacar nas dificuldades e contribuir no desempenho organizacional (PAAUWE, 2004) Dessa forma, a gestão de pessoas necessita estar preparada para propor inovações, procedimentos e abordagens para destacar nas dificuldades e contribuir no desempenho organizacional (PAAUWE, 2004) As inovações surgem quando se pensa nas mudanças de um contexto ou produto e são percebidas como novidades, o que é crucial para as companhias no momento de fazer interferências e administrar. Gestão de Pessoas Os principais responsáveis pelo surgimento de inovações nas organizações são as pessoas, por isso, o RH deve se centralizar nos processos sobre as atividades transacionais e administrativos intrínseco ao setor (MAESTRO et al., 2013). As novas gerações estão entrando no mundo do trabalho, e as organizações precisam gerenciar a mudança, aumentando o comprometimento dos colaboradores com a empresa. São necessárias ferramentas que ajudem a superar esses desafios, como coordenar o envolvimento ou favorecer a transição para a era digital (QUISHIDA et al., 2017). Todavia, ao encarar as mudanças culturais e permitir iniciativas de inovação, é preciso fazê-lo de forma ativa e constante, com impacto comedido nas organizações, para não abalar os funcionários que a compõe. A gestão de pessoas deve propiciar a efetivação desses empreendimentos, oportunizado uma cultura organizacional com foco na renovação e engenhosidade (MAESTRO et al., 2013). A cultura organizacional sofre forte influência do mundo externo e passa a apreciar as transformações e as revoluções com foco no futuro da organização. As transformações são aceleradas, dinâmicas, sem acesso com o passado e trazem um ambiente com denso e imprevisível. A pessoa deve ter capacidade de lidar com novos problemas e auxiliar a empresa a superá-los (CHIAVENATO, 2014) Avanços & Olhares, ISSN: 2595-2579, Nº 9, Barra do Garças - MT 9 Uma empresa, para alcançar o sucesso, necessita que a finalidade da gestão de pessoas seja visível e concreta. É a motivação dos funcionários que garantirá a qualidade das atividades desenvolvidas. Na atualidade um dos desafios é promover a qualidade de vida, que visa, além da eficiência, o bem-estar social (SOVIENSKI; STIGAR 2008). Chiavenato (2000) aponta a necessidade de investir nos colaboradores, pois aplicação do capital só será bem-sucedida quando for ágil, bem aplicado e dirigido. Para isso, gerir pessoas deve ser antes, durante e depois da administração do capital ou de qualquer outro recurso empresarial. As empresas que se destacaram pelo sucesso perceberam que seus funcionários alavancam os resultados Para Stadler (2009) gerir pessoas significa atrair, ambientar, acrescentar, averiguar e assegurar colaboradores na empresa. Isso significa apresentar situações organizacionais para que o ser humano possa discutir e aprimorar suas capacidades, alinhando os objetivos organizacionais aos seus pessoais. Significa também usar conhecimentos técnicos, gerenciais e de relacionamentos. A técnica é o dia a dia do trabalho, a administração é a tomada de decisão do gestor e a interpessoal é o que diferencia os profissionais. Impactos da Pandemia na gestão de pessoas Muitos dos grandes líderes das companhias, além do próprio Gestor, foram os responsáveis pela Gestão de Pessoas, ficando responsáveis por gerir e administrar os funcionários. O gestor tem como uma das suas competências reconhecer um profissional e saber onde o coloca para mostrar o seu potencial, Drucker (2006, p.28) enfatiza: “Não se gerencia pessoas, a tarefa é liderar pessoas”. Motta (2001) complementa que a liderança é a principal função do gestor, uma vez que a liderança estimula os outros a envolverem-se na procura de objetivos em comum. Esse aspecto é a identidade do gestor, a capacidade de conhecer-se e o papel da organização. Gestores de recursos humanos são ótimos líderes e sabem lidar com conflitos interpessoais de forma adequada. Hunter enfatiza essa capacidade de liderança quando afirma que a eficiência do líder está no seu desenvolvimento, habilidades e qualidades morais que o habilitam a instigar e instigar um grupo de pessoas. Valorizar o indivíduo verdadeiramente, é liderar sabendo identificar e reconhecer as suas necessidades legítimas do outro (HUNTER, 2009). Em síntese, a definição de liderança não tem relação com a hierarquia ou com a Avanços & Olhares, ISSN: 2595-2579, Nº 9, Barra do Garças - MT 10 posição ocupada, sendo uma questão de potencializar o profissional, o que é crucial para o progresso das organizações. Chiavenato (2000) defende a necessidade de potencializar o investimento em pessoas. A pandemia alterou os conceitos de liderança e a capacidade de pessoas de serem potenciais colaboradores nas empresas, uma vez que as necessidades de afastamento social causaram diversos problemas. A principal mudança veio relacionado à forma de contratação. Dado que as companhias tiveram uma grande mudança na forma de contratar, precisaram desenvolver novas formas de avaliar o desempenho e a capacidade do futuro funcionário. Para efetivar contratação em época de pandemia, muitas empresas necessitaram inovar, visando sempre encontrar o profissional adequado e certo para a vaga (MARCOS e CRUZ, 2022). A partir da mudança no modelo de negócios, as companhias precisam se reinventar em um cenário que não para de se alterar. Sendo assim, para que essas mudanças fossem efetivadas, as equipes tiveram que se envolver num procedimento organizado e fundamentado em estratégias de gestão de mudanças (CORREIA NETO; ALBUQUERQUE, 2021). Foi necessário repensar os processos de seleção, para que surgissem novas contratações de colaboradores, como os chatbots7 ou algoritmos de triagem de currículos. 7 Os chatbots são programas de computador capazes de gerar conversas através de regras de linguagem de programação ou inteligência artificial in CORREIA. Mafalda Pinto. A Evolução dos Chatbots e os seus benefícios para marcas e consumidores. Dissertação (mestrado). Universidade Católica Portuguesa. 2018. Disponível em: https://repositorio.ucp.pt/bitstream/10400.14/27105/1/Tese%20Mafalda%20Correia%20CD.pdf Acesso em: 23 mar. 2023 Impactos da Pandemia na gestão de pessoas A segurança dos funcionários nos cuidados de saúde de que necessitavam se tornou um fator crucial para as pessoas e uma grande vantagem para as organizações em relação ao contexto atual (ATHAR, 2020). As empresas devem assegurar a execução das políticas, incentivando a diversidade em contexto diversificados. Elaborar programas de entendimento e ferramentas para administrar o tempo. É crucial que a mudança do empreendimento e a capacitação do pessoal estejam de acordo com as finalidades determinadas pelas companhias e sejam atingidas para aumentar a motivação e o desenvolvimento dos funcionários (VIDAL, 2020). Enquanto as grandes companhias já utilizavam tecnologias para o setor de Recursos Humanos, na pandemia a propensão é que continue na pós-pandemia. A expansão do teletrabalho fez com fossem estendidas para todo âmbito empresarial, possibilitando ao RH de qualquer organização automatizarem a maioria dos seus processos. Avanços & Olhares, ISSN: 2595-2579, Nº 9, Barra do Garças - MT 11 De acordo com Bueno (2018), quando os líderes elaboram um ambiente em que os indivíduos possam se sentir bem consigo mesmas, com orgulho do seu papel dentro da organização, se sentem bem no trabalho. Geralmente, as pessoas que estão contentes focam no desenvolvimento de boas experiências para o cliente, o que é um passo importante para um líder no mercado de trabalho. Os desafios na gestão de pessoas, em especial, é a acentuada inquietude dos empresários com o crescimento da liderança, podem ser descritos como resultado das demandas oriundas da criação de novas estratégias e composições organizacionais internamente e externamente (CARVALHO NETO; SANT’ANNA, 2013). As companhias têm consciência de que uma boa experiência do funcionário indica elevado sucesso financeiros. Empresas bem-sucedidas trabalham em parceria com os seus funcionários para criar experiências únicas, autênticas que atendam ao objetivo de aumentar o desenvolvimento individual, da equipe e da instituição. O RH tem a função primordial na capacitação dos funcionários (RODRIGUES, 2020). Além das alterações nas contratações os trabalhos em boa parte passaram a ser Home Office que: [...] foi inventado em 1857 por J. Edgar Thompson, através das atividades de telégrafo. Atualmente é definida como uma modalidade de trabalho em que se determina regras para se trabalhar fora do ambiente corporativo, sendo o Home Office, uma variação do teletrabalho, em que os colaboradores realizam suas atividades especificamente em casa. Impactos da Pandemia na gestão de pessoas Com o avanço da tecnologia o formato ganhou ainda mais força e solidez, uma vez que as ferramentas tecnológicas de hoje permitem que esse tipo de trabalho seja autossuficiente e o colaborador consiga exercer sua total capacidade através dele (MACEDO, 2022, p.18). [...] foi inventado em 1857 por J. Edgar Thompson, através das atividades de telégrafo. Atualmente é definida como uma modalidade de trabalho em que se determina regras para se trabalhar fora do ambiente corporativo, sendo o Home Office, uma variação do teletrabalho, em que os colaboradores realizam suas atividades especificamente em casa. Com o avanço da tecnologia o formato ganhou ainda mais força e solidez, uma vez que as ferramentas tecnológicas de hoje permitem que esse tipo de trabalho seja autossuficiente e o colaborador consiga exercer sua total capacidade através dele (MACEDO, 2022, p.18). No ano de 2011, a legislação brasileira reconheceu home office como novo formato de relação de trabalho, pela Lei n° 12.551, que, no seu artigo 6.º, não distingue o trabalho na empresa realizado no domicílio e a distância, desde que sigam os requisitos da relação de emprego, vale ressaltar que com a reforma trabalhista, o Home Office foi regulamentado, mas já era praticado por empresas privadas. O Home Office foi usado por milhares de organizações para dar prosseguimento aos trabalhos de forma não presencial na pandemia. O uso desse tipo de formato gerou alguns benefícios para a organização e o empregado, como ampliação na produtividade, flexibilidade na jornada de trabalho, redução de custos e melhor qualidade de vida, dentre outros. Essas Avanços & Olhares, ISSN: 2595-2579, Nº 9, Barra do Garças - MT 12 alterações tiveram um impacto direto no setor de gestão de pessoas, que passou a elaborar novos processo de adaptação. Segundo Luedke (2021, p.15), “O home office era e é uma opção de formato de trabalho para muitas empresas, que surgiu como “prática” viável e adaptável para a maioria das pessoas (e única, para algumas), no momento da crise COVID-19”. No entanto, em alguns casos não tivemos um home office, dividido entre as partes com planejamento e fluxos transparentes. O que ocorreu foi de forma aleatória, rápida e implantada não por opção, mas sim pela necessidade. A implantação do Home Office e a capacitação para esse tipo de trabalho gerou grandes desafios na área de RH, no período da pandemia, como treinar os funcionários para usar ferramentas e manter a motivação. Impactos da Pandemia na gestão de pessoas O obstáculo para os líderes de equipes foi trabalharem à distância, mantendo o envolvimento e a motivação, sendo assim, um desafio para a gestão de pessoas. Segundo o Great Place to Work (2022), 59% dos líderes de RH, o maior obstáculo enfrentado na pandemia, foi o de adotar novas políticas de trabalho, depois manter uma boa comunicação interna 49,2%, e desenvolver lideranças 39,8%. Outro problema levantado foi a manutenção/disseminação da cultura organizacional, a contratação de profissionais com qualificação, experiência do funcionário, criação de programas e benefícios para saúde mental, a transformação digital dos processos e ferramentas de RH. O setor também teve que criar um perfil de funcionário, com competências e habilidades que se tornaram fundamentais para esse período, outra questão era estimular os colaboradores com treinamentos e palestras. As dispensas para cortes de custos ou por questões estratégicas se tornaram outro problema e o setor precisou reestruturar a organização, mantendo funcionários que possuíam as habilidades necessárias para o momento em questão. CONSIDERAÇÕES FINAIS Para qualquer organização alcançar sucesso depende de diversos fatores, sobretudo, os que dizem respeito às atividades dos Recursos Humanos. São esses os grandes desafios que surgem devido às mudanças que estão ocorrendo no mundo globalizado. É preciso adquirir novas técnicas, sobretudo, para podermos planejar e gerir o RH em momentos de adversidades e inconstância. É importante para as organizações modernas desenvolverem e implementam processos de liderança com uma inclinação encorajadora, baseada num novo Avanços & Olhares, ISSN: 2595-2579, Nº 9, Barra do Garças - MT 13 perfil de habilidade que possibilite reconsiderar sua função e repensar muitas de suas metas, nas quais os recursos humanos são fundamentais. A liderança é um caminho para o progresso nas relações de trabalho e na gestão de pessoas. Os métodos para estimular as equipes pretendem, principalmente, fornecer ferramentas para criar líderes verdadeiros, pessoas que não precisam da hierarquia ou influência para atingir suas metas e objetivos. Líderes empresariais ao depararem com pandemia na Covid-19, tiveram dificuldades para entender o cenário, já houveram mudanças profundas na sociedade e nos indivíduos. É notório a mudança que ocorreu em relação ao trabalho remoto, na realocação dinâmica de recursos, na aceleração da digitalização e na automação para atender às necessidades individuais e organizacionais em constante evolução. Em geral, as companhias enfrentam ainda os desafios causados pela pandemia. No entanto, o sistema de gestão com base na hierarquia que resolve com uniformidade, burocracia e controle, não é mais eficaz. 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https://openalex.org/W4385666840	https://www.repository.cam.ac.uk/bitstreams/18b0e813-8bef-4645-a64e-3f7d6732e4eb/download	English	null	Defining the spatial distribution of extracellular adenosine revealed a myeloid-dependent immunosuppressive microenvironment in pancreatic ductal adenocarcinoma	Journal for immunotherapy of cancer	2,023	cc-by	18,327	WHAT THIS STUDY ADDS Correspondence to Dr Vincenzo Graziano; vincenzo.graziano@astrazeneca. com Dr Duncan I Jodrell; Duncan.Jodrell@cruk.cam.ac.uk © Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY. Published by BMJ. For numbered affiliations see end of article. Correspondence to Dr Vincenzo Graziano; vincenzo.graziano@astrazeneca. com Dr Duncan I Jodrell; Duncan.Jodrell@cruk.cam.ac.uk © Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY. Published by BMJ. Abstract To cite: Graziano V, Dannhorn A, Hulme H, et al. Defining the spatial distribution of extracellular adenosine revealed a myeloid-dependent immunosuppressive microenvironment in pancreatic ductal adenocarcinoma. Journal for ImmunoTherapy of Cancer 2023;11:e006457. doi:10.1136/ jitc-2022-006457 WHAT IS ALREADY KNOWN ON THIS TOPIC Background The prognosis for patients with pancreatic ductal adenocarcinoma (PDAC) remains extremely poor. It has been suggested that the adenosine pathway contributes to the ability of PDAC to evade the immune system and hence, its resistance to immuno-oncology therapies (IOT), by generating extracellular adenosine (eAdo). ﬁ The adenosine pathway generates extracellular adenosine and its components are known to be overexpressed in tumors. Extracellular adenosine is recognized as an immune suppressive molecule. CD73, the member of the pathway that enables the final step of conversion of AMP to adenosine, is highly expressed on the cancer cell surface in many tumors, including pancreatic ductal adenocarcino- ma (PDAC). ﬁ The adenosine pathway generates extracellular adenosine and its components are known to be overexpressed in tumors. Extracellular adenosine is recognized as an immune suppressive molecule. CD73, the member of the pathway that enables the final step of conversion of AMP to adenosine, is highly expressed on the cancer cell surface in many tumors, including pancreatic ductal adenocarcino- ma (PDAC). Methods Using genetically engineered allograft models of PDAC in syngeneic mice with defined and different immune infiltration and response to IOT and autochthonous tumors in KPC mice we investigated the impact of the adenosine pathway on the PDAC tumor microenvironment (TME). Flow cytometry and imaging mass cytometry (IMC) were used to characterize the subpopulation frequency and spatial distribution of tumor- infiltrating immune cells. Mass spectrometry imaging (MSI) was used to visualize adenosine compartmentalization in the PDAC tumors. RNA sequencing was used to evaluate the influence of the adenosine pathway on the shaping of the immune milieu and correlate our findings to published data sets in human PDAC. Defining the spatial distribution of extracellular adenosine revealed a myeloid-dependent immunosuppressive microenvironment in pancreatic ductal adenocarcinoma Vincenzo Graziano ‍ ‍ ,1,2 Andreas Dannhorn,3 Heather Hulme,3 Kate Williamson,4 Hannah Buckley,1 Saadia A Karim,5 Matthew Wilson,6 Sheng Y Lee,1 Brajesh P Kaistha,1 Sabita Islam,7 James E D Thaventhiran,4 Frances M Richards,1 Richard Goodwin,3 Rebecca Brais,8 Jennifer P Morton,5,9 Simon J Dovedi,6 Alwin G Schuller,10 Jim Eyles,6 Duncan I Jodrell1,7 To cite: Graziano V, Dannhorn A, Hulme H, et al. Defining the spatial distribution of extracellular adenosine revealed a myeloid-dependent immunosuppressive microenvironment in pancreatic ductal adenocarcinoma. Journal for ImmunoTherapy of Cancer 2023;11:e006457. doi:10.1136/ jitc-2022-006457 Original research Original research Open access Defining the spatial distribution of extracellular adenosine revealed a myeloid-dependent immunosuppressive microenvironment in pancreatic ductal adenocarcinoma WHAT THIS STUDY ADDS ﬁ Here, we show that in murine models of PDAC, the adenosine pathway is overexpressed by a popula- tion of myeloid immune cells. We visualized ade- nosine spatially in the tumor microenvironment of a relevant, preclinical model of PDAC, identifying that its distribution is mostly confined to myeloid-rich, hypoxic areas. We discovered that protumorigenic myeloid populations (in particular, M2 macrophages) represent the target of adenosine stimulation (Adora2a expressing) and therefore it is responsible for the formation and maintenance of an immune suppressive microenvironment. We were able to generate a specific, transcriptomic signature from our preclinical experiments that predicts survival in patients with PDAC. Finally, we demonstrated that inhibiting the adenosine pathway improved response to cytotoxic and immunotherapy drugs in murine PDAC models. ﬁ Here, we show that in murine models of PDAC, the adenosine pathway is overexpressed by a popula- tion of myeloid immune cells. We visualized ade- nosine spatially in the tumor microenvironment of a relevant, preclinical model of PDAC, identifying that its distribution is mostly confined to myeloid-rich, hypoxic areas. We discovered that protumorigenic myeloid populations (in particular, M2 macrophages) represent the target of adenosine stimulation (Adora2a expressing) and therefore it is responsible for the formation and maintenance of an immune suppressive microenvironment. We were able to generate a specific, transcriptomic signature from our preclinical experiments that predicts survival in patients with PDAC. Finally, we demonstrated that inhibiting the adenosine pathway improved response to cytotoxic and immunotherapy drugs in murine PDAC models. Results We demonstrated high expression of adenosine pathway components in tumor-infiltrating immune cells (particularly myeloid populations) in the murine models. MSI demonstrated that extracellular adenosine distribution is heterogeneous in tumors, with high concentrations in peri-necrotic, hypoxic regions, associated with rich myeloid infiltration, demonstrated using IMC. Protumorigenic M2 macrophages express high levels of the Adora2a receptor; particularly in the IOT resistant model. Blocking the in vivo formation and function of eAdo (Adoi), using a combination of anti-CD73 antibody and an Adora2a inhibitor slowed tumor growth and reduced metastatic burden. Additionally, blocking the adenosine pathway improved the efficacy of combinations of cytotoxic agents or immunotherapy. Adoi remodeled the TME, by reducing the infiltration of M2 macrophages and regulatory T cells. RNA sequencing analysis showed that genes related to immune modulation, hypoxia and tumor stroma were downregulated following For numbered affiliations see end of article. Introduction Survival for patients with pancreatic ductal adenocarci- noma (PDAC) has not changed significantly in the last 50 years and remains poor (https://www.cancerresearchuk. org/health-professional/cancer-statistics-for-the-uk).1 There is a need for new treatments, given that the current standard of care for patients with metastatic disease is asso- ciated with poor outcomes, with less than 10% of patients living for more than 2 years.2 In addition to relative resis- tance to conventional therapies, cancer immunotherapy (immuno-oncology therapy, IOT) is also ineffective in this disease, except in a small group of patients (1–2%) with microsatellite instability/mismatch repair deficient (MSI-H/dMMR) tumors.3 Several authors consider that the reason for this resistance can be ascribed to the low mutational burden of this neoplasm, which leads to lymphocyte exclusion and anergy.4 5 However, the tumor microenvironment (TME) in PDAC has also been shown to be populated by a rich variety of immune cells, most of which demonstrate immune suppressive features, which contribute to the resistance to immunotherapy.6 p g yp Some recent publications link the adenosine pathway to the biology and aggressiveness of PDAC. PDAC has been shown to have an increased adenosine pathway RNA signature associated with a worse prognosis,18 and genes encoding for the receptors for eAdo as well as CD73 have been found to be overexpressed in bulk-RNA sequencing (RNAseq) when comparing tumors to normal pancreatic tissue.8 A role for eAdo in shaping myeloid response to PDAC has recently been suggested. Using genetic manipu- lation of CD73 in cancer cells and mice, Jacoberger-Foissac and colleagues demonstrated that CD73 can be overex- pressed in a percentage of tumor-infiltrating myeloid cells other than cancer cells, contributing to infiltration of M2 macrophages in KPC mice.36 King and colleagues highlighted that genetic alteration of CD73 impaired the secretion of GM-CSF (Granulocyte-macrophage colony- stimulating factor), reducing myeloid-derived suppressive cell infiltration in PDAC mouse models.37 Also, it has been suggested that genetic alteration of CD73 in cancer cells induces a modest sensitivity to gemcitabine treatment in vitro. However, little is known about the complex mecha- nism generated by the adenosine pathway resulting in the immunosuppressive characteristics of pancreatic cancer microenvironment and stroma, in particular the role that the adenosine pathway and its therapeutic inhibition have in shaping the immune infiltration of this disease. Open access results in inhibition of T cells/natural killer (NK) cell activation.24 25 Adoi and a specific adenosine signature derived from this is associated with a poorer prognosis in patients with PDAC. Adoi and a specific adenosine signature derived from this is associated with a poorer prognosis in patients with PDAC. Conclusions The formation of eAdo promotes the development of the immunosuppressive TME in PDAC, contributing to its resistance to conventional and novel therapies. Therefore, inhibition of the adenosine pathway may represent a strategy to modulate the PDAC immune milieu and improve therapy response in patients with PDAC. The myeloid populations play a pivotal role in the aggressiveness of many cancer types and in particular, PDAC. For instance, the presence of protumorigenic populations of macrophages26 27 and MDSCs28 infiltrating the microenvironment, is associated with poor survival and correlates with immune exclusion of PDAC. Macro- phages can elicit the secretion of cytokines which can on the one hand, favor the proliferation and invasiveness of cancer cells while interacting with cancer-associated fibroblasts,29–31 and on the other hand induce anergy and physical exclusion of adaptive immune cells.32–34 Targeting macrophages in a preclinical pancreatic cancer model has been demonstrated to be effective to obtain tumor regression and reduce metastatic formation.35 Unfortunately, this approach has not translated into clin- ical benefit, which in part can be explained by the fact that the global reduction of the tumor-infiltrating macro- phages can be biologically different from reprogramming distinct tumor associated macrophage subtypes.31 HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY ﬁ This study unveils a previously unknown myeloid- dependent axis of immunosuppression in PDAC and could inform future clinical trials that will evaluate inhibitors of the adenosine pathway. Such studies might improve outcomes for patients with PDAC, a major unmet clinical need. 1 Graziano V, et al. J Immunother Cancer 2023;11:e006457. doi:10.1136/jitc-2022-006457 Open access Introduction py The adenosine pathway is an immunosuppressive axis which has gained much attention in cancer immunology for its role in suppressing the immune activation associ- ated with cytotoxic treatments (chemotherapy, targeted therapy and radiotherapy).7–9 This has led to the clinical evaluation of inhibitors of the pathway in combination with more conventional approaches.10 The adenosine pathway involves conversion of extracellular ATP, a powerful immune activator, to extracellular adenosine (eAdo) by the ectonucleotidases CD39 and CD73.11 eAdo has been linked to cancer in several studies that have demon- strated that its concentration in different tumor tissues is several folds higher than in normal tissues.7 12 CD39 is overexpressed in a subpopulation of exhausted tumor- infiltrating T cells13 14 and its expression correlates with another marker of immunosuppression (Programmed cell death protein 1 or PD-1 expression).14 CD39 and CD73 have roles in the aggressiveness of adult glioblas- toma,15 where they are expressed on infiltrating macro- phages.16 17 The adenosine signature recently published by Sidders and colleagues18 shows that this pathway correlates with resistance to immunotherapies and is associated with other genetic features of tumor aggres- siveness, such as p53 mutations. The abundant presence of eAdo in the microenvironment can dampen immune activation through the stimulation of a protumorigenic stroma. This is mostly orchestrated by macrophages and myeloid derived suppressive cells (MDSCs),19 20 favoring a tolerogenic function of dendritic cells (DCs)21–23 which p g Here, we propose a model where the tumor-infiltrating immune cell populations of PDAC generate an axis of immunosuppression, where eAdo produced mostly in hypoxic regions of the tumor (identified using mass spectrometry imaging, MSI), enriched for the myeloid cell infiltration, stimulates protumorigenic M2 macro- phages. The axis described is expressed preferentially by the IOT-resistant model when compared with the IOT-responsive one. Therefore, blocking the adenosine pathway in the IOT-resistant PDAC model, strongly suppresses the formation of eAdo and reshapes the Graziano V, et al. J Immunother Cancer 2023;11:e006457. doi:10.1136/jitc-2022-006457 2 Open access team. Tumors were detected by palpation followed by ultrasound imaging by the Genome Editing Core. Tumor tissues, spleens and mesenteric and inguinal lymph nodes from KPC mice were provided once tumor dimensions or health status rendered them unsuitable for therapeutic studies. KPC mice were killed when showing clinical signs of the disease (swollen abdomen, loss of body condi- tioning resembling cachexia, reduced mobility). immune microenvironment, favoring disease control when combined with cytotoxic treatments and immuno- therapies. Methods and materials Cell lines and chemicals KrasLSL-G12D/+; Trp53LSL-R172H/+; Pdx1-Cre; Rosa26YFP/YFP (KPCY)-derived cell lines 2838c3, 6499c4, 6620c1 (IOT- responsive), 6419c5, 6694c2 and 6422c1 (IOT-resistant) were a kind gift from Ben Stanger (University of Penn- sylvania). The cell lines were obtained from single cell cloning strategy, as described previously, and were gener- ated from tumors developed in KPCY mice on a C57BL/6 background.38 PANC-1 was used for in vitro experiments as a representative human PDAC cell line. Cells were grown up to 20 passages in DMEM (Dulbecco's modified eagle medium medium with pyruvate, L-glutamine and D-glucose; Gibco, #41966029) supplemented with 5% fetal bovine serum (FBS) (Gibco, #10270106). All the cell lines were analyzed for short tandem repeat (STR) finger- printing and tested for Mycoplasma routinely. Introduction Bulk RNAseq gene analysis confirms the role of the myeloid-dependent adenosine pathway in PDAC survival, underpinning the importance of our results for understanding the biological complexity and the clinical utility of the adenosine pathway inhibition. g g y KPC mice used for the interventional study were bred at the CRUK Beatson Institute and maintained on a mixed background. All work was performed under UK Home Office license and approved by the University of Glasgow Animal Welfare and Ethical Review Board (ref. n. PP8411096). Mice of both sexes, in similar propor- tions, were used in all cohorts. Mice suspected to have PDAC following palpation were anesthetized in 0.2 L/ min medical air and isoflurane and then underwent three-dimensional (3D) ultrasound imaging using the VisualSonics Vevo 3100 ultrasound system with MX550D 40 µm resolution transducer (Fujifilm). Mice were randomly assigned to one of the four treatment groups (A: vehicles+isotype; B: AZD6738+gemcitabine; C: AZD4635+2c5mIgG1; D: AZD6738+gemcitabine + AZD4635+2c5mIgG1) once tumors were confirmed by imaging, and follow-up scans were performed weekly until endpoint was reached. Schedule of the treatments are specified in the results and figures sections. Mice were culled when exhibiting moderate symptoms of pancreatic cancer (see above). Statistical assessment of survival was carried out by Kaplan-Meier and log-rank analysis. Anal- ysis of ultrasound images was performed using Vevo Lab software (V.3.1.1) from VisualSonics. In 3D mode, stacked images of the tumor were imported and the tumor border annotated, allowing a 3D construct to be formed. Mice and in vivo experiments Tumor allograft experiments were performed in the animal facility (Biological Resource Unit, BRU) of the CRUK Cambridge Institute, in accordance with the UK Animals Scientific Procedures Act 1986, with approval from the CRUK Cambridge Institute Animal Ethical Review and Welfare Body (ref. n. PP6047951 and PPL7008363). 8–12 weeks old female C57BL/6 mice were used for in vivo experiments and were purchased from Charles River (UK). g When indicated the following drugs were used: AZD6738 (ATRi; 25 mg/kg daily for 4 days), AZD4635 (Adora2ai; 50 mg/kg two times a day), 2c5mIgG1 (anti- CD73; 10 mg/kg two times a week), AB740080 D265A (anti-PD-L1: 10 mg/kg two times a week), NIP228 mouse IgG1 control kappa (isotype; 10 mg/kg two times a week) and NIP228 muIgG1 D265A (isotype; 10 mg/kg two times a week) were provided by AstraZeneca. The antibody anti- CD73 2c5mIgG1 is a murine IgG1 with minimal Fc medi- ated activity39; gemcitabine hydrochloride (Tocris, 3259) was used at 100 mg/kg two times a week; InVivoPlus anti- CD40 (clone FGK4.5/FGK45; Bio X Cell BE0016-2) and InVivoPlus rat IgG2a isotype control, anti-trinitrophenol (clone 2A3; Bio X Cell BE0089) were used as a single injection of 100 µg. InVivoPlus anti-CTLA-4 (clone 9H10; Bio X Cell BP0131) or InVivoPlus isotype control poly- clonal Syrian hamster IgG (Bio X Cell BP0087): 200 µg/ dose×three times. Tumor allograft studies were performed with technical assistance from CRUK-CI BRU staff. Mice were subcu- taneously injected in the right flank with 1×106 KPCY- derived cells in 50% phosphate-buffered saline (PBS) and 50% Matrigel basement membrane matrix (#354234, Corning). In the interventional experiments, mice were treated as indicated, starting 12–14 days from tumor cell implantation, to allow the microenvironment to estab- lish. Tumor volume was calculated using the formula; (π/6)×(width)2×length. Tumor response was defined based on the % of change of the longest diameter from start of therapy (stable disease (SD) <20% increase and <30% decrease of target lesion according to the response evaluation criteria in solid tumours or RECIST v. 1.1). Mice were then killed at specific endpoints (eg, 14 days from start of treatment) or when the tumor reached 2000 mm3 (or before in case of appearance of clinical signs). Human cell line viability assay For viability experiments, 3000 PANC-1 cells/well were plated into a 96-well plate overnight and then dosed with IC50 for each compound (gemcitabine 0.5 µM; oxal- iplatin 30 µM; docetaxel 0.5 nM; 5-FU 52 µM; cisplatin 20 µM) either alone or in combination with oleclumab at 1 nM. For combination and pretreatment experiments, cells were either pretreated with oleclumab at 1 nM for indicated time (2 hours or 24 hours) before addition of chemotherapies at IC50s. Viability was assessed using CellTiter-Glo as per manufacturer’s instructions at either 3 or 7 days and expressed as percentage of cells in the treated wells over control. Oleclumab (MEDI9447) was kindly provided by AstraZeneca. Incucyte time lapse imaging KPCY-derived cells were plated at 2500 cells/well density in a 96-well black-wall plate (at least three wells per condi- tion). Cells were grown in cell culture medium supple- mented with the indicated concentration of anti-CD73 (2c5mIgG1) or isotype (NIP228 mouse IgG1 control kappa) antibody in triplicate. Images were acquired with 10× objective, every 3 hours from three different fields per well using Incucyte Live cells imaging microscope (Essen BioScience). Confluence was calculated as the average of the three fields using the Incucyte algorithm. Experiments were repeated at least three times. Flow cytometry y y Single cell suspensions were aliquoted in a round-bottom 96-well/plate (CoStar, 3879) and stained with live/dead fixable stain (Invitrogen, L34962; 1:100 in PBS) for 10 min at room temperature. After washing in MACS buffer, cells were FC-blocked with anti-CD16/32 antibody (BioLegend Cat# 101320, RRID:AB_1574975; 1:100) for 5 min. Then, antibodies for surface staining were added and incubated at 4°C for 30 min. After washing, cells were fixed with FACS (fluorescence-activated cell sorting) fix buffer (PBS+1% formaldehyde+0.02 g/mL glucose+0.02% sodium azide) for 10 min and then washed and resuspended in MACS buffer for FACS analysis. For intracellular staining, cells were fixed with fixation/perme- abilization buffer (Invitrogen 00-5123-43 and 00-5223-56) for 15 min, then washed in perm buffer and stained with the relevant antibody in perm buffer for 60 min. Cells were then washed and resuspended in MACS buffer for FACS analysis. Samples were acquired using BD-Sym- phony flow cytometer and the generated FCS files were analyzed using FlowJo V.10 software (RRID:SCR_008520). The following antibodies were used and gating strat- egies are shown in the Online supplemental figures (myeloid populations and lymphoid population; online supplemental figure1A,B respectively): BV786-CD45 (BD Biosciences Cat# 564225, RRID:AB_2716861; 1:200), APC/Fire750-CD3 (BioLegend Cat# 100248, RRID:AB_2572118; 1:50), BV650-CD8 (BioLegend Cat# 100741, RRID:AB_11124344; 1:100), BV711-CD4 (BioLegend Cat# 100549, RRID:AB_11219396; 1:200), APC-Foxp3 (Invitrogen Cat# 17-5773-82, RRID:AB_469457; 1:100), FITC-CD19 (BioLegend Cat# 115505, RRID:AB_313640; 1:200), BV510- CD11b (BioLegend Cat# 101245, RRID:AB_2561390; 1:200), PerCP/Cy5.5-CD44 (BioLegend Cat# 103031, RRID:AB_2076206; 1:200), BV421-PD-1 (BioLegend Cat# 135218, RRID:AB_2561447; 1:100), BV421- PD-L1 (BioLegend Cat# 124315, RRID:AB_10897097; 1:100), PE/Cy7-CD39 (BioLegend Cat# 143805, RRID:AB_2563393; 1:100), PE-CD73 (BioLegend Cat# 127206, RRID:AB_2154094; 1:100), FITC-F4/80 (BioLegend Cat# 123108, RRID:AB_893502; 1:200), PE/ Cy7-CD206 (BioLegend Cat# 141719, RRID:AB_2562247; 1:100), PerCP/Cy5.5-Ly6C (BioLegend Cat# 128012, RRID:AB_1659241; 1:100), APC/Cy7-Ly6G (BioLegend Cat# 127624, RRID:AB_10640819; 1:100), AF700- MHC-II (BioLegend Cat# 107629, RRID:AB_2290801; 1:200), PE-CD11c (eBioscience Cat# 12-0114-83, RRID:AB_465553; 1:200), BV605-NKp46 (BioLegend Cat# 137619, RRID:AB_2562452; 1:25), APC-Adora2a Clonogenic assay KPCY-derived cells were seeded at 200 cells/well in a 6-well plate. After 24 hours, cells were treated with different concentrations of anti-CD73 (2c5mIgG1) anti- body (1, 10, 100 µg/mL) or isotype (NIP228 mouse IgG1 g KrasLSL-G12D/+; Trp53LSL-R172H/+; Pdx1-Cre (KPC) mice for tumor phenotyping, were obtained from a breeding colony maintained by the CRUK-CI Genome Editing Core 3 Graziano V, et al. J Immunother Cancer 2023;11:e006457. doi:10.1136/jitc-2022-006457 Open access lysis buffer (1 mL; 0.15M ammonium chloride; 10 mM potassium hydrogen carbonate; 0.1 mM EDTA; pH 7.4 (adjusted with KOH) was then used to resuspend spleno- cytes, 3 min at room temperature and then washed with MACS buffer and centrifuged at 300 g for 5 min. Samples were eventually resuspended in 200–400 µL of MACS buffer. control kappa) for 8 days. Antibodies were added every 3 days. On day 8, colonies were stained following the sulforhodamine (SRB) protocol previously described.40 Images were taken and analyzed using GelCount (Oxford Optronix). Colony forming efficiency was calculated as a ratio between the number of colonies and number of plated cells. Surviving fractions were calculated as the ratio between wells treated with anti-CD73 antibody and the ones treated with isotype. At least three wells per condition were plated for each of the three replicates per experiment. control kappa) for 8 days. Antibodies were added every 3 days. On day 8, colonies were stained following the sulforhodamine (SRB) protocol previously described.40 Images were taken and analyzed using GelCount (Oxford Optronix). Colony forming efficiency was calculated as a ratio between the number of colonies and number of plated cells. Surviving fractions were calculated as the ratio between wells treated with anti-CD73 antibody and the ones treated with isotype. At least three wells per condition were plated for each of the three replicates per experiment. Single cell suspension preparation For experiments in subcutaneous (s.c.) allografts, tumors were weighed and placed in RPMI and finely minced with scissors in a 2 mL tube, which was then washed with up to 2.5 mL of digestion buffer (Tumor Dissociation Kit, Miltenyi, 130-096-730) plus deoxyribonuclease I (300 µg/ mL, Sigma, DN25-1G). Dissociation was performed using the protocol suggested by Miltenyi. For KPC tumors, a trypsin inhibitor (250 µg/mL, Sigma, T6522) was added to the digestion buffer. Following the digestion, the samples were passed through a 70 µM strainer filter (Greiner Bio- One, 542–070) washed with MACS buffer (PBS+0.1% FBS and 2 nM EDTA). Mouse spleens, inguinal and mesenteric lymph nodes were mashed on a 100 µM filter (Greiner Bio-One, 542–000) over a 50 mL tube, using a syringe plug and the filter was washed with MACS buffer and centrifuged (at 4°C as for all the following centrifugation). Red cell 4 Graziano V, et al. J Immunother Cancer 2023;11:e006457. doi:10.1136/jitc-2022-006457 Open access (Novus Biotech Cat# NBP1-39474APC; 1:150), APC-CD86 (BioLegend Cat# 105012, RRID:AB_493342; 1:100). Gating strategy for immune subpopulations is shown in online supplemental figure 1A,B. For CD73 in vitro staining of KPCY-derived cell lines, cells were stained as described above with live/dead fixable stain and PE-CD73 antibody and analyzed with BD-Symphony flow cytometer. For in vitro treatment, cells were treated with anti-CD73 (2c5mIgG1) or isotype (NIP228) antibody at a concen- tration of 10 µg/mL for 24 hours. Competitive staining was performed before this experiment to confirm there was no competition between 2c5mIgG1 and TY11.8 (PE- CD73) clones (data not shown). data processing was performed in SCiLS Lab (V.2021b, Bruker Daltonik, Bremen, Germany). data processing was performed in SCiLS Lab (V.2021b, Bruker Daltonik, Bremen, Germany). , , y) MALDI-MSI analysis was performed on a rapifleX Tissuetyper instrument (Bruker Daltonik, Bremen, Germany) operated in negative detection mode. 9-Aminoacridine prepared in 80:20 methanol:water was used as an MALDI matrix and spray deposited using an automated spray system (M3-Sprayer, HTX technologies, Chapel Hill, North Carolina, USA). MALDI experiments were performed with a spatial resolution of 50 µm. A total of 400 laser shots were summed up per pixel to give the final spectra. For all experiments the laser was oper- ated with a repetition rate of 10 kHz. All raw data were directly uploaded and processed in SCiLS lab (V.2021b) software packages. Imaging mass cytometry IMC was performed on a slide which had already been analyzed by DESI-MSI. Antibodies used for IMC staining are shown in online supplemental table 1. Untagged antibodies were tagged in house, using Fluidigm Maxpar Antibody Labeling Kit, according to manufacturer’s instructions. Following DESI-MSI analysis, the slide was fixed with 4% paraformaldehyde in PBS for 10 min. The slide was washed 3×5 min in PBS, permeabilized for 5 min with 1:1000 dilution of Triton X-100 in casein solution, washed 3×5 min in PBS, and blocked for 30 min with casein solution. Antibodies were diluted to an appropriate concentration in casein solution and the slide incubated overnight with the antibody solution at 4°C. The slide was washed 3×5 min in PBS and nuclei were stained with DNA intercalator-iridium at a dilution of 1:400 in PBS for 30 min. The slide was washed 3×5 min in PBS, 30 s in deionized water, then dried for storage at room tempera- ture until analysis. A region for IMC analysis was selected using consecutive H&E stained sections and the DESI-MSI results. A box with approximately 2×1.8 mm area was selected for analysis to include necrotic, necrotic margin and viable tumor regions. IMC analysis was performed using a Hyperion instrument (Fluidigm Corporation, San Francisco, California, USA) with an ablation energy of 6 db an ablation frequency of 200 Hz. IMC images were produced using MCD viewer (V.1.0, Fluidigm) and analysis was performed using HALO (Indica labs). Tissue regions were classified using random forest with all markers included. Cells positive for each marker were manually optimized by setting a cell intensity threshold. Values for the numbers of positive cells for markers of interest were exported for analysis in GraphPad Prism V.8 (RRID:SCR_002798) Mass spectrometry imaging DESI-MSI analysis was performed on a Q-Exactive mass spectrometer (Thermo Scientific, Bremen, Germany) equipped with an automated two-dimensional-DESI ion source (Prosolia, Indianapolis, Indy, USA) operated in negative ion mode, covering the applicable mass range up to an m/z of 1000, with a nominal mass resolution of 70,000. The injection time was fixed to 150 ms resulting in a scan rate of 3.8 pixel/s. The spatial resolution was adapted between experiments to allow acquisition of the data for all directly compared samples within a single experiment of 48 hours, with pixel sizes ranging from 35 to 75 µm. A home-built Swagelok DESI sprayer was oper- ated with a mixture of 95% methanol, 5% water deliv- ered with a flow rate of 1.5 µL/min and nebulized with nitrogen at a backpressure of 6 bar. The resulting .raw files were converted into .mzML files using ProteoWizard msConvert42 (V.3.0.4043) and subsequently compiled to an .imzML file (imzML converter43 V.1.3). All subsequent Single cell suspension preparation All DESI and MALDI data and images were normalized to the total ion current to compensate for signal variation across the course of the experiments. Data segmentation pipeline is shown in online supple- mental materials and methods. Tissue preparation for mass spectrometry imaging and imaging mass cytometry analysis PDAC mouse tumors were snap frozen in liquid nitrogen immediately after resection and the tissues were embedded in a hydroxypropyl methylcellulose (HPMC)/polyvin- ylpyrrolidone (PVP) hydrogel as previously described.41 Sectioning was performed on a CM3050 S cryostat (Leica Biosystems, Nussloch, Germany) at a section thickness of 10 µm and the tissue sections were immediately thaw mounted and dried under a stream of nitrogen and sealed in vacuum pouches to preserve the metabolic integrity of the sections. Tissue sections for DESI (Desorption Elec- trospray Ionization)-MSI and imaging mass cytometry (IMC) were thaw-mounted onto Superfrost microscope slides (Thermo Scientific Waltham, Massachusetts, USA), while sections prepared for MALDI (Matrix-Assisted Laser Desorption Ionization)-MSI were thaw mounted onto conductive ITO coated slides (Bruker Daltonik, Bremen, Germany). PVP (MW 360 kDa) and HPMC (viscosity 40–60 cP, 2% in H2O (20 C) were purchased from Merck (Darmstadt, Germany). Methanol, water, isopentane and isopropyl alcohol were obtained from Fisher Scientific (Waltham, Massachusetts, USA). Open access Open access CI. Briefly, tissues were removed from the mouse at the endpoint and immediately formalin-fixed for 24 hours. Fixed tissues were then processed, embedded in paraffin and sectioned (3 µm sections). Following dewaxing and rehydration, as standard, antigen retrieval was performed using Leica’s Epitope Retrieval Solution 2 (Tris EDTA) at 100°C for 20 min. Additional protein block from Dako (X090930-2) was applied. The staining using anti-mouse CD8 (Cell Signaling, #98941), anti-mouse Foxp3 (Affyme- trix. #14–5773) and anti-mouse p53 (Novocastra; #NCL- L-p53-CM5p) antibodies, was performed on Leica’s automated BOND-III platform in conjunction with their Polymer Refine Detection System (DS9800) and a modi- fied version of their standard template. Slides were dehy- drated and cleared in xylene on Leica’s automated ST5020 before sections were mounted on Leica’s coverslipper, CV5030 (mounting media: DPX Mountant for histology; Sigma-Aldrich, 06 522–500 ML) and scanned using a ScanScopeAT2 (Aperio Leica Biosystems). Quantifica- tion of viable tumor tissue was performed after exclusion of necrotic area using the Halo software V.3.3.2541.405 (Indica Labs). Cell density was calculated as the number of positive cells×mm2 of tumor tissue analyzed. Sections of mouse spleen were used on each slide as internal control. group), sample 23,729 showed minimal necrosis, low peri- necrotic adenosine and a high ATP/AMP ratio suggesting a very high energetic state. This identified the sample as a potential outlier which was confirmed on visual inspec- tion of a PCA (Principal Component Analysis) plot (online supplemental figure 1C). It was excluded prior to downstream analysis. y For the remaining 11 samples, initially genes were filtered to maintain only genes that were expressed at a reasonable level in >5 treatment conditions using the filterByExpr() command from the edgeR package (V.3.36.0). Differential gene expression analyses were performed on raw read counts of the combined data object of all 11 samples. To identify significantly expressed genes between Adoi and control groups, we used a Wald test within the DESeq2 package (RRID:SCR_000154, V.1.34.0). Genes were considered differentially expressed when the anal- ysis resulted in an adjusted p value (corrected for multiple testing using the Benjamini and Hochberg method) below 0.05. The volcano plot was generated using the EnhancedVolcano package (V.1.12.0) with the addition of custom code. RNA sequencing RNA was extracted from 12 s.c. allograft tumor tissues (six mice of the vehicle+isotype group or control and six mice of the AZD4635+2c5mIgG1 group or Adoi) weighing up to 30 mg. Tissues were first disrupted and homogenized using TissueLyser II and then RNA was extracted using a Qiagen RNAeasy kit, according to the manufacturer’s instructions. RNA was then quantified using a Qubit 3.0 (Life Technologies) and purity and quality were assessed using an Agilent 4150 (G2992AA) TapeStation system (Agilent). Library construction was followed by paired-end 50 bp sequencing on a NovaSeq 6000 sequencer. Open access Gene set enrichment analysis of 712 genes identified as differentially expressed with p adjusted ≤0.05 and log2 (fold change) ≤−0.58 was performed via the Enrichr (RRID:SCR_001575) (online supplemental table 2) server database for Kyoto Encyclopedia of Genes and Genomes (KEGG) (https://www.genome.jp/kegg/) pathways and Gene Ontology (GO): Biological processes (http://gene- ontology.org/). Subsequently, enriched terms ranked for significance for each database were downloaded and are reported in online supplemental table 4 and 5. Terms of interest were selected from the top 15 ranks in each table. Genes from this study which were shown to be enriched in these terms of interest were then selected to be displayed in a heatmap. Raw counts were normalized with DESeq2 (RRID:SCR_000154) prior to visualization of gene expres- sion levels with pheatmap (V.1.0.12). Please refer to the online supplemental methods and materials references section for all of the above. For the analysis of the lung metastatic burden of any individual mouse, the four right lobes and the left lobe were cut into multiple pieces and together fixed and then embedded, then treated as above. p53 staining was used for helping the detection of smaller lesions (minimum of five cells). Analysis was performed using Halo software and expressed as % of metastatic areas/total lung area analyzed. Mice with intra-abdominal/thoracic organs direct infiltration were excluded from the analysis. Analysis of human PDAC available data sets and generation of PDAC-specific adenosine signature In order to the evaluate the correlation of the adenosine- related gene expression profile to human PDAC we analyzed 712 genes which had at least a 50% decreased expression (log2 fold change <−0.58) following adenosine inhibition treatment, of which 561 had a human ortholog (online supplemental table 2 and 3). Immunohistochemistry Immunohistochemistry (IHC) was performed as previ- ously described40 in the histopathology core at the CRUK Graziano V, et al. J Immunother Cancer 2023;11:e006457. doi:10.1136/jitc-2022-006457 5 Bioinformatics analysis Sequencing files in FASTQ format were aligned against the GRCh38 mouse genome using HISAT2 (RRID:SCR_015530) with default parameters. Samtools (RRID:SCR_002105) was used to create, index and merge BAM files of reads from different lanes belonging to indi- vidual samples. FeatureCounts (RRID:SCR_012919) was used to quantify gene-level expression of transcripts. All downstream analyses were completed in R V.4.1.2. Prior to analysis, MSI data for sequenced samples were exam- ined. From the vehicle/isotype-treated arm (control For the analysis of the adenosine-related gene expres- sion in Bailey44 PDAC subtypes (ADEX, immunogenic, squamous and pancreatic progenitor) we derived z-scores for the 517 genes analyzed in the data set (out of the 561 genes) for the 97 patients with RNAseq data and subtype information (https://www.cbioportal. org/, RRID:SCR_014555). The z-score of all genes were summed per patient and the total number represented as Graziano V, et al. J Immunother Cancer 2023;11:e006457. doi:10.1136/jitc-2022-006457 6 Open access the adenosine pathway gene score as previously shown18 and in online supplemental materials and methods. the adenosine pathway gene score as previously shown18 and in online supplemental materials and methods. CD39 and Adora2a (online supplemental figure 2B,C). Exposing cells to an anti-CD73 antibody (2c5mIgG1) reduced significantly the detection of CD73 in all the cells after only 24-hour treatment (p<0.05 in all cell lines, figure 1B), but this did not translate into inhibition of cell growth after short or long exposure at high concentra- tions. In addition, confluency experiments showed that the treatment did not affect the proliferation of any of the cell lines over a period of 72 hours (figure 1C and online supplemental figure 2D,E), and colony-forming experiments performed on 2838c3 (IOT-responsive) and 6419c5 (IOT-resistant) demonstrated no differences in terms of number or size of the colonies formed after 8 days of continuous treatment (figure 1D,E and online supplemental figure 2F). Accordingly, in vitro inhibition of CD73 was performed on human PDAC cell line PANC-1 using anti-human CD73 oleclumab (online supplemental figure 2G-I). Oleclumab did not reduce tumor cell growth when used alone or in combination with multiple cyto- toxic agents, either as a concomitant treatment (online supplemental figure 2G) or when used as a pretreatment (online supplemental figure 2H,I). In order to evaluate whether a direct effect of anti-CD73 exposure affects cell proliferation, reducing adenosine formation, we cultured KPCY-derived cell lines with increasing concentrations of AMP and 5’-N-(Ethylcarboxamido)adenosine (a stable form of adenosine). Bioinformatics analysis We were able to demonstrate that adenosine and AMP have no effect on the proliferation capacity of these cell lines (online supplemental figure 2G,H), corroborating the hypothesis of a non-cancer cell direct effect of anti-CD73 therapeutic approaches. pp For the generation of a PDAC-specific adenosine signa- ture and application of this to PDAC survival, from the list of 561 human ortholog genes, we manually curated the ones-related without ambiguity to the major biolog- ical processes implicated in PDAC pathogenesis and indicated by pathway analysis (hypoxia, immunity and extracellular matrix organization). Of these genes, only those that correlated positively or negatively to survival in PDAC (https://kmplot.com)45 and were significantly co-expressed with CD73 and/or Adora2a in public data sets (Bailey et al or The Cancer Genome Atlas, TCGA) were selected. A final list of 52 genes was analyzed (online supplemental table 6). Using PDAC specific data from TCGA46 available in https://www.cbioportal.org/, we derived the z-score of these 52 genes for each patient with known disease- specific survival (DSS), progression-free survival (PFS) and disease-free survival (DFS). The z-scores for all genes were summed up for each patient and were deemed high adenosine score if >0 or low adenosine score if <0, as previously shown.18 Comparison between KPCY-derived cell lines allograft and KPC autochthonous tumors Despite the lack of activity in our cell line experiments, preliminary responses have been reported in early phase clinical trials when quemliclustat (a CD73 small molecule inhibitor) or oleclumab were combined with PD-(L)1 [Programmed cell death-(ligand) 1] inhibition and standard-of-care therapy.49–51 As we hypothesized that this might be the result of impact on the TME, we investigated the expression of this pathway on the tumor-infiltrating immune cells for different murine PDAC models, including KPCY-derived cell line allografts (with differen- tial response to IOT) and autochthonous KPC tumors. In order to understand the complexity and similarities of the immune system in these models we first compared the immune infiltration of the cell line allografts to the KPC model. Statistics GraphPad Prism V.8 (RRID:SCR_002798) was used for statistical analyses. Analysis and comparisons of two groups was performed with two-tailed unpaired Student’s t-test when assuming Gaussian distribution or Mann-Whitney test. Analysis of three or more groups was performed with one-way analysis of variance with Tukey’s multiple comparisons post-test analysis unless otherwise specified. Kaplan-Meier analysis with log-rank Mantel-Cox test was used to evaluate difference in survivals. Differences were considered significant when p<0.05. Data availability statement The data generated in this study are available within the article and its online supplemental data files or from the corresponding authors on reasonable request. Code for differential expression analysis and visualization of RNAseq data is available via GitHub https://github.com/ ka-lw/AdenoPDAC.47 Results (A) Representative histogram of CD73 expression on KPCY-derived cell line (2838c3) in flow cytometry. (B) CD73 expression was evaluated on KPCY-derived cell lines treated with 10 µg/mL of NIP228 (IgG isotype) or 2c5mIgG1 (anti-CD73 neutralizing antibody) for 24 hours. (C) 2838c3 (left) and 6419c5 (right) cell lines were grown with increasing concentration of anti-CD73 or isotype (100 µg/ mL) and confluency was evaluated using Incucyte time lapse imaging for up to 72 hours. For each experiment, three different wells per condition were used per experiment. (D–E) Representative images (D) and graphs (E) showing survival fraction of cells (2838c3 left, 6419c5 right) from the colony-forming experiment following 8-day treatment with anti-CD73 or isotype. For each experiment, three different wells per condition were used. All data are presented as mean±SEM from experiments repeated three times. Statistical analysis was performed with two-tailed unpaired Student’s t-test (B) mixed-effect model (C) and one- way analysis of variance with post-test analysis for multiple comparisons; p values are shown in the graphs when considered significant (p<0.05). IOT, immuno-oncology therapy; FMO, fluorescence minus one; MFI, mean fluorescence intensity. double-expressing immune cells, when compared with secondary lymphoid organs (spleen and nodes). In partic- ular, the majority (65–91% in tumor vs 36–56% in spleen) of tumor-infiltrating CD11b+ myeloid cells express the two receptors, due to an increase in expression of CD73, given that those cells are normally CD39+ (figure 2A,B). Similar results were shown for Tregs and CD8+ T cells, which are normally CD73+ and displayed an increase in expression of CD39 in tumor, compared with the secondary lymphoid organs counterparts (online supple- mental figure 4A; p<0.05). There was no significant differ- ence in these findings when comparing the two models, despite their differential response to IOT. We then confirmed these findings in KPC autochthonous tumors and found similar results, with a significant increase of CD39/CD73 double expressing CD11b+ myeloid cells infiltrating the tumors compared with spleens (mean 77% vs 36% p<0.0001, figure 2C,D), harvested from the same mice. Of note, four KPC mice had synchronous metastases (three liver and one spleen), and in three of figure 3A and have a similar total CD4+ T cells (online supplemental figure 3C) and regulatory T-cell (Tregs) infiltration (mean 3.2% vs 1% vs 0.7%, online supple- mental figure 3B) to IOT-resistant tumors. Moreover, KPC tumors showed greater heterogeneity regarding myeloid infiltrating populations (online supplemental figure 3D–H). Results Adenosine pathway expression on KPCY-derived cell lines Human PDAC tumor cells express CD73 and demon- strate weak sensitivity to the targeting of CD73 in vitro.36 48 For this reason, we sought to investigate whether murine KPCY-derived cell lines (which are associated with contrasting ability to generate IOT-resistant or respon- sive tumors when reimplanted in syngeneic mice) express the proteins of the canonical adenosine pathway (CD39, CD73 and Adora2a). We found that, as in human cells, mouse PDAC cell lines express CD73 (from 72% to 99% of cells; figure 1A and online supplemental figure 2A) but demonstrate negligible or no expression of The immunosuppressive characteristics identified in autochthonous KPC tumors, appear to be more aligned with those of the IOT-resistant model. In particular regarding lymphocyte populations, KPC tumors are usually infiltrated by a low number of CD8+ T cells (mean number of CD45+ cells for 2838c3 is 6.9%, 0.9% for 6419c5 and 1.9% for KPC tumors), which express lower levels of PD-1, a known marker of activation/exhaustion (mean 70% vs 13% vs 10%) as shown in online supplemental Graziano V, et al. J Immunother Cancer 2023;11:e006457. doi:10.1136/jitc-2022-006457 7 Figure 1 Expression of CD73 on KPCY-derived cell lines and response to anti-CD73 in vitro inhibition. (A) Representative histogram of CD73 expression on KPCY-derived cell line (2838c3) in flow cytometry. (B) CD73 expression was evaluated on KPCY-derived cell lines treated with 10 µg/mL of NIP228 (IgG isotype) or 2c5mIgG1 (anti-CD73 neutralizing antibody) for 24 hours. (C) 2838c3 (left) and 6419c5 (right) cell lines were grown with increasing concentration of anti-CD73 or isotype (100 µg/ mL) and confluency was evaluated using Incucyte time lapse imaging for up to 72 hours. For each experiment, three different wells per condition were used per experiment. (D–E) Representative images (D) and graphs (E) showing survival fraction of cells (2838c3 left, 6419c5 right) from the colony-forming experiment following 8-day treatment with anti-CD73 or isotype. For each experiment, three different wells per condition were used. All data are presented as mean±SEM from experiments repeated three times. Statistical analysis was performed with two-tailed unpaired Student’s t-test (B) mixed-effect model (C) and one- way analysis of variance with post-test analysis for multiple comparisons; p values are shown in the graphs when considered significant (p<0.05). IOT, immuno-oncology therapy; FMO, fluorescence minus one; MFI, mean fluorescence intensity. Figure 1 Expression of CD73 on KPCY-derived cell lines and response to anti-CD73 in vitro inhibition. Results These results suggest that our IOT-resistant and responsive models stand out as the extreme clonotypes which can arise from the complex and heterogeneous biology found in KPC autochthonous tumors. The adenosine pathway is enriched in immune cells infiltrating PDAC models PDAC, pancreatic ductal adenocarcinoma; Tregs, regulatory T cells; dLN, draining lymph node. classification and segmentation approaches were driven by tissue-defining metabolic patterns. Areas characterized by a high energetic state defined by a high abundance of ATP and ADP and a low abundance of depleted high energy phosphates such as AMP, were called viable tumor. In contrast, areas of tumor adjacent to necrosis (termed necrotic margin) were characterized by high abundance of lactate, products of ribonucleotide catabolism (ie, xanthine and hypoxanthine) and other metabolites asso- ciated with tissue hypoxia and an overall energy-deprived state (figure 3A,B). We found that adenosine is present in high concentrations in the microenvironment of PDAC murine models, although showing a heterogenous distri- bution, with high abundance in the hypoxic necrotic margin areas (figure 3C and online supplemental figure 4D). The IOT-resistant model 6419c5 shows a higher abun- dance of adenosine, mostly due to the paucity of necrotic areas in this 2838c3 IOT-responsive model (figure 3A–C). This trend of adenosine expression was then evaluated in other KPCY-derived allografts, confirming the lower these, myeloid cells infiltrating the metastases were also enriched for CD73+CD39+ double expression (online supplemental figure 4C) when compared with secondary lymphoid organs. Significant increased co-expression was also observed for Tregs and CD8+ T cells in KPC tumors when compared with mesenteric or inguinal lymph nodes (online supplemental figure 4B). An increased percentage of CD39/CD73 double expressing CD8 T cells and Tregs was noted in the spleens (online supplemental figure 4B) if compared with what was found in non-tumor bearing mice (not shown) or s.c. tumor bearing mice, suggesting trafficking of immune lymphoid populations between primary lesions and closer lymphoid organs. The adenosine pathway is enriched in immune cells infiltrating PDAC models We hypothesized that the adenosine pathway might have a more impactful role in the TME, as opposed to a cell autonomous effect. For this reason, we investigated the expression of the adenosine pathway components on tumor-infiltrating immune cells which represent a significant proportion of cells seen in PDAC lesions. We showed a highly significant enrichment in both the IOT- resistant and IOT-responsive models for CD39+CD73+ Graziano V, et al. J Immunother Cancer 2023;11:e006457. doi:10.1136/jitc-2022-006457 8 Open access Open access Figure 2 The adenosine pathway members are expressed on PDAC-infiltrating immune cells. (A) Representative flow cytometry plots showing expression of CD39 and CD73 on myeloid population (left), Tregs (middle) and CD8+ T cells (right) from KPCY-cell line derived tumor (upper) and matched spleen (lower) (N=5 mice per group). (B) Box and whisker graph showing CD39+CD73+ double expression on CD11b+ cells for 2838c3 (N=5) and 6419c5 (N=5) in tumors, matched spleens and tumor draining lymph nodes. (C–D) Representative flow cytometry plots (C) and box and whisker graph (D) showing CD39+CD73+ double expression on CD11b+ cells infiltrating autochthonous KPC tumors (N=13). All data are presented as interleaved box and whiskers. Statistical analysis was performed using one-way analysis of variance with post hoc test analysis for multiple comparisons (B) and two-tailed unpaired Student’s t-test (D) p values are shown in the graphs when considered significant (p<0.05). PDAC, pancreatic ductal adenocarcinoma; Tregs, regulatory T cells; dLN, draining lymph node. ne pathway members are expressed on PDAC-infiltrating immune cells. (A) Representative flow Figure 2 The adenosine pathway members are expressed on PDAC-infiltrating immune cells. (A) Representative flow cytometry plots showing expression of CD39 and CD73 on myeloid population (left), Tregs (middle) and CD8+ T cells (right) from KPCY-cell line derived tumor (upper) and matched spleen (lower) (N=5 mice per group). (B) Box and whisker graph showing CD39+CD73+ double expression on CD11b+ cells for 2838c3 (N=5) and 6419c5 (N=5) in tumors, matched spleens and tumor draining lymph nodes. (C–D) Representative flow cytometry plots (C) and box and whisker graph (D) showing CD39+CD73+ double expression on CD11b+ cells infiltrating autochthonous KPC tumors (N=13). All data are presented as interleaved box and whiskers. Statistical analysis was performed using one-way analysis of variance with post hoc test analysis for multiple comparisons (B) and two-tailed unpaired Student’s t-test (D) p values are shown in the graphs when considered significant (p<0.05). Adenosine distributes primarily in the hypoxic areas surrounding necrosis As shown, necrosis was present in only one 2838c3 sample at 21 days post-implantation. Error bars represent SD. (C) MSI analysis showing relative abundance of adenosine in the different areas in 6419c5 and 2838c3 allografts at day 21 post-implantation. Bars represent means. (D–E) Representative plots (E) and summary graph (F) from flow cytometry analysis showing Adora2a expression on protumorigenic M2 macrophages in allografts (upper) derived from 6419c5 (left) and 2838c3 (right) implantation. Same expression is shown in M2 macrophages in matched spleens (lower) (8–9 mice per group were used). (F–G) Flow cytometry plots (G) and graph (H) showing expression of Adora2a in M2 macrophages comparing KPC (n=8) autochthonous tumors and matched spleens. (H) Box and whisker plot of the percentage of M2 macrophages positive for Adora2a comparing two allografts (6419c5 and 2838c3) and KPC tumors. Statistical analysis was performed using one-way analysis of variance with post hoc test analysis for multiple comparisons (E,H) and two-tailed unpaired Student’s t-test (G) p values are shown in the graphs when considered significant (p<0.05). IOT, immuno-oncology therapy; TAM, tumor associated macrophage; MFI, mean fluorescence intensity. distribution is spatially heterogeneous and targets myeloid subpopulations. (A) Mass spectrometry Figure 3 Adenosine distribution is spatially heterogeneous and targets myeloid subpopulations. (A) Mass spectrometry imaging (MSI) representative images showing adenosine expression and distribution in pancreatic ductal adenocarcinoma subcutaneous allografts (four mice per group) at day 21 post-implantation. Classification was obtained based on metabolites expression and is represented as follows: viable tumor (red), necrotic margins (green). (B) Relative tissue composition differences of viable tumor, necrotic margin and necrotic areas for 6419c5 (N=4) and 2838c3 (N=4) allografts at day 21 post- implantation. As shown, necrosis was present in only one 2838c3 sample at 21 days post-implantation. Error bars represent SD. (C) MSI analysis showing relative abundance of adenosine in the different areas in 6419c5 and 2838c3 allografts at day 21 post-implantation. Bars represent means. (D–E) Representative plots (E) and summary graph (F) from flow cytometry analysis showing Adora2a expression on protumorigenic M2 macrophages in allografts (upper) derived from 6419c5 (left) and 2838c3 (right) implantation. Same expression is shown in M2 macrophages in matched spleens (lower) (8–9 mice per group were used). (F–G) Flow cytometry plots (G) and graph (H) showing expression of Adora2a in M2 macrophages comparing KPC (n=8) autochthonous tumors and matched spleens. Adenosine distributes primarily in the hypoxic areas surrounding necrosis Given the enrichment of the adenosine pathway in the TME of PDAC models, we anticipated that eAdo might have been abundant in the TME. Using MSI we evalu- ated the presence and the distribution of the purinergic system in the TME of IOT-resistant tumors. The tissue 9 Graziano V, et al. J Immunother Cancer 2023;11:e006457. doi:10.1136/jitc-2022-006457 n access 3 Adenosine distribution is spatially heterogeneous and targets myeloid subpopulations. (A) Mass spectrometry g (MSI) representative images showing adenosine expression and distribution in pancreatic ductal adenocarcinoma aneous allografts (four mice per group) at day 21 post-implantation. Classification was obtained based on metabolites sion and is represented as follows: viable tumor (red), necrotic margins (green). (B) Relative tissue composition nces of viable tumor, necrotic margin and necrotic areas for 6419c5 (N=4) and 2838c3 (N=4) allografts at day 21 post- tation. As shown, necrosis was present in only one 2838c3 sample at 21 days post-implantation. Error bars represent MSI analysis showing relative abundance of adenosine in the different areas in 6419c5 and 2838c3 allografts at day 2 mplantation. Bars represent means. (D–E) Representative plots (E) and summary graph (F) from flow cytometry analysis g Adora2a expression on protumorigenic M2 macrophages in allografts (upper) derived from 6419c5 (left) and 2838c3 mplantation. Same expression is shown in M2 macrophages in matched spleens (lower) (8–9 mice per group were (F–G) Flow cytometry plots (G) and graph (H) showing expression of Adora2a in M2 macrophages comparing KPC (n= thonous tumors and matched spleens. (H) Box and whisker plot of the percentage of M2 macrophages positive for 2a comparing two allografts (6419c5 and 2838c3) and KPC tumors. Statistical analysis was performed using one-way s of variance with post hoc test analysis for multiple comparisons (E,H) and two-tailed unpaired Student’s t-test (G) p Open access ure 3 Adenosine distribution is spatially heterogeneous and targets myeloid subpopulations. (A) M Figure 3 Adenosine distribution is spatially heterogeneous and targets myeloid subpopulations. (A) Mass spectrometry imaging (MSI) representative images showing adenosine expression and distribution in pancreatic ductal adenocarcinoma subcutaneous allografts (four mice per group) at day 21 post-implantation. Classification was obtained based on metabolites expression and is represented as follows: viable tumor (red), necrotic margins (green). (B) Relative tissue composition differences of viable tumor, necrotic margin and necrotic areas for 6419c5 (N=4) and 2838c3 (N=4) allografts at day 21 post- implantation. Adenosine distributes primarily in the hypoxic areas surrounding necrosis (H) Box and whisker plot of the percentage of M2 macrophages positive for Adora2a comparing two allografts (6419c5 and 2838c3) and KPC tumors. Statistical analysis was performed using one-way analysis of variance with post hoc test analysis for multiple comparisons (E,H) and two-tailed unpaired Student’s t-test (G) p values are shown in the graphs when considered significant (p<0.05). IOT, immuno-oncology therapy; TAM, tumor associated macrophage; MFI, mean fluorescence intensity. 10 Graziano V, et al. J Immunother Cancer 2023;11:e006457. doi:10.1136/jitc-2022-006457 Open access abundance of adenosine in the IOT-responsive models and higher expression in one other IOT-resistant tumor (6694c2), particularly in the necrotic margins. A third IOT- resistant model (6422c1) showed levels of adenosine similar to the IOT-responsive tumors, suggesting that the expression of the adenosine pathway might not be a unique feature in the generation of immunosuppres- sion in murine PDAC (online supplemental figure 4E,F). The autochthonous KPC tumor model showed a higher degree of complexity in their histological composition (online supplemental figure 4G). MSI analysis demon- strated a primarily hypoxia-driven metabolic phenotype in which adenosine and other metabolites such as succinate, lactate and metabolites associated with purine metabo- lism were elevated. The hypoxic phenotype, and there- fore adenosine, was not limited to the margin around established necrosis, but found throughout the samples independent of established necrosis. This limited the ability to delineate the necrotic margin as it was possible in the KPCY-derived s.c. tumors, but confirmed the importance of hypoxia in the generation of adenosine. When we investigated the cell population distribution in the different areas using IMC in IOT-resistant tumors, we noted in the necrotic margin areas a 2.7-fold increase in the number of infiltrating CD11b+ myeloid cells (mean 1970 in the necrotic margin vs 730 in viable tumor CD11b+ cells/mm2), that led to a significant decrease of the ratio between cancer cells/myeloid cells (online supplemental figure 5A). This again suggests that myeloid cells have an instrumental role in the generation of adenosine in this aggressive model of PDAC. more, these findings were confirmed in KPC tumors where Adora2a was found to be increased in M2 macrophages infiltrating the lesions when compared with matched spleens (figure 3F,G). The KPC model demonstrated once again the heterogeneity of pancreatic lesions, which in terms of M2 macrophages, positive for the Adora2a receptor, covers the entire range of expression seen in the two s.c. models used (figure 3H). Adenosine distributes primarily in the hypoxic areas surrounding necrosis Notably, of three KPC mice where metastatic nodules were found, Adora2a expression was found retained in the M2 macrophages infiltrating the secondary lesions (online supplemental figure 5D, top panel). Lymphoid cells were negative for Adora2a both in tumors and in metastases (online supple- mental figure 5D, bottom panel). g p In addition to protumorigenic macrophages, Adora2a expression was found enriched in other myeloid immune populations infiltrating the tumors. In particular CD11b− DCs, CD11b+ DCs (online supplemental figure 5E), M1 macrophages (online supplemental figure 5F), and monocytic MDSCs (mo-MDSCs) (online supplemental figure 5G) for both models and granulocytic MDSCs (gMDSCs) for IOT-resistant tumors (online supplemental figure 5H) express significantly higher Adora2a amount when compared with matched spleens. This expression differs significantly between IOT-responsive and resistant models in CD11b+ DCs (mean MFI 2080±174 vs 3190±636, respectively; p=0.007), M1 macrophages (4520±983 vs 6940±1690; p=0.02) and mo-MDSCs (1890±479 vs 6110±1870; p=0.001) (online supplemental figure 5E–H). Targeting adenosine pathway delays tumor growth of murine pancreatic cancer representing a combinational therapeutic opportunity Expression of ADORA2A receptor on myeloid subpopulations of pancreatic cancer models Our data suggest a mechanism by which the myeloid popu- lation contributes to the protumorigenic functionality of the pancreatic cancer microenvironment, where eAdo generated by the myeloid cell populations and cancer cells would target and stimulate further the myeloid cell subpopulations, in particular the protumorigenic M2 macrophages. Therefore, we inhibited in vivo eAdo formation and function, using an antibody against CD73 (2c5mIgG1) and a small molecule inhibitor of Adora2a (AZD4635), a combination (Adoi) which would maxi- mize the inhibition of the axis. The 14-day treatment was started after the microenvironment was allowed to estab- lish (12–14 days after implantation) in the IOT-resistant allografts (figure 4A). The anti-CD73 was extremely effec- tive in reducing the expression of CD73 on the surface of all live cells (online supplemental figure 6A). MSI data confirmed marked reduction in adenosine forma- tion in the TME (figure 4B,C). In particular, adenosine was completely abolished in the viable tumor areas, while a small amount remained in the necrotic margins, accounting for a 95% decrease (figure 4C), highlighting the importance to block residual adenosinergic signaling downstream CD73 inhibition, co-targeting adenosine receptors. The effectiveness of the treatment on the extra- cellular purinergic pathway was also supported by the Having shown in these models that in the PDAC micro- environment, immunosuppressive adenosine is present abundantly, we then investigated which cells within the microenvironment might be responsive to this. We inves- tigated the expression of the adenosine A2a receptor (Adora2a, the receptor with the highest affinity for adenosine) that has been found to be frequently overex- pressed in human tumors, by flow cytometry. We found that Adora2a was highly expressed by tumor-infiltrating myeloid population when compared with the spleen (online supplemental figure 5B,C) and this expres- sion was significantly higher in the IOT-resistant model in terms of MFI (mean fluorescence intensity 10,000 vs 6700, p<0.0001) and % of Adora2a+ myeloid cells (15% vs 11%, p=0.009). In contrast, lymphoid popula- tions infiltrating the tumors were negative for Adora2a expression (online supplemental figure 5B, bottom). When comparing different subpopulations, protumor- igenic M2 macrophages, infiltrating both IOT-resistant and IOT-responsive PDAC showed high positivity for the receptor. The IOT-resistant model had higher expression of Adora2a compared with the IOT-responsive model (figure 3D,E; p<0.0001) and percentage of Adora2a+ M2 positivity (72% vs 43%; p<0.0001) (figure 3H). Once 11 Graziano V, et al. J Immunother Cancer 2023;11:e006457. Figure 4 continued decrease of molecules downstream of adenosine (adenine and inosine in viable tumor and necrotic margin areas) (online supplemental figure 6B,C), and the increase of upstream and alternative pathway molecules as AMP (in the necrotic margin, online supplemental figure 6D) and xanthine (in both viable tumor and necrotic margin, online supplemental figure 6E) respectively. There was no change in the distribution of ATP, ADP and hypoxan- thine (online supplemental figure 6F–H). decrease of molecules downstream of adenosine (adenine and inosine in viable tumor and necrotic margin areas) (online supplemental figure 6B,C), and the increase of upstream and alternative pathway molecules as AMP (in the necrotic margin, online supplemental figure 6D) and xanthine (in both viable tumor and necrotic margin, online supplemental figure 6E) respectively. There was no change in the distribution of ATP, ADP and hypoxan- thine (online supplemental figure 6F–H). p g ) These data suggest that targeting myeloid related, eAdo formation and effect would have an effect on tumor growth, making this approach a candidate for combina- torial therapeutic studies. Indeed, when combined with cytotoxic treatment (AZD6738, an ATR inhibitor and gemcitabine; figure 4F,G) or IOT [anti-CD40 agonist (F), anti-CTLA-4 (C) and anti-PD-L1 (P),FCP; online supplemental figure 7D], the adenosine modulation reduced further the tumor growth rate of the aggressive IOT-resistant 6419c5 tumor model. AZD6738/gemcit- abine alone was able to significantly slow the growth of the IOT-resistant model (2/7 SD (28.5%), but the addi- tion of the adenosine blocking (AZD4635+2c5mIgG1) led to further stabilization of the tumor growth in a 2-week regimen (p=0.003 vs AZD6738/gem alone; 4/7 SD (57.1%); figure 4G,H). These data supported the investigation of the same combination in autochthonous tumors in KPC mice (figure 4I) to assess whether the addition of Adoi to cytotoxic treatment (AZD6738+gem- citabine) prolonged survival in this model, considered a gold standard in this disease. As figure 4J shows, the combination of Adoi and ATRi/gem induced a threefold increase in median overall survival (mOS) in KPC mice compared with control groups (vehicles+isotype 12.5 days vs combo 41 days, p=0.0006). The four-drug regimen is significantly better than single schedule arms (mOS: Adoi 19 days and AZD6738+gemcitabine 13 days). A weekly ultrasound revealed a tendency for tumor stabilization in mice treated with cytotoxic therapeutics plus adenosine inhibition, when compared with the control arm during the first 2 weeks of treatment (figure 4K). Figure 4 continued adenosine expression and distribution in PDAC allografts treated with vehicle+isotype or Adoi, at day 14 from treatment start (six mice per treatment group). Classification was obtained based on metabolites expression and is represented as follows: viable tumor (green line), necrotic margins (yellow line). (D) Tumor growth ratio (left) and weight (right) of 6419c5 allografts in C57Bl/6 mice treated with anti-CD73+AZD4635 (N=16) or vehicle+isotype (N=15). (E) Representative image (left) and graph (right) showing % of area of the lung analyzed occupied by metastasis (met/lung areas×100) in vehicle+isotype (N=12) and anti-CD73+AZD4635 (N=13), evaluated for the presence of spontaneous occurrence of lung metastases. Every dot represents a single mouse. Tissues were stained with an anti-p53 antibody to highlight the presence of cancer cells. A group of more than five p53-positive cells was counted as metastasis. (F) Schedule of 6419c5 tumor allografts 14-day treatment as following (N=7 mice per group): vehicles+isotype, AZD6738+gemcitabine, anti-CD73+AZD4635, AZD6738+gemcitabine + anti-CD73+AZD4635. (G) Tumor growth ratio (14 days) of 6419c5 tumor allografts treated as above (H) percentage change in the long diameter length following 14 days of treatment per group. Number of mice with stable disease (SD, <20% increase and <30% decrease) are shown at the bottom. (I) Schedule of KPC mice treatment as following: vehicles+isotype (12 mice), AZD6738+gemcitabine (11 mice), anti-CD73+AZD4635 (7 mice), AZD6738+gemcitabine + anti-CD73+AZD4635 (combo, 12 mice). Adenosine inhibition was administered until endpoint, while AZD6738+gemcitabine was allowed up to 3 weeks. (J) Survival analysis of KPC mice treated as above, including median overall survival (mOS). (K) Percentage of tumor volume increased in vehicles versus combo group at day 7 and 14 of treatment. All data are presented as mean±SEM. Statistical analysis was performed using Mann-Whitney test (D–E,K) mixed-effect model (F) and one-way analysis of variance with post hoc test analysis for multiple comparisons (C). Log-rank Mantel-Cox test to evaluate difference in survivals (J) p values are shown in the graphs and considered significant when p<0.05. IOT, immuno-oncology therapy; IP, intraperitoneal. Figure 4 continued of the mice and blocking adenosine strongly reduced the metastatic burden (median of % mets/lung area 0.77% vs 2.6%; p=0.016; figure 4E). Graziano V, et al. J Immunother Cancer 2023;11:e006457. doi:10.1136/jitc-2022-006457 Expression of ADORA2A receptor on myeloid subpopulations of pancreatic cancer models doi:10.1136/jitc-2022-006457 Open access Figure 4 In vivo modulation of the adenosine pathway reduces tumor growth and metastasis and improves the efficacy of cytotoxic treatment. (A) Schedule of adenosine inhibition (Adoi) treatment. Treatment was started following 12–14 days from implantation and continued for 2 weeks. Antibody anti-CD73 (2c5mIgG1, murine IgG1) was dosed two times per week intraperitoneally at 10 mg/kg. Adora2a inhibitor (AZD4635) was given by oral gavage two times a day at 50 mg/kg. (B–C) Mass Figure 4 In vivo modulation of the adenosine pathway reduces tumor growth and metastasis and improves the efficacy of cytotoxic treatment. (A) Schedule of adenosine inhibition (Adoi) treatment. Treatment was started following 12–14 days from implantation and continued for 2 weeks. Antibody anti-CD73 (2c5mIgG1, murine IgG1) was dosed two times per week intraperitoneally at 10 mg/kg. Adora2a inhibitor (AZD4635) was given by oral gavage two times a day at 50 mg/kg. (B–C) Mass spectrometry imaging (MSI) representative images (B) and MSI analysis graph with relative abundance (a.u.) (C) showing Figure 4 continued on next page 12 Graziano V, et al. J Immunother Cancer 2023;11:e006457. doi:10.1136/jitc-2022-006457 Open access Figure 4 continued (C) Heatmap showing genes regulated during treatment (light gray for controls, dark gray for Adoi) which are part of the significantly different pathways according to KEGG. (D) Table (left) showing genes associated with innate immunity that are downregulated by Adoi treatment. Validation of RNAseq data (right graphs) through flow analysis of PD-L1 and CD206 expression (MFI ratio) on intratumoral live cells (8 mice in the control and 10 in the Adoi arms). The samples analyzed for validation were from different mice from the ones used for RNAseq. GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; RNAseq, RNA sequencing; PDL1, Programmed death-ligand 1; MFI; mean fluorescence intensity; ECM, extracellular matrix. Figure 5 Adenosine inhibition remodels the pancreatic ductal adenocarcinoma tumor microenvironment (A) Volcano plot related Figure 5 Adenosine inhibition remodels the pancreatic ductal adenocarcinoma tumor microenvironment. (A) Volcano plot related to 6419c5 allografts. Genes overexpressed in vehicle+isotype arm are on the left side (blue dots), and genes overexpressed in the anti-CD73+AZD4635 arm are on the right side (Adoi, orange dots). (B) Enrichment bar plot of significantly overexpressed pathways in the vehicle+isotype group (adenosine high), according to KEGG and GO biological processes. (C) Heatmap showing genes regulated during treatment (light gray for controls, dark gray for Adoi) which are part of the significantly different pathways according to KEGG. (D) Table (left) showing genes associated with innate immunity that are downregulated by Adoi treatment. Validation of RNAseq data (right graphs) through flow analysis of PD-L1 and CD206 expression (MFI ratio) on intratumoral live cells (8 mice in the control and 10 in the Adoi arms). The samples analyzed for validation were from different mice from the ones used for RNAseq. GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; RNAseq, RNA sequencing; PDL1, Programmed death ligand 1; MFI; mean fluorescence intensity; ECM extracellular matrix Figure 5 Adenosine inhibition remodels the pancreatic ductal adenocarcinoma tumor microenvironment. (A) Volcano plot related to 6419c5 allografts. Genes overexpressed in vehicle+isotype arm are on the left side (blue dots), and genes overexpressed in the anti-CD73+AZD4635 arm are on the right side (Adoi, orange dots). (B) Enrichment bar plot of significantly overexpressed pathways in the vehicle+isotype group (adenosine high), according to KEGG and GO biological processes. (C) Heatmap showing genes regulated during treatment (light gray for controls, dark gray for Adoi) which are part of the significantly different pathways according to KEGG. Figure 4 continued The Adoi approach led to a 30% reduction of tumor growth ratio (mean of 8 vs 5.5-folds increase from the baseline, p=0.003, figure 4D and online supplemental figure 7A) and tumor weight (median 1.24 vs 0.78 g, 0.039 figure 4A). Single agent AZD4635 induced a similar tumor control to the full combination. However, the ther- apeutic effect was delayed (online supplemental figure 7B), with a 30% growth reduction in the combination arm when compared with Adora2ai inhibition alone with single agent AZD4635, over the first 4 days of treatment (p=0.01). For biological reasons, given also that AZD4635 is a competitive inhibitor and its efficacy is dependent on the amount of eAdo, we chose to use it in combina- tion with anti-CD73 antibody to maximize the blocking on eAdo effects. These data support previous findings showing the same combination had greater antitumor immune effect.39 In contrast, targeting the adenosine pathway with Adoi in 2838c3 allografts where necrosis and consequently peri-necrotic adenosine are low, and M2 macrophages express lower level of Adora2a, did not translate into tumor growth reduction (online supple- mental figure 7C). The adenosine pathway has been shown to control the metastatic process and several authors have shown that inhibiting this axis can reduce the metastatic burden in mouse models.52 53 However, we were able to show for the first time that blocking adenosine generation and function can significantly reduce the occurrence of spontaneous metastasis in an IOT-resistant preclinical model of PDAC. The 6419c5 s.c. model spontaneously develops lung metastases in 100% Combining adenosine blockade (Adoi) and immu- notherapy (FCP) reduced significantly tumor growth of the IOT-resistant allograft model when compared with control treatment (65% tumor growth reduc- tion; p=0.002) and FCP (35% tumor growth reduction; 13 Graziano V, et al. J Immunother Cancer 2023;11:e006457. doi:10.1136/jitc-2022-006457 Open access p=0.021) arms (online supplemental figure 7D–F). Data from two separate experiments with the same controls growth alone or in combination we analyzed the changes in immune infiltration following anti-CD73 and AZD4635 Figure 5 Adenosine inhibition remodels the pancreatic ductal adenocarcinoma tumor microenvironment. (A) Volcano plot related to 6419c5 allografts. Genes overexpressed in vehicle+isotype arm are on the left side (blue dots), and genes overexpressed in the anti-CD73+AZD4635 arm are on the right side (Adoi, orange dots). (B) Enrichment bar plot of significantly overexpressed pathways in the vehicle+isotype group (adenosine high), according to KEGG and GO biological processes. Figure 4 continued To assess whether the combination of Adoi plus ATRi/gemcitabine had an effect on the immune infiltration of the TME of IOT-resistant model (6419c5 allografts), we performed IHC staining for CD8 and Foxp3 and showed that the quadruple combina- tion almost tripled the ratio CD8/Tregs (median 0.70 vs 0.25 of vehicles+isotype group; p=0.04; figure 6G,H). The four-drug combination produced an enhanced increase of the CD8+ cell infiltration (median 4.5 cells/mm2 vs 2.7 in the control group, 3.9 in the AZD6738/gem group and 2.8 in the AZD4635/αCD73 group) and reduction of Foxp3+ cell infiltration (median 6.5 cells/mm2 vs 8.1 in the control group, 9.3 in the AZD6738/gem group and 7.2 in the AZD4635/αCD73 group) (online supple- mental figure 8K). Similarly, combination with IOT (Adoi+FCP) determined a further increase of the intratu- moral CD8/Tregs ratio induced by FCP treatment (ratio means control 0.28 vs FCP/Adoi 6.10 p=0.008; online supplemental figure 8L) mostly due to the repression of the Treg recruitment induced by IOT alone. Furthermore, the adenosine signaling has previously been associated with the presence of hypoxia54 55; here we show for the first time that the presence of a functional eAdo pathway is responsible for the expression of several genes related to hypoxia (including Hif1a) suggesting the presence of a positive feedback loop. p p p Considering that the RNAseq analysis indicated that the innate immune system was affected by adenosine inhibition, we analyzed the changes in immune infiltra- tion following anti-CD73 and AZD4635 treatment. Flow cytometry analysis confirmed the findings highlighted by the RNAseq analysis. Following treatment, tumors were less likely to be infiltrated by M2 macrophages (median approximately 35,000 vs 23,000 cells/100 mg; p=0.004; figure 6A,B), in particular PD-L1+ ones (median 79% vs 65%; p=0.004; figure 6B, right). IMC analysis also revealed a trend towards reduction of M2 macrophages following treatment (F4/80+ CD206+ (figure 6C–E) or CD68+ CD206+ (figure 6D–F)) and the trend was more pronounced in viable tumor compared with necrotic margin, but the difference was not significant. Notably this reduced trend in infiltration is present in areas other than those where adenosine is abundant, suggesting that adenosine could be stimulating the production of factors affecting recruitment of macrophages in the viable tumor areas. Figure 4 continued Finally, IMC data also showed a trend towards a reduction (not significant, ns) of total macrophages in the treated tumors as shown by flow cytometry, again more evident in the viable tumor regions (figure 6C,D and F4/80 and CD68 panels and online supplemental figure 8H). IMC also showed a trend towards global reduction (ns) of M1 macrophages (F4/80+ CD206– MHC-II+) (online supple- mental figure 8I,J) which was not apparent in the flow analysis. Further studies would shed light on the relevance of these results, analyzing the contribution of adenosine abundance and spatial distribution to modeling of tumor infiltration by distinct macrophage subpopulations beyond M1-M2 dichotomy. relative expression of 712 genes decreased by ≥50% (log fold <−0.58; figure 5A). KEGG and GO biological process pathway analysis revealed that eAdo has a broad impact on TME of the IOT-resistant model of PDAC (figure 5B and online supplemental figure 8A). Genes associated with hypoxia response and vasculogenesis (eg, Hif1a, Slc2a1, Hilpda, Adm, Vegfa, Vegfd), immunity and immune suppression (eg, Cd274, Cd209, Mrc1, Cd200, Il1a, Il6, Ptgs2) and tumor stroma/extracellular matrix (ECM) organization (eg, Col5a3, Col6a3, Itga2, Mmp13, Mmp3, Mmp9, Ereg, Pthlh) were significantly downregulated by the treatment (figure 5B,C). ( g ) Of note, several functional and structural downregu- lated genes were associated with the innate immune system (figure 5D left), particularly with M2 polarized macro- phages (eg, Cd209, Mrc1, Mrc2, CD163). RNAseq analysis also showed a significant decrease of the expression of Cd274 (PD-L1) gene, suggesting a strong rationale for the use of adenosine inhibition as combination for immuno- therapy studies involving immune checkpoint inhibitors. The downregulation of Mrc1 (CD206) and Cd274 (PD- L1) was validated analyzing their protein expression on tumor-infiltrating live cells following Adoi treatment. As shown in figure 5D (right), the proteins of these genes were strongly downregulated in the adenosine inhibition arm (38% and 41% reduction of CD206 and PD-L1 on live cells respectively). y y Considering the reduction of the immune suppres- sion in the TME following Adoi, we explored changes associated with the adaptive immune system following combination of adenosine inhibition and cytotoxic or immunotherapy therapeutics. Figure 4 continued (D) Table (left) showing genes associated with innate immunity that are downregulated by Adoi treatment. Validation of RNAseq data (right graphs) through flow analysis of PD-L1 and CD206 expression (MFI ratio) on intratumoral live cells (8 mice in the control and 10 in the Adoi arms). The samples analyzed for validation were from different mice from the ones used for RNAseq. GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; RNAseq, RNA sequencing; PDL1, Programmed death-ligand 1; MFI; mean fluorescence intensity; ECM, extracellular matrix. p=0.021) arms (online supplemental figure 7D–F). Data from two separate experiments with the same controls showed that adding Adoi to FCP remains the best combi- nation in controlling tumor growth (online supplemental figure 7G–I). These data suggest again that targeting the adenosine pathway in PDAC offers a new strategy to modulate the antitumor immune response. p=0.021) arms (online supplemental figure 7D–F). Data from two separate experiments with the same controls showed that adding Adoi to FCP remains the best combi- nation in controlling tumor growth (online supplemental figure 7G–I). These data suggest again that targeting the adenosine pathway in PDAC offers a new strategy to modulate the antitumor immune response. growth alone or in combination we analyzed the changes in immune infiltration following anti-CD73 and AZD4635 treatment. The TME of PDAC is an intricate structure that relies on the presence of multiple non-malignant cells. This TME is recapitulated in preclinical models of PDAC.38 In order to investigate the broader effect of Adoi in the 6419c5 PDAC model, given the effect we have seen on tumor growth when used alone or in combination, we performed a bulk RNAseq analysis of the 6419c5 model treated with Adoi or control (five vs six mice, see methods). Following the 14-day treatment with AZD4635 and anti-CD73 the Targeting the adenosine pathway results in reprogramming of the Tme in the IOT-resistant pancreatic cancer mouse model To understand the role of adenosine modulation in reprogramming the TME, given the effect on tumor 14 Graziano V, et al. J Immunother Cancer 2023;11:e006457. doi:10.1136/jitc-2022-006457 Open access declined in F4/80+ macrophages (p=0.02) but not in DCs following treatment (online supplemental figure 8E). Of note, the combination of 2c5mIgG1 and AZD4635 is required to reduce the M2 macrophage infiltration (p=0.03, online supplemental figure 8F) and the ratio of M2/M1 macrophages (p=0.04; online supplemental figure 8G) in the tumor, while single agents fail to do so. Figure 4 continued Blocking the axis, also led to a reduced frequency of infiltrating Tregs (mean 42% vs 27%; p=0.03, online supplemental figure 8B,C) and a reduction of PD-L1 expression for all live cells within the tumor, more prom- inently CD45+ cells (p=0.008) as shown in online supple- mental figure 8D. However, the expression of PD-L1 Adenosine-related gene expression is associated with phenotype and survival in human pancreatic cancer To evaluate the importance of this pathway in the context of human PDAC and highlight the role in the formation of TME we scored our adenosine-related gene expression set against the PDAC subtypes published by Bailey.44 Of our 712 genes with a 50% decrease, 561 had a human ortholog and of these the z-scores of 517 genes were summed in each of the 97 patients with RNAseq data in Bailey et al to obtain a score. We were able to show that the adenosine-related gene expression is mostly expressed in the aggressive squamous subtype (figure 7A), that has been associated with a poorer prognosis. The score 15 Graziano V, et al. J Immunother Cancer 2023;11:e006457. doi:10.1136/jitc-2022-006457 Open access Figure 6 Immunosuppressive immune subpopulations are modulated following adenosine pathway blockade. (A) Representative flow cytometry plots showing tumor associated macrophages infiltrating 6419c5 allografts following 14 days of treatment with vehicle+isotype (left panel) or anti-CD73+AZD4635 (right panel). (B) M2 macrophage allograft infiltration (left, number of cells/100 mg of tumor) and (right) percentage of M2 macrophages positive for PD-L1 (8 and 10 mice per group analyzed). (C–D) Representative IMC image of F4/80 and CD206 (C) and CD68 and CD206 (D) positive cells infiltrating a 6419c5 allograft merged (first panel of C and D) or not with segmentation following Adoi (lower panels) or control (upper). The tissue segmentation of the IMC image was performed by Random Forest Classification using all markers analyzed. The scale bar on the IMC image is 200 µm. Segmentation shows viable tumor (green), necrosis (yellow), necrotic margin (blue) and off-tissue (red). (E–F) The bar plots show cell density (number of cells per mm2) of F480+CD206+ cells (E) and CD68+CD206+ (F) per segment area (six mice per treatment group). (G–H) Immunohistochemistry analysis showing 6419c5 tumor-infiltrating CD8+/Foxp3+ ratio (G) at day 14 of the following treatments: Vehicles+isotype (N=7), AZD6738+gemcitabine (N=7), aCD73+AZD4635 (N=6) and AZD6738+gemcitabine + aCD73+AZD4635 (N=7). (H) Representative immunohistochemistry images of the latter. All data are represented as box and whisker plots. Figure 7 RNA gene expression demonstrates a crucial role for the adenosine pathway in correlation to human PDAC prognosis. (A–B) Human ortholog genes with a 50% downregulation following 14 days of Adoi, were scored using z-score derived from Bailey’s44 subtypes data set. Genes scores comparing Adex, immunogenic, squamous and pancreatic progenitor (A) or squamous versus non-squamous (B) are shown. Each dot represents a single patient. (C) PDAC-specific gene signature of 52 genes used for analysis of the TCGA human PDAC data set, related to the main pathways implied (immunity, hypoxia response and tumor stroma). (D–E) Gene signature applied to PDAC TCGA data set (D). Kaplan-Meier curves show progression- free survival (PFS, 176 patients) and (E) disease-specific survival (DSS, 170 patients). (F) Gene signature validation applied to PDAC QCMG Bailey’s data set. Kaplan-Meier curve shows overall survival (OS, 95 patients). Three genes (Pecam1, Trarg1 and Vegfd) were not assessed in Bailey’s data set, thus the signature is composed of 49 genes. Data in A–B are presented as mean±SEM. Statistical analysis was performed using one-way analysis of variance with post hoc test analysis for multiple comparisons (A) two-tailed unpaired Student’s t-test (B) and log-rank Mantel-Cox test to evaluate difference in survivals (D–F) p values are shown in the graphs when considered significant (p<0.05). eAdo, extracellular adenosine; mOS, median overall survival; PDAC, pancreatic ductal adenocarcinoma; mPFS, median progression-free survival; TCGA, The Cancer Genome Atlas; mDSS, median disease-specific survival. Figure 7 RNA gene expression demonstrates a crucial role for the adenosine pathway in correlation to human PDAC prognosis. (A–B) Human ortholog genes with a 50% downregulation following 14 days of Adoi, were scored using z-score derived from Bailey’s44 subtypes data set. Genes scores comparing Adex, immunogenic, squamous and pancreatic progenitor (A) or squamous versus non-squamous (B) are shown. Each dot represents a single patient. (C) PDAC-specific gene signature of 52 genes used for analysis of the TCGA human PDAC data set, related to the main pathways implied (immunity, hypoxia response and tumor stroma). (D–E) Gene signature applied to PDAC TCGA data set (D). Kaplan-Meier curves show progression- free survival (PFS, 176 patients) and (E) disease-specific survival (DSS, 170 patients). (F) Gene signature validation applied to PDAC QCMG Bailey’s data set. Kaplan-Meier curve shows overall survival (OS, 95 patients). Three genes (Pecam1, Trarg1 and Vegfd) were not assessed in Bailey’s data set, thus the signature is composed of 49 genes. Figure 4 continued Statistical analysis was performed using Mann-Whitney test (B–H) and one-way analysis f i ith t h t t l i f lti l i (E H) l h i th h h id d Open access Fi 6 I i i b l i d l d f ll i d i h bl k d Figure 6 Immunosuppressive immune subpopulations are modulated following adenosine pathway blockade. (A) Representative flow cytometry plots showing tumor associated macrophages infiltrating 6419c5 allografts following 14 days of treatment with vehicle+isotype (left panel) or anti-CD73+AZD4635 (right panel). (B) M2 macrophage allograft infiltration (left, number of cells/100 mg of tumor) and (right) percentage of M2 macrophages positive for PD-L1 (8 and 10 mice per group analyzed). (C–D) Representative IMC image of F4/80 and CD206 (C) and CD68 and CD206 (D) positive cells infiltrating a 6419c5 allograft merged (first panel of C and D) or not with segmentation following Adoi (lower panels) or control (upper). The tissue segmentation of the IMC image was performed by Random Forest Classification using all markers analyzed. The scale bar on the IMC image is 200 µm. Segmentation shows viable tumor (green), necrosis (yellow), necrotic margin (blue) and off-tissue (red). (E–F) The bar plots show cell density (number of cells per mm2) of F480+CD206+ cells (E) and CD68+CD206+ (F) per segment area (six mice per treatment group). (G–H) Immunohistochemistry analysis showing 6419c5 tumor-infiltrating CD8+/Foxp3+ ratio (G) at day 14 of the following treatments: Vehicles+isotype (N=7), AZD6738+gemcitabine (N=7), aCD73+AZD4635 (N=6) and AZD6738+gemcitabine + aCD73+AZD4635 (N=7). (H) Representative immunohistochemistry images of the latter. All data are represented as box and whisker plots. Statistical analysis was performed using Mann-Whitney test (B–H) and one-way analysis of variance with post hoc test analysis for multiple comparisons (E–H) p values are shown in the graphs when considered significant (p<0.05). IMC, imaging mass cytometry; PD-L1, Programmed death-ligand 1. pressive immune subpopulations are modulated following adenosine pathway blockade. 16 Graziano V, et al. J Immunother Cancer 2023;11:e006457. doi:10.1136/jitc-2022-006457 Open access Open access Figure 7 RNA gene expression demonstrates a crucial role for the adenosine pathway in correlation to human PDAC prognosis. (A–B) Human ortholog genes with a 50% downregulation following 14 days of Adoi, were scored using z-score derived from Bailey’s44 subtypes data set. Genes scores comparing Adex, immunogenic, squamous and pancreatic progenitor (A) or squamous versus non-squamous (B) are shown. Each dot represents a single patient. Figure 4 continued (C) PDAC-specific gene signature of 52 genes used for analysis of the TCGA human PDAC data set, related to the main pathways implied (immunity, hypoxia response and tumor stroma). (D–E) Gene signature applied to PDAC TCGA data set (D). Kaplan-Meier curves show progression- free survival (PFS 176 patients) and (E) disease specific survival (DSS 170 patients) (F) Gene signature validation applied Data in A–B are presented as mean±SEM. Statistical analysis was performed using one-way analysis of variance with post hoc test analysis for multiple comparisons (A) two-tailed unpaired Student’s t-test (B) and log-rank Mantel-Cox test to evaluate difference in survivals (D–F) p values are shown in the graphs when considered significant (p<0.05). eAdo, extracellular adenosine; mOS, median overall survival; PDAC, pancreatic ductal adenocarcinoma; mPFS, median progression-free survival; TCGA, The Cancer Genome Atlas; mDSS, median disease-specific survival. 17 Graziano V, et al. J Immunother Cancer 2023;11:e006457. doi:10.1136/jitc-2022-006457 Open access remained significantly higher even when comparing the squamous subtypes with the grouped non-squamous (figure 7B). treatment of several types of cancer, and has dramati- cally changed the life expectancy of many patients with metastatic disease.57 However, this has not been true for patients with PDAC which is associated with a very low response rate to immunotherapy, usually confined to MSI-H/dMMR tumors, found only rarely in this disease.3 In order to create a PDAC-specific adenosine signature and to evaluate its performance in PDAC-specific outcomes, we created a signature using the 561 human orthologous genes with a ≥50% decrease in expression following Adoi treatment. Of these, 52 genes were selected for the signa- ture (figure 7C), that according to our RNAseq data set were clearly associated with the pathway areas depen- dent on adenosine (hypoxia response, immunity, tumor stroma), were associated positively or negatively with PDAC prognosis, and were significantly co-expressed with CD73 and/or Adora2a in the PDAC genome data sets (TCGA or Bailey). Of the 176 and 170 patients with available PFS and DSS outcome, respectively, we found that the presence of a high adenosine signature is associated with higher prob- ability of PDAC progression (median progression-free suvival, mPFS high Ado 13.05 vs low Ado 18.25, p=0.02; figure 7D) and a poorer PDAC-specific survival (median disease-specific survival, mDSS high Ado 19.66 vs low Ado NR, p=0.01; figure 7E), suggesting again that the presence of a functional adenosine pathway has a detrimental role in human PDAC. The signature was validated using the PDAC QCMG Bailey’s data set (49 genes were assessed, see figure legend) confirming a shorter overall survival in patients with high adenosine signature tumors (figure 7F p=0.003). It is worthy to consider that patients in the TCGA data set are predominantly non-metastatic, and the presence of the adenosine signature seems to become relevant for PDAC associated death 20 months after diag- nosis. The presence of a high adenosine signature appears to be associated with shorter DFS, (median disease-free survival, mDFS high Ado 23.5 months vs low Ado 49.7 months, p=0.12; online supplemental figure 8M). y y The role of the innate immune system in the genera- tion of an immune suppressive/protumorigenic micro- environment in PDAC is well known. The presence of marked infiltration of macrophages has been identified as an independent predictive factor of the aggressiveness and prognosis of PDAC, in patients.26 27 Only recently, three phase I clinical trials in patients with PDAC have shown that targeting the innate immune system can have an impact in patients with PDAC. A phase I trial published on Lancet Oncology, showed that a combination of anti PD-1 and CD40 agonist, added to gemcitabine and nab- paclitaxel, led to 60% of response rate, with some durable responses, although the clinical benefit of adding a CD40 agonist to an anti PD-1 plus chemotherapy as first-line in patients with metastatic PDAC, was not confirmed in a phase 2 randomized trial.58 59 In addition, the inhibi- tion of the CXCL12/CXCR4 axis has been demonstrated to modify the immunosuppressive TME of patients with PDAC and colorectal cancer.60 61 The eAdo pathway has also been shown to influence the TME fostering the immune suppression provided by some innate immune subpopulations (as myeloid and NKs) and inhibiting the function of the adaptive immune system, in particular, T cells.7 9 52 By activating its recep- tors, eAdo is able to increase the intracellular concentra- tion of cyclic adenosine monophosphate (cAMP) which leads to the induction of an M2 phenotype of macro- phages and block the secretion of interleukin (IL)-1β increasing the release of CXCL1, IL-6, IL-10 and IL-8 among others from myeloid population which are known to orchestrate immune exclusion.11 62 eAdo also favors the formation and maintenance of Treg cells,11 which are known to favor cancer progression and IOT resistance . Discussion Overall our data highlight that the generation of the eAdo is instrumental for the innate immune system in shaping a protumorigenic, immune suppressive microen- vironment in PDAC, in the context of a hypoxic milieu. We can speculate that this unfavorable environment may create the condition for a more aggressive PDAC pheno- type which would then translate into the ability to escape the immune system, be resistant to cytotoxic treatment and metastasize readily. A recent publication, shows that genomic targeting on mouse PDAC cells of CD73 leads to a reduced in vivo tumor formation and change in the circulating and infil- trating immune system.37 However, to date, little was known about the expression of the adenosine pathway in the context of the innate and adaptive immune system in PDAC, how the eAdo is gener- ated and what are the targets of adenosine also in regard to its spatial distribution and formation of adenosine. PDAC is projected to become the second highest cause of cancer-related death in the USA within 10 years,56 and represents one of the major unmet needs of cancer treat- ment. Despite extensive efforts by laboratory and clinical scientists in the last 50 years, only 1% of patients diagnosed with PDAC today will survive for 10 years (https://www. cancerresearchuk.org/health-professional/cancer-statis- tics-for-the-uk).1 The response rate following standard treatments is poor, usually short-lasting, and associated with significant treatment-related toxicity.2 In the past few years, immunotherapy has provided new hope in the Our results show that the mechanism of generation of eAdo in pancreatic cancer TME is finely orchestrated by tumor infiltrating myeloid cells and tumor cells, due to the expression of high level of CD39 in infiltrating myeloid cells and CD73 on both cell types. A recent paper demonstrated the presence of CD73 on MDSCs and macrophages.36 We identify that more broadly, ~90% of tumor-infiltrating myeloid cells in s.c. KPCY-derived tumors express CD73. We also validated this finding using an autochthonous tumor model as the KPC mouse. We 18 Graziano V, et al. J Immunother Cancer 2023;11:e006457. doi:10.1136/jitc-2022-006457 Open access have also shown that the pathway can be overexpressed in T cells infiltrating the tumors, regardless of their acti- vation status (figures 1 and 2 and online supplemental figure 2 and 4). Discussion The distribution of eAdo is spatially heterogeneous and a high level of eAdo correlates with the presence of a hypoxic environment and is favored by the presence of necrosis, where the myeloid population is enriched (figure 3). Necrosis is common in human PDAC and related to poor prognosis for all stages.63 The enrichment of a CD39+ CD73+ double population, poten- tially able to independently produce adenosine, does not seem to correlate with IOT-resistant or responsive tumor models, but there is a difference when the target of adenosine (Adora2a receptor), is considered. Adora2a on myeloid populations, in particular in protumorigenic M2 macrophages but also in antigen presenting cells, is differentially expressed in regard of IOT response pheno- type, with the resistant tumors abundantly overexpressing the receptor in these populations (figure 3 and online supplemental figure 4 and 5). Future analysis of human PDAC fresh specimens are needed to confirm the mech- anism we propose in the context of human disease. The bulk RNAseq analysis of tumor treated with adenosine inhibition revealed indeed a broader role for adenosine in PDAC TME (figure 5). Genes related to immunosup- pression and innate immunity recruitment (Cd274, Csf2, Cxcl2, Ccl3, Ccl4, Ccl12, Il1a, Osm, Il6), angiogenesis (Vegfa, Vegfd, Adm2, Flt1, Pgf, Egln1) and cell-ECM interaction (Adam19, Adamts14, Adamts4, Adamts5, Col5a3, Col6a, Itga2, Itga7, Mmp3, Mmp9, Mmp12) indicated the targeting of population of cells responsible for the acquisition of a protumorigenic, pro-metastatic, pro-fibrotic and immune resistant phenotype. Further, the downregulation of genes associated with hypoxia (Hif1a, Hilpda, Nos2, Hk1, Hk2, Egln3) following treatment shows not only that the adenosine pathway is induced during hypoxia (eg, CD73 and Adora2b), but that the hypoxia response is also dependent on the presence of the adenosine pathway in what we can speculate is a positive feedback loop. The inhibition of adenosine led to a reduced infiltration of M2 macrophages further from the hypoxic regions where adenosine is most abundant, suggesting that the effect of adenosine is stimulating the secretion of factors that are recruiting monocytes into the TME, replenishing macro- phage infiltration (figure 6 and online supplemental figure 8). Notably, targeting the pathway can reduce tumor growth in an IOT-resistant model, improving the response to cytotoxic and immunotherapy combina- tions (figure 4 and online supplemental figure 7) even in historically therapy-resistant models such as the KPC mice. Discussion Targeting adenosine would represent an alternative strategy to reduce the infiltration of protumorigenic spares a subpopulation of macrophages characterized by the expression of Cd274 (PD-L1), Vegfa, Hilpda, Bhlhe40, Mmp12, Cebpb, Hmox1 among others.31 Given that all of these genes are among the immune suppressive and vasculogenic molecules that seem to be strongly down- regulated by adenosine inhibition and our data show a reduction of some subpopulation of PD-L1+ macro- phages, we can speculate that adenosine inhibition could potentially target these populations of pro-angiogenic, immune suppressive macrophages. Information provided by the PDAC specific adenosine signature, indicate that the adenosine pathway appears to play a role in progression and survival of human PDAC, due to the ability of the adenosine inhibition to profoundly reprogram the TME in PDAC models (figures 5 and 6 and online supplemental figure 8). Gath- ering more information on the role of this pathway in human cancers should be a priority, retrospectively eval- uating and then prospectively stratifying the patients on the basis of histopathological/radiological features and the spatial distribution of adenosine. In summary, we have shown for the first time that tumor-infiltrating myeloid immune cells contribute to the generation of eAdo in the context of PDAC, correlating with the presence of hypoxia. Macrophages in particular, express high levels of Adora2a receptor in PDAC models and targeting the adenosine/myeloid axis remodels the TME. Data from IMC, flow cytometry and RNAseq suggest that the adenosine pathway is fundamental to the formation of a protumorigenic, immunosuppressive TME, and its expression is associated with an aggres- sive phenotype and poor survival in human PDAC. The remodeling of the TME caused by the inhibition of the adenosine pathway, translates into a delay of PDAC tumor growth. Finally, targeting eAdo should be considered as an adjunct to improve the efficacy of cytotoxic and IOT in future PDAC-specific clinical trials. Author affiliations 1Cancer Research UK Cambridge Institute, University of Cambridge, Cambridge, UK 2Cancer Research UK Cambridge Centre, University of Cambridge, Cambridge, UK 3Imaging and Data Analytics, Clinical Pharmacology and Safety Sciences (CPSS), AstraZeneca R&D, Cambridge, UK 4Medical Research Council Toxicology Unit, University of Cambridge, Cambridge, UK 5Cancer Research UK Beatson Institute, Glasgow, UK 6Oncology R&D, Research and Early Development, AstraZeneca R&D, Cambridge, UK 7Department of Oncology, University of Cambridge, Cambridge, UK 8Department of Pathology, Cambridge University Hospitals NHS Foundation Trust, Cambridge, UK 9School of Cancer Sciences, University of Glasgow, Glasgow, UK 10Oncology, AstraZeneca R&D Boston, Waltham, Massachusetts, USA Graziano V, et al. J Immunother Cancer 2023;11:e006457. doi:10.1136/jitc-2022-006457 Open access Co-expression of Cd39 and Cd103 identifies tumor-reactive Cd8 T cells in human solid tumors. Nat Commun 2018;9:2724. 14 Simoni Y, Becht E, Fehlings M, et al. Bystander Cd8(+) T cells are abundant and Phenotypically distinct in human tumour infiltrates. Nature 2018;557:575–9. i Competing interests AD, HH, BPK, MW, FMR, RG, SJD, AGS and JE are AstraZeneca employees and own company stocks and shares. VG has been an AstraZeneca employee since May 2023. HB holds a studentship partially funded by AstraZeneca. DIJ has received support for clinical and laboratory studies from AstraZeneca. DIJ is a consultant for Ellipses Pharma and Sentinel Oncology. 15 Ott M, Tomaszowski K-H, Marisetty A, et al. Profiling of patients with glioma reveals the dominant immunosuppressive axis is refractory to immune function restoration. JCI Insight 2020;5:e134386. 16 Takenaka MC, Gabriely G, Rothhammer V, et al. Author correction: control of tumor-associated Macrophages and T cells in glioblastoma via AHR and Cd39. Nat Neurosci 2019;22:1533. Patient consent for publication Not applicable. Patient consent for publication Not applicable. Ethics approval Not applicable. 17 Goswami S, Walle T, Cornish AE, et al. Immune profiling of human tumors identifies Cd73 as a Combinatorial target in glioblastoma. Nat Med 2020;26:39–46. Provenance and peer review Not commissioned; externally peer reviewed. 18 Sidders B, Zhang P, Goodwin K, et al. Adenosine signaling is Prognostic for cancer outcome and has predictive utility for Immunotherapeutic response. Clin Cancer Res 2020;26:2176–87. Data availability statement Data are available in a public, open access repository. Data are available upon reasonable request. All data relevant to the study are included in the article or uploaded as supplementary information. The data generated in this study are available within the article and its supplementary data files or from the corresponding authors upon reasonable request. Code for differential expression analysis and visualization of RNAseq data is available via Github https://github.com/ka-lw/AdenoPDAC. p p 19 Cekic C, Day Y-J, Sag D, et al. Myeloid expression of adenosine A2A receptor suppresses T and NK cell responses in the solid tumor Microenvironment. Cancer Res 2014;74:7250–9. 20 Sorrentino C, Miele L, Porta A, et al. Myeloid-derived Suppressor cells contribute to A2B adenosine receptor-induced VEGF production and angiogenesis in a mouse Melanoma model. Oncotarget 2015;6:27478–89. Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Open access i 5 Balachandran VP, Łuksza M, Zhao JN, et al. Identification of unique Neoantigen qualities in long-term survivors of Pancreatic cancer. Nature 2017;551:512–6. 6 Ho WJ, Jaffee EM, Zheng L. The tumour Microenvironment in Pancreatic cancer - clinical challenges and opportunities. Nat Rev Clin Oncol 2020;17:527–40. l UU_00025/12). SAK and JPM were supported by Cancer Research UK core funding to the Beatson Institute (A17196 and A31287) and to JPM Lab (A29996). The KPCY-derived cell lines were a kind gift of Ben Stanger (University of Pennsylvania). AZD4635, AZD6738 and the antibodies anti-CD73 (2C5, murine IgG1-Fc), anti- PD-L1 (AB740080 D265A), NIP228 muIgG1 isotype, NIP228 muIgG1 D265A isotype, were kindly provided by AstraZeneca. The results shown here are in part based on data generated by the TCGA Research Network: https://www.cancer.gov/tcga. 7 Antonioli L, Blandizzi C, Pacher P, et al. Immunity, inflammation and cancer: a leading role for adenosine. Nat Rev Cancer 2013;13:842–57. 8 Allard B, Allard D, Buisseret L, et al. The adenosine pathway in Immuno-oncology. Nat Rev Clin Oncol 2020;17:611–29. 9 Allard D, Allard B, Stagg J. On the mechanism of anti-Cd39 immune Checkpoint therapy. J Immunother Cancer 2020;8:e000186. Contributors VG and DIJ conceptualized the project and are the study guarantors. VG, AD, HH, KW, JPM, JE and DIJ designed the experiments. VG, AD, HH, KW, HB, SAK, MW, SYL and BPK conducted the experiments. VG, AD, HH, KW, SI, FMR, RB, JPM, SJD, JE and DIJ analyzed the data. JEDT, RG, JPM, SJD, AGS, JE and DIJ provided supervision and VG, AD, HH, KW, SI, JPM, SJD, AGS, JE and DIJ discussed the results. VG and DIJ wrote the manuscript. All authors read, reviewed, edited and approved the final manuscript. 10 Augustin RC, Leone RD, Naing A, et al. Next steps for clinical translation of adenosine pathway inhibition in cancer Immunotherapy. J Immunother Cancer 2022;10:e004089. 11 Cekic C, Linden J. Purinergic regulation of the immune system. Nat Rev Immunol 2016;16:177–92. 12 Goodwin KJ, Gangl E, Sarkar U, et al. Development of a Quantification method for adenosine in tumors by LC-MS/MS with Dansyl chloride Derivatization. Anal Biochem 2019;568:78–88. Funding This study was funded by Cambridge Trust (N/A); University of Cambridge Experimental Medicine Training Initiative and AstraZeneca (EMI-AZ) (N/A); Medical Research Council (MC_UU_00025/12); Cancer Research UK (A17196, A29996, A31287, C14303/A17197, C9545/A29580, C96/A25238, C9685/A27444). 13 Duhen T, Duhen R, Montler R, et al. Open access in particular Research Instrumentation & Cell Services (RICS), Flow Cytometry, Histopathology, Biological Resource Unit (BRU), Genomics and Genome editing cores. VG would like to thank Maike de la Roche (CRUK Cambridge Institute - University of Cambridge) and Klaus Okkenhaug (Department of Pathology - University of Cambridge) for helpful suggestions and advice. This study was also supported by Cancer Research UK Precision Panc grant C96/A25238. HB holds a PhD studentship at the University of Cambridge which is supported jointly by the University of Cambridge Experimental Medicine Training Initiative (EMI) program in partnership with AstraZeneca (EMI-AZ) and the NIHR Biomedical Research Center and SYL is founded by the Cambridge Trust (Cambridge International Scholarship). Work by JEDT/KW is funded by a core grant award from the MRC (MC_ UU_00025/12). SAK and JPM were supported by Cancer Research UK core funding to the Beatson Institute (A17196 and A31287) and to JPM Lab (A29996). The KPCY-derived cell lines were a kind gift of Ben Stanger (University of Pennsylvania). AZD4635, AZD6738 and the antibodies anti-CD73 (2C5, murine IgG1-Fc), anti- PD-L1 (AB740080 D265A), NIP228 muIgG1 isotype, NIP228 muIgG1 D265A isotype, were kindly provided by AstraZeneca. The results shown here are in part based on data generated by the TCGA Research Network: https://www.cancer.gov/tcga. in particular Research Instrumentation & Cell Services (RICS), Flow Cytometry, Histopathology, Biological Resource Unit (BRU), Genomics and Genome editing cores. VG would like to thank Maike de la Roche (CRUK Cambridge Institute - University of Cambridge) and Klaus Okkenhaug (Department of Pathology - University of Cambridge) for helpful suggestions and advice. This study was also supported by Cancer Research UK Precision Panc grant C96/A25238. HB holds a PhD studentship at the University of Cambridge which is supported jointly by the University of Cambridge Experimental Medicine Training Initiative (EMI) program in partnership with AstraZeneca (EMI-AZ) and the NIHR Biomedical Research Center and SYL is founded by the Cambridge Trust (Cambridge International Scholarship). Work by JEDT/KW is funded by a core grant award from the MRC (MC_ 2 Conroy T, Desseigne F, Ychou M, et al. FOLFIRINOX versus Gemcitabine for metastatic Pancreatic cancer. N Engl J Med 2011;364:1817–25. i 3 Le DT, Durham JN, Smith KN, et al. Mismatch repair deficiency predicts response of solid tumors to PD-1 blockade. Science 2017;357:409–13. i 4 Hu ZI, Shia J, Stadler ZK, et al. Evaluating mismatch repair deficiency in Pancreatic adenocarcinoma: challenges and recommendations. Clin Cancer Res 2018;24:1326–36. ORCID iD ORCID iD Vincenzo Graziano http://orcid.org/0000-0001-7656-824X 27 Steele NG, Carpenter ES, Kemp SB, et al. Multimodal mapping of the tumor and peripheral blood immune landscape in human Pancreatic cancer. Nat Cancer 2020;1:1097–112. Open access Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise. 21 Li L, Huang L, Ye H, et al. Dendritic cells Tolerized with adenosine A₂AR agonist attenuate acute kidney injury. J Clin Invest 2012;122:63170:3931–42.:. 22 Borodovsky A, Barbon CM, Wang Y, et al. Small molecule Azd4635 inhibitor of A2Ar signaling Rescues immune cell function including Cd103(+) Dendritic cells enhancing anti-tumor immunity. J Immunother Cancer 2020;8:e000417. 23 Wilson JM, Kurtz CC, Black SG, et al. The A2B adenosine receptor promotes Th17 differentiation via stimulation of Dendritic cell IL-6. J Immunol 2011;186:6746–52. Open access This is an open access article distributed in accordance with the Creative Commons Attribution 4.0 Unported (CC BY 4.0) license, which permits others to copy, redistribute, remix, transform and build upon this work for any purpose, provided the original work is properly cited, a link to the licence is given, and indication of whether changes were made. See https://creativecommons.org/ licenses/by/4.0/. 24 Romio M, Reinbeck B, Bongardt S, et al. Extracellular purine metabolism and signaling of Cd73-derived adenosine in murine Treg and teff cells. Am J Physiol Cell Physiol 2011;301:C530–9. 25 Cekic C, Linden J. Adenosine A2A receptors intrinsically regulate Cd8+ T cells in the tumor Microenvironment. Cancer Res 2014;74:7239–49. i 26 Ino Y, Yamazaki-Itoh R, Shimada K, et al. Immune cell infiltration as an indicator of the immune Microenvironment of Pancreatic cancer. Br J Cancer 2013;108:914–23. ORCID iD Vincenzo Graziano http://orcid.org/0000-0001-7656-824X 1 UK cancer research. 2022 Available: https://www.cancerresearchuk. org/health-professional/cancer-statistics/statistics-by-cancer-type/ pancreatic-cancer Graziano V, et al. J Immunother Cancer 2023;11:e006457. doi:10.1136/jitc-2022-006457 Discussion Author affiliations 1Cancer Research UK Cambridge Institute, University of Cambridge, Cambridge, UK 2Cancer Research UK Cambridge Centre, University of Cambridge, Cambridge, UK 3Imaging and Data Analytics, Clinical Pharmacology and Safety Sciences (CPSS), AstraZeneca R&D, Cambridge, UK 4Medical Research Council Toxicology Unit, University of Cambridge, Cambridge, UK 5Cancer Research UK Beatson Institute, Glasgow, UK 6Oncology R&D, Research and Early Development, AstraZeneca R&D, Cambridge, UK 7Department of Oncology, University of Cambridge, Cambridge, UK 8Department of Pathology, Cambridge University Hospitals NHS Foundation Trust, Cambridge, UK 9School of Cancer Sciences, University of Glasgow, Glasgow, UK 10Oncology, AstraZeneca R&D Boston, Waltham, Massachusetts, USA 9School of Cancer Sciences, University of Glasgow, Glasgow, UK 10Oncology, AstraZeneca R&D Boston, Waltham, Massachusetts, USA Present affiliations The present affiliation of Brajesh P Kaistha is: Oncology R&D, Research and Early Development, AstraZeneca, Cambridge, UK and Frances M Richards is: Oncology R&D, Translational Medicine, AstraZeneca, Cambridge, UK. 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https://openalex.org/W2165073988	https://zenodo.org/records/2409838/files/article.pdf	German	null	Elemente und Ephemeride von 1910 LD	Astronomische Nachrichten	1,911	public-domain	877	80 80 4469 79 Ephemeride des Fayeschen Kometen 1910 e gerechnet fur l z h M. Z. Berlin mit den Elementen aus Lick Bull. 187. 191 I Jan. 30 37 E'ebr. I 3 4 5 6 7 8 9 2 I 0 I 1 I 2 I 3 I 4 '5 16 7 7 18 I9 2 0 2 1 2 2 2 3 24 25 26 2 7 a vera 1 vera 4h 1 8 ~ 9= 19 32 2 o 55 2 2 I9 2 3 44 2 5 I 0 26 36 2 8 3 29 31 3' 0 32 29 33 59 35 3 0 37 1 38 33 40 6 41 39 43 '3 44 4 8 46 23 47 59 49 36 51 7 3 5 2 51 54 29 56 8 57 48 4 59 2 8 5 1 9 + 6" 3 2.'7 6 40.2 6 47.6 6 55.0 7 2.5 7 10.0 7 17.4 7 24.9 7 32.4 7 47.3 7 3 9 3 7 54.8 8 2 . 2 8 9.6 8 17.0 8 31.6 8 38.8 8 46.0 9 0 . 2 9 7.2 9 14.2 9 2 1 . 1 9 28.0 9 34.8 9 41.5 9 48.1 +9 54.7 8 24.3 8 53.1 log Y 0.2736 0.2778 0 . 2 8 2 I 0.2864 0.2907 0.2952 0.2996 0.3041 log n 0.0996 0.1170 0. I 3 4 I 0. I 508 0.1673 0.1836 0.1996 0.2 I 53 - w I 2m9 ~~ 13.0 13.1 I 3.2 13.3 13.4 '3.5 13.6 1911 Febr. 80 2 7 28 Marz I 3 4 5 6 7 8 9 2 I 0 I 1 I 2 I 3 14 I 5 16 I 7 18 I9 2 0 2 1 2 2 23 24 2 5 26 2 7 5 h I m 95 2 50 4 32 6 I4 7 56 9 39 '3 5 14 49 16 33 18 18 2 0 3 2 1 49 23 35 2 5 2 1 2 7 7 28 54 30 41 32 28 34 I 5 36 3 37 50 39 39 41 27 43 16 45 5 46 54 48 43 5 50 33 I 1 2 2 d Vera f 9" 5417 1 0 1.2 10 7.6 10 13.9 10 26.3 I 0 32.4 10 38.4 1 0 44.3 10 50.1 10 55.8 1 1 1.4 11 7.0 I 1 12.4 11 17.8 I I 23.0 11 28.2 I 1 33.2 11 38.2 11 43.0 1 1 47.7 I I 52.3 I I 56.8 1 2 5.5 1 2 9.7 1 2 13.8 1 2 17.7 f 1 2 21.6 I 0 2 0 . 2 I 2 1.2 Die Helligkeit H beruht auf einer Schatzung von Prof. A. Abetti 1910 Dez. 31 Kiel, Bureau der Astr. Nachr., 1911 Jan. 28. ( 1 2 ~ ) log Y 0.304 I 0.3086 0.3131 0.3176 0.3222 0.3268 0.33'3 0.3358 log n 0.2153 0.2307 0.2458 0.2607 0 . 2 7 5 2 0.2894 0.3032 0.3168 N I 3m6 - 13.7 I 3.8 13.9 14.0 14.1 14.2 '4.3 md dem r2n2-Gesetz. M. Ebell. Ephemeride des Fayeschen Kometen 1910 e echnet fur l z h M. Z. Berlin mit den Elementen aus Lick Bull. 187. 1 Genaherte aquatoreale Kreisbahnelernente aus Dez. 2 2 , 2 8. Epoche 1910 Dez. 2 5 . 5 M. 2. Gr. log Y = 0.3359 p = 18.~535 211 = 102"41!7 fi' = 348 2 2 . 8 24 47.7 i' = Ephemeride. r z h M. Z. Gr. 1911 Jan. 2 1 a = 5h38m2 d = +2.3'21' Febr. o 5 34.4 + 2 2 54 I 0 5 34.5 + 2 2 32 Notiz betreffend einen roten Stern. Auf einer Platte von 1910 Sept. 11 mit z Stunden Expositionszeit und +34"46!9 Die erste I'latte ist etwas weniger einer andern von 1910 Okt. ~ ~~ I n h a l t zu Nr. 4469. CV. ZurhcZZen. Ein Beitrag zur Kenntnis des Stundenwinkelfehlers astrophotographischer Aufnahmen. 65. - 0, Mezyner. Die gunstigste Beobachtungszeit in Potsdam. 75. - W. Ceraski. Trois variables nouvelles. 77. - M. EbeZZ. Ephemeride des Fayeschen Kometen 1910 e. 79. - N. Haponowicz. Elemente und Ephemeride von 1910 LD. 79. - M. MiindZer. Notiz betreffend einen roten Stern. 79. Geschlossen I ~ T I Febr. I . Heraiisgeber H. K O b o l d . Druck von C. Schaidt (Georg Oheim, Alfred Oheim). Expedit I Febr. I . Heraiisgeber H. K O b o l d . Druck von C. Schaidt (Georg Oheim, Alfred Oheim). Expedition: Kiel, Moltkestr. So. 80 13 mit I Stunde Expositionszeit ist der Stern BD +34"56 (8m1) oh 1 9 ~ 5 1 % (1855.0) nicht zu sehen, obwohl beide Platten die Sterne bis etwa 10715 enthalten. gut fokussiert als die zweite. Es scheint sich um einen sehr roten Stern zu handeln.) Mundenheim, 191 I Jan. 26. Max Mundler. ) Der Stern ist Nr. 39 in Kriigers Katalog farbiger Sterne (Farbe 8c.9, Spektrum IV) = Birm. Esp. 10 = Kii 165 (GrOOe 7m87, Nach Haw. Ann. 55.88 fand Prof. 0. C. Wendell in 4 Nachten 1906 Febr. 10 bis Okt. 2 durch photometrische Beobachtungen eine rotlich). Helligkeitsschwankung von om36. Red.
https://openalex.org/W3085966047	https://joe.uobaghdad.edu.iq/index.php/main/article/download/2464/1545	English	null	BLUNDER DETECTION TECHNIQUES IN ADJUSTMENT COMPUTATIONS	Maǧallaẗ al-handasaẗ/Journal of engineering	2,007	cc-by	6,501	BLUNDER DETECTION TECHNIQUES IN ADJUSTMENT COMPUTATIONS Mr. Bashar Saleem Dept.of Surveying. College of Engineering. University of Baghdad Mr. Jareer Jabir Dept.of Surveying. College of Engineering. University of Baghdad Mr. Hussein Alwan Dept.of Surveying. College of Engineering. University of Baghdad Volume 13 June 2007 Volume 13 June 2007 Journal of Engineering Number 2 ABSTRACT In adjustment of geodetic and photogrammetric networks, the surveying engineer faces many problems, such as errors of blunder nature in the observations (when comparing the homogeny of precision) make these observations odd from the result, and effecting directly on observation values after adjustment, and the statistical results after analysis as, adjusted coordinates of points, standard deviations, and ellipse of errors. The research is deal with some of the most common usage of statistical methods to detect these odds observations to confirm which best method is, by studying the advantages and disadvantages of each method to geodetic network. g Three statistical methods will use in the analysis, these are:- Three statistical methods will use in the analysis, these are:- Three statistical methods will use in the analysis, these are:- 1. Standardized residuals method 2. F-t test 3. Robust estimation method 3 The adjustments were accomplished by preparing a Matlab program with the three blunder detection methods and the results were evaluated and some scientific conclusions were reached. It was found that the robust estimation method represent the better blunder detection technique due to its The adjustments were accomplished by preparing a Matlab program with the three blunder detection methods and the results were evaluated and some scientific conclusions were reached. The adjustments were accomplished by preparing a Matlab program with the three blunder detection methods and the results were evaluated and some scientific conclusions were reached. It was found that the robust estimation method represent the better blunder detection technique due to its ability in what is called (multi-blunder detection) , and the resulted higher accuracy indices. It was found that the robust estimation method represent the better blunder detection technique due to its ability in what is called (multi-blunder detection) , and the resulted higher accuracy indices. UN C ON O S The used methods for blunder detection will be explained theoretically and mathematically as follows: The used methods for blunder detection will be explained theoretically and mathematically as follows: The used methods for blunder detection will be explained theoretically and mathem INTRODUCTION When computing and adjusting geodetic networks, It is quiet important to ensure that the considered observations are free of blunders and systematic errors, and therefore the results are only affected by the random errors which presented in every measuring process. Obviously, precaution in the observation process has to be the first step to avoid undesirable error appearance. Moreover, an adequate data filtering previous to the adjustment, checking reciprocal observations, and closures even with more detailed schemes is a suitable routine to be adopted in order to detect and eliminate the wrong observations. Blunders or mistakes could be defined as obviously incorrect data points or results that are not reasonably close to the expected value. Some blunder or systematic errors may slip into the adjustment process. So it would be interested to deal with the subject that deals with the blunder detection. KEY WORDS EY WORDS under detection, Geodetic Networks, Robust Estimation, Standardized Residual. Blunder detection, Geodetic Networks, Robust Estimation, Standardized Residual. Blunder detection, Geodetic Networks, Robust Estimation, Standardized Residual. ABSTRACT ا عىذ تصحيح انشبكاث انجيُديسيت َشبكاث انتخهيج انجُي تعتشض مٍىذس انمساح ت جمهت مه األ مرُس مى ٍرا َجرُد طاءراا راث غرالط هءر ف لعط االسصاداث (عىذ مقاسوت تجاوس انذقت) تجعم مه ٌزي االسصاداث شارة عه انبقيت ,َترثحش لصرُسة مباشرشة عهرم قري االسصراداث لعذ ان ت صحيح َانىتائج االحصائيت لعذ انتحهيم ك اإلحذاحياث ا نمصححً نهىقاغَ االوحشافاث انمعياسيت اظافت انرم انشركم االٌهيهيجر ن ألاءراا .انىاتجت يتىاَل انبحج مجمُع ت مه ا نءشق اإلحصائيت انشائعت َانمستخذمت نهكشر عره ٌرزي انشصرذاث انشرارة القرشاس انءشيقرت االفعرم مره ار ل دساست محاسه َ مساَئ كم غشيقت نشبكت جيُديسيت .حيج ااتيشث ح ح ت غشق: احصائيت متمخهت لاالت 1. غشيقت انبُاق انقياسيت (Standardized residuals method) 2غ يقت ااتبا(F t) test .انىاتجت يتىاَل انبحج مجمُع ت مه ا نءشق اإلحصائيت انشائعت َانمستخذمت نهكشر عره ٌرزي انشصرذاث انشرارة القرشاس انءشيقرت االفعرم مره ار ل دساست محاسه َ مساَئ كم غشيقت نشبكت جيُديسيت .حيج ااتيشث ح ح ت غشق: احصائيت متمخهت لاالت 1. غشيقت انبُاق انقياسيت (Standardized residuals method) ا غ قت ا يتىاَل انبحج مجمُع ت مه ا نءشق اإلحصائيت انشائعت َانمستخذمت نهكشر عره ٌرزي انشصرذاث انشرارة القرشاس انءشيقرت االفعرم مره ار ل دساست محاسه َ مساَئ كم غشيقت نشبكت جيُديسيت .حيج ااتيشث ح ح ت غشق: احصائيت متمخهت لاالت ش س ش ش 1. غشيقت انبُاق انقياسيت (Standardized residuals method) 2. غشيقت ااتباس (F-t) test 3. غشيقت انتخميه انمتي ه (Robust estimation method) َلاعذاد لشوامج لهغت (Matlab) ت َ اجشاا انتصحيح تخمريه انىترائج ن هحصرُل عهرم لعرط االسرتىتاجاث انعهميرت َا نكشر عره انشصرذاث انشارة لتءبيق انءشق انخ ث طع ي. 1485 B. Saleem Blunder Detection Techniques in Adjustment Computations H. Alwan J. Jabir َقذ َجذ ان غشيقت انتخميه انمتيه (Robust estimation method ) تمخرم طفعرم تقىيرً فر انكشر عره انشصرذاث انشرارة اسرتىادا نرم قالهيت انءشيقت ف انكش انمتعذد نهشصذاث انشارة (multi –blunder detection) َلسبب انذقت انعانيت انت تُفشٌا ٌزي انءشيقت مقاسورت مط انءشيقتيه ا ألاشييه B. Saleem H. Alwan J. Jabir Blunder Detection Techniques in Adjustment Computations B. Saleem H. Alwan J. Jabir Blunder Detection Techniques in Adjustment Computations Blunder Detection Techniques in Adjustment Computations َقذ َجذ ان غشيقت انتخميه انمتيه (Robust estimation method ) تمخرم طفعرم تقىيرً فر انكشر عره انشصرذاث انشرارة اسرتىادا نرم قالهيت انءشيقت ف انكش انمتعذد نهشصذاث انشارة (multi –blunder detection) َلسبب انذقت انعانيت انت تُفشٌا ٌزي انءشيقت مقاسورت مط انءشيقتيه ا ألاشييه. Since a computed parameter divided by its standard deviation which is a random normal variable, we can compute a (t) value as: Standardized Residual The detection of blunder among the observations was treated using a technique pioneered by the geodesist Baarda (1968). In Baarda’s method a statistical test which is called as Standardized residuals used to detect blunder [Baarda, 1968]. Blunders will affect the observations badly and produce incorrect estimations of the unknowns and their covariance matrix. If the blunders are detected by a statistical test, then those contaminated observations are removed, the network is re-adjusted, and we obtain the final results. This method of standardized residuals, detect one blunder in every iteration and remove it from the observations, and readjust the network with the original observations, the iterations continued until remove all blunders and until the chi square test (2) passed depend upon the significance level, variance and the degree of freedom. The method also depends on computing the variance-covariance matrix of residuals vv  . The standardized residuals are computed as: ii i i q v v  ii i i q v v  (1) (1) Where iv is the standardized residual, Where iv is the standardized residual, iv is the residual, and ii q is the diagonal element of the vv  matrix. ii q is the diagonal element of the vv  matrix. Since a computed parameter divided by its standard deviation which is a random normal variable, we can compute a (t) value as: 1486 1486 Volume 13 June 2007 Number 2 Volume 13 June 2007 Journal of Engineering 0 0 S v S v q S v t i v i ii i i i    (2) However, this equation should be based on "good "value for the reference variance since a blunder automatically affects the value of 0 S , the method summarized as follows: asurements that qualify for rejection. 1) Locate all measurements that qualify for rejection. 1) Locate all measurements that qualify for rejection. 1) Locate all measurements that qualify for rejection. 2) Reject the single observation with the largest standardized residuals. 2) Reject the single observation with the largest standardized residuals. 3) Repeat adjustment 1& 2 until all observations qualify for rejection are rejected, and until (goodness of fit) pass. Robust estimation method The method is the latest method for blunders detection, that was obtained by scientist Huber in 1981 [Huber, 1981] and developed in 1990's [Francis, 2004]. The basics of this method are depending upon the original weights of observations. The method is the latest method for blunders detection, that was obtained by scientist Huber in 1981 [Huber, 1981] and developed in 1990's [Francis, 2004]. The basics of this method are depending upon the original weights of observations. Ordinary Least square adjustment is not sensitive to the blunder in the observation. From the Figure (1) below, it could be noticed how the regression line of least square dropped to the blunder points ,and affect the regression line by this amount ,but if we use the method of robust estimation that resistant to blunder the problem will be very different . The regression line by both ordinary least square method, and robust line fit shown, and it could be noticed how the blunder point could not affect the regression line of the robust estimation method, and how affects on the least square line regression. Robust estimation method will treat the blunder from a new view of point, which will compute a weight through a special function upon the scientific function which detect blunder and give a correct result, then treat the blunder. Robust estimation method will treat the blunder from a new view of point, which will compute a weight through a special function upon the scientific function which detect blunder and give a correct result, then treat the blunder. One can notice how the blunder points dropped the least square line towards the blunder points, how the regression line of robust estimation is canceled the blunder point and how resistant the dropping of the blunder point and did not let it to affect the line of regression. 1487 1487 B. Saleem Blunder Detection Techniques in Adjustment Computations H. Alwan J. Jabir Fig (1). robust regression line and ordinary least square regression line (with one blunder). Blunder Detection Techniques in Adjustment Computations Fig (1). robust regression line and ordinary least square regression line (with one blunder). Specifies a weight function, a tuning constant, and the presence or absence of a constant term. The weight function can be any of the names listed in the following Table (1). Table (1). Weight function used in robust estimation function. tune: the tuning constant from table (1), s: an estimate of the standard deviation of the error term. s = MAD/0.6745, and Volume 13 June 2007 Number 2 Volume 13 June 2007 Number 2 Volume 13 June 2007 Journal of Engineering 4) If the value of ir is greater than rejection level, then this observation is considered as a blunder, and it must be down weighted to a new weight according to the weight function in the table above. F-T TEST METHOD It is one of the traditional methods for detecting blunders; it was presented by the researcher Xu Pieliang. The basic of this method depends upon the tests F-t. Initially F test used for the global test and then t test to check for each blunder immediately after an F test. The U statistics here is actually the 2 statistics given by Stefanovic [Xu Pieliang, 1987]. Compared with an F test or (2) test, this method is convenient for testing each blunder after the global test, and makes it possible to discuss the relationship of significance levels between the global test and the test of each blunder. Let the mathematical model be: V B L    Robust estimation method Tune constant Meaning Weight function 1.339 r r r abs W / ) sin( )) ( (   Andrew 4.685 2 2) 1( )) ( ( r r abs W    Bisquare 2.385 ) 1 /( 1 2r W   Cauchy 1.400 )) ( 1 /( 1 r abs W   Fair 1.345 )) ( /( 1 r abs W  Huber 1.205 r r W /) tanh(  Logistics 2.795 )) ( /( 1 r abs W  Talwar 2.985 ) exp( 2   r W Welsech Table (1). Weight function used in robust estimation function. Procedure for robust estimation method could be summarized as follow: Procedure for robust estimation method could be summarized as follow 1) Solve as ordinary least square method 2) Solve for hat matrix Hˆ (covariance of observations) Where: t t B B B B H     1 ) ( 3) Compute r t t B B B B H     1 ) ( (3) 3) Compute r 3) Compute r ) - 1( ii i i h s tune V r    (4) Where: iV : the residuals of observation i, iV : the residuals of observation i, tune: the tuning constant from table (1), tune: the tuning constant from table (1), tune: the tuning constant from table (1), s: an estimate of the standard deviation of the error term. s = MAD/0.6745, and ii h : the vector of leverage values (diagonal element of the hat matrix H). ii h : the vector of leverage values (diagonal element of the hat matrix H). ii h : the vector of leverage values (diagonal element of the hat matrix H ii h : the vector of leverage values (diagonal element of the hat matrix H). The quantity MAD is the median absolute deviation of the residuals. The constant 0.6745 makes the estimate unbiased for the normal distribution. 1488 Number 2 V B L    ) (      vv t t B B W B B W (16) 2 1 - 1 1 1 1 - ] ) ( [  t i t i i vv B B W B B W    (17) ) ˆ( ) ( 2 2 l D l D vv      2 2 1 1 1 1 2 1 2 . ) (      vv t t B B W B B W 2 1 - 1 1 1 1 - ] ) ( [  t i t i i vv B B W B B W    (16) (17) To test if blunders exist in 2ˆV we test the zero hypotheses: To test if blunders exist in 2ˆV we test the zero hypotheses: (18) 0 2   L Ho 0 2   L Ho (18) When a priori value of the unit weight variance is known, the quadratic form: ) ( ~ ˆ ˆ 2 2 1 2   V V U vv t    (19) If eq. (18) is correct the noncentral parameter (δ) of U is equal to zero; otherwise, 1 ) ˆ ( L V D Lt    ) ( ~ ˆ ˆ 2 2 1 2   V V U vv t    (19) If eq. (18) is correct the noncentral parameter (δ) of U is equal to zero; otherwis 1 ˆ 2 2 1 2 ) ˆ ( L V D Lt      . 2 2 1 2 ) ˆ ( L V D Lt      . (20) 2 2 2 tics U is Stefanovic’s 2 tests. In fact statistics U is Stefanovic’s 2 tests. In fact statistics U is Stefanovic’s 2 tests. V B L    (5) (5) Where B: the design matrix, L: the observational vector with the weight matrix W, L: the observational vector with the weight matrix W, g Δ: the vector with u unknown parameters, and p V: the normally distributed error vector. V: the normally distributed error vector. If there are blunder in (l ), eq. (5) is rewritten as: If there are blunder in (l ), eq. (5) is rewritten as: If there are blunder in (l ), eq. (5) is rewritten as: 1 1 1 V B l    1 1 1 V B l    (6) 2 2 2 2 V l B l      (7) h (6) 2 2 2 2 V l B l      2 2 2 2 V l B l      (7) Where: Where: 1 B : a matrix with full column rank, 1 B : a matrix with full column rank, 1l : a no-blunder observational error, 1l : a no-blunder observational error, 2l : may be considered to be a vector containing some blunders ( 2l  ) after the initial identification, and V1 and V2 are the normally distributed error vectors of ( 1l ) and ( 2l ), respectively. The ordinary least square solutions are: The ordinary least square solutions are: ) ( ) ( 1 1 1 1 1 1 1 l W B B W B t t    (8) xx  = 1 - 1 1 1 ) ( B W Bt 2  (9) ) ( ˆ 1 1 1 2 u m n V W V t     (10) 1 1 1 l B V    (11) ) ( ) ( 1 1 1 1 1 1 1 l W B B W B t t    xx  = 1 - 1 1 1 ) ( B W Bt 2  ) ( ˆ 1 1 1 2 u m n V W V t     1 1 1 l B V    (9) (10) (11) 1489 B. Saleem Blunder Detection Techniques in Adjustment Computations H. Alwan J. Jabir B. Saleem Blunder Detection Techniques in Adjustment Computations H. Alwan J. V B L    ˆ l W B B W B B B l t t     (12) For each element ( 2ˆl ) of ( 2l ) (i > n-m): 1 ) ( ˆ l W B B W B B l t t   For each element ( 2ˆl ) of ( 2l ) (i > n-m): 1 1 1 1 1 1 1 ) ( ˆ l W B B W B B l t t i i   Where 2 2 1 1 1 1 1 1 1 ) ( ˆ l W B B W B B l t t i i   (13) Where iB : a row vector of 2 B . The predicted residuals vector is therefore (denoted by 2ˆV ) The predicted residuals vector is therefore (denoted by 2ˆV ) The predicted residuals vector is therefore (denoted by 2ˆV )    2 2 2 2 2 - ˆ - ˆ B l l l V (14) For each element of 2ˆV    2 2 2 2 2 - ˆ - ˆ B l l l V (14) For each element of 2ˆV For each element of 2ˆV For each element of 2 V   i i i B l V - ˆ (15)   i i i B l V - ˆ   i i i B l V - ˆ (15)   i i i B l V - (15) If ( 1l ) and ( 2l ) are unrelated, ( 2l ) is unrelated to Δ, thus we can get from eq. (14) and (15) ( ) If ( 1l ) and ( 2l ) are unrelated, ( 2l ) is unrelated to Δ, thus we can get from eq. (14) and (15) ( ) If ( 1l ) and ( 2l ) are unrelated, ( 2l ) is unrelated to Δ, thus we can get from eq. (14) and (15) ) ˆ( ) ( 2 2 l D l D vv      2 2 1 1 1 1 2 1 2 . V B L    Jabir Where 1 W : the weight matrix of ( 1l ), and n: total number of observations, The predicted vector ( 2ˆl ) of ( 2l ) is: 1 1 1 1 1 1 1 2 2 2 ) . . ( ˆ. ˆ l W B B W B B B l t t     (12) For each element ( 2ˆl ) of ( 2l ) (i > n-m): 1 1 1 1 1 1 1 ) ( ˆ l W B B W B B l t t i i   (13) Where Blunder Detection Techniques in Adjustment Computations Where 1 W : the weight matrix of ( 1l ), and n: total number of observations, The predicted vector ( 2ˆl ) of ( 2l ) is: 1 1 1 1 1 1 1 2 2 2 ) . . ( ˆ. ˆ l W B B W B B B l t t     (12) For each element ( 2ˆl ) of ( 2l ) (i > n-m): 1 1 1 1 1 1 1 ) ( ˆ l W B B W B B l t t i i   (13) Where Where 1 W : the weight matrix of ( 1l ), and n: total number of observations, ˆ The predicted vector ( 2ˆl ) of ( 2l ) is: 1 1 1 1 1 1 1 2 2 2 ) . . ( ˆ. ˆ l W B B W B B B l t t     The predicted vector ( 2ˆl ) of ( 2l ) is: 1 1 1 1 1 1 1 2 2 2 ) . . ( ˆ. 1) Initial identification of blunders. 1) Initial identification of blunders. 2) Division of model in eq. (5) into two parts–the adjustment model in eq.(6) and the prediction model in eq.(7) according to the initial identification and then solving for Δ in eq. (8), the predicted residuals vector 2ˆV from eq. (16) and its variance-covariance matrix. 3) Use of the F test for the global test. If the zero hypotheses are rejected, further investigation (a t test) is needed to determine which ( il ) is responsible. 3) Use of the F test for the global test. If the zero hypotheses are rejected, further investigation (a t test) is needed to determine which ( il ) is responsible. V B L    Now we further establish two statistics, denoted by F and t, respectively [Xu Pieliang, 1987]: 2 2 / ˆ / /   m U F  2 2 1 2 1 1 1 1 2 1 - 2 ˆ / ˆ ] ) (  m V B B W B B W t t  -m-t) 2 2 1 2 1 1 1 1 2 1 - 2 2 ˆ / ˆ ] ) ( [ ˆ  m V B B W B B W V F t t t   ~F (m, n-m-t) 2 2 1 2 1 1 1 1 2 1 - 2 2 ˆ / ˆ ] ) ( [ ˆ  m V B B W B B W V F t t t   (21) ( , ) Using eq. (21) we can conveniently test whether ( 2l ) contain some blunders. When the zero hypotheses are rejected, further testing is needed to determine which ( 2l ) is responsible. Using eq. (21) we can conveniently test whether ( 2l ) contain some blunders. When the zero hypotheses are rejected, further testing is needed to determine which ( 2l ) is responsible. Therefore establish the zero hypotheses for each elements of ( 2l ): Therefore establish the zero hypotheses for each elements of ( 2l ): Therefore establish the zero hypotheses for each elements of ( 2l ): 1490 Volume 13 June 2007 Number 2 Volume 13 June 2007 Number 2 Journal of Engineering 0 2   L Ho (22) It is clear that when eq. (21) is correct, 2 2 / ˆ / ˆ   vv v t   ~t (n-m-t) 2 2 / ˆ / ˆ   vv v t   (23) ~t (n-m-t) 2 2 / ˆ / ˆ   vv v t   (23) ~t (n-m-t) 2 2 / ˆ / ˆ   vv v t   (23) After the F test, the t test can therefore be used to detect blunder in ( 2l ). It is not difficult to see that a (t) test makes it easy to test each element ( il ) of ( 2l ). The steps according to this method may be summarized as follow: 1) Initial identification of blunders. APPLICATION TO ACTUAL NETWORK: For mathematical verification, a combined (Hybrid) geodetic net were chosen, which was adjusted by [Ghilani, 1994]. The systematic errors are corrected and the observed distances are reduced to mean see level. Goodness of fit test for this net after ordinary least square adjustment was failed which means that blunders exist, so the net was suitable to check our methods for blunders detection. For mathematical verification, a combined (Hybrid) geodetic net were chosen, which was adjusted by [Ghilani, 1994]. The systematic errors are corrected and the observed distances are reduced to mean see level. Goodness of fit test for this net after ordinary least square adjustment was failed which means that blunders exist, so the net was suitable to check our methods for blunders detection. specifications of this network were as follows [Ghilani, 1994]; (see appendix A) T l i (2000 2001) The specifications of this network were as follows [Ghilani, 1994]; (see appendix A) -Two control points (2000, 2001). -11 unknown points. p -19 distance observations, and 17 angles. Fig (2) shows the configuration of the net. 1491 B. Saleem Blunder Detection Techniques in Adjustment Computations H. Alwan J. Jabir Blunder Detection Techniques in Adjustment Computations Fig (1). Configuration of the geodetic net Fig (1). Configuration of the geodetic net CONCLUSIONS Among all the weight functions used in robust estimation method it is highly recommended to use the (Cauchy) weight function. RESULTS AND DISCUSSIONS RESULTS AND DISCUSSIONS After performing the least square adjustment, each method was checked for blunders detection in the observation. The results had been summarized in the Table (2) Table (2) Comparison between blunder detection methods used for adjustin ison between blunder detection methods used for adjusting the geodetic network. Table (2) Comparison between blunder detection methods used for adjusting the geodetic network. he adj ust me nt of the abo ve net work by using 1st method (Standardized residuals) was done by [Ghilani, 1994], while this research adjust the same network by using the two other methods. Method Iteration 2 test ±σ Blundered observations Redundancy Standardized residuals method 3 Pass 1.162 2 12 Robust estimation method 1 (One step) Pass 0.983 2 14 F-t test method 1 (Two steps) Pass 1.165 2 12 T work by using 1st method (Standardized residuals) was done by [Ghilani, 1994], while this research adjust the same network by using the two other methods. work by using 1st method (Standardized residuals) was done by [Ghilani, 1994], while this research adjust the same network by using the two other methods. 1492 Volume 13 June 2007 Number 2 Journal of Engineering It was found that the robust estimation method represent the better blunder detection technique due to its ability in what is called (multi-blunder detection), and the resulted higher accuracy indices, this is obvious from the comparing the values of ( ±σ). CONCLUSIONS It could be concluded the following: 1. It is clear that standardized residuals method, Robust estimation method and F-t test method are all effective in blunder detection. 1. It is clear that standardized residuals method, Robust estimation method and F-t test method are all effective in blunder detection. 2. A disadvantage on standardized residuals method was noticed, in that it is an iterative procedure or a single blunder detection technique. Robust estimation and F-t test has the advantage in that it is a multiple blunder detection techniques. 2. A disadvantage on standardized residuals method was noticed, in that it is an iterative procedure or a single blunder detection technique. Robust estimation and F-t test has the advantage in that it is a multiple blunder detection techniques. 3. From the two applications discussed, it is quite evident that Robust estimation technique gives a lower value for the final variance of unit weight ( 2 0ˆ) and also for the standard deviations of the adjusted unknown, which indicates that Robust estimation is the best blunder detection method. This lower value of variance could be explained in that the redundancy (n-u) remain fixed while in the standardized residuals method and F-t test method the redundancy decreased each time a 3. From the two applications discussed, it is quite evident that Robust estimation technique gives a lower value for the final variance of unit weight ( 2 0ˆ) and also for the standard deviations of the adjusted unknown, which indicates that Robust estimation is the best blunder detection method. g ( 0 ) adjusted unknown, which indicates that Robust estimation is the best blunder detection method. This lower value of variance could be explained in that the redundancy (n-u) remain fixed while in the standardized residuals method and F-t test method the redundancy decreased each time a blunder detected and removed This lower value of variance could be explained in that the redundancy (n-u) remain fixed while in the standardized residuals method and F-t test method the redundancy decreased each time a blunder detected and removed This lower value of variance could be explained in that the redundancy (n-u) remain fixed while in the standardized residuals method and F-t test method the redundancy decreased each time a blunder detected and removed 4. Among all the weight functions used in robust estimation method it is highly recommended to use the (Cauchy) weight function. 4. REFERENCES Baarda, W. (1968), A Testing Procedure for Use in Geodetic Networks, Netherlands Geodetic Commission Publ. On Geodesy, New series 2, No. 5, Delft 1968 Francis, W.O.Adoul (2004), Robust Geodetic Parameter Estimation under Least Square through Weighting on the Basis of the Mean Square Error, Netherlands Geodetic Commission, publication on Geodesy ,New Series ,Vol.2,No.5,Delft, 2004. Francis, W.O.Adoul (2004), Robust Geodetic Parameter Estimation under Least Square through Weighting on the Basis of the Mean Square Error, Netherlands Geodetic Commission, publication on Geodesy ,New Series ,Vol.2,No.5,Delft, 2004. Huber, P.J., (1981), Robust Statistics, John Wiley& Sons, New York. Wolf & Ghilani (1994), Adjustment Computations, John Wily & Sons, New York. Wolf, Paul R.(1982), Elements of Photogrammetry, 2nd edition, John Wily & Sons, New York Xu Pieliang (1987) A test method for many outliers, ITC journal, No.4, 1987. SOLUTION Adjusted stations Error ellipse confidence level at 0.950 Station X Y Sx Sy Su Sv t ================================================================================ 1 2,477,236.78 420,351.57 26.138 27.582 95.745 40.382 137.21° 2 2,477,500.02 419,949.06 18.862 32.883 95.031 41.414 158.93° 3 2,477,835.61 420,206.18 22.972 41.726 124.575 38.036 155.07° 4 2,478,007.59 420,410.17 28.839 47.264 145.573 41.617 151.30° 5 2,477,631.63 420,566.15 32.636 36.320 127.266 40.399 138.75° 6 2,477,667.20 420,320.89 26.259 37.620 118.995 39.741 147.78° 102 2,476,455.42 419,742.35 9.931 6.023 27.726 15.494 75.95° 103 2,476,731.25 419,918.44 14.445 13.310 40.980 34.721 120.10° 201 2,476,576.61 419,589.00 8.329 9.077 27.541 19.397 37.62° 202 2,476,948.76 419,330.06 12.410 16.494 46.100 32.569 16.98° 203 2,477,465.47 419,816.79 15.649 30.287 85.777 36.510 163.29° Adjusted Distance Observations Station Station Occupied Sighted Distance V S Std.Res. Red.# ======================================================================= 1 3 616.23 0.495 5.356 26.05 0.746 1 2 480.95 0.243 5.921 13.29 0.689 3 1 616.23 0.495 5.356 26.05 0.746 3 4 266.81 -31.287 6.525 -1802.59 0.622* 3 6 203.77 1.789 6.889 106.83 0.579 3 5 413.76 3.317 5.059 171.47 0.773 3 2 422.77 0.069 5.459 3.67 0.736 5 2 630.97 1.394 6.012 76.87 0.679 5 1 449.39 -1.281 7.192 -79.16 0.541 5 6 247.83 1.225 7.100 74.85 0.553 5 4 407.04 9.153 7.819 614.84 0.458 5 3 413.76 3.297 5.059 170.43 0.773 102 103 327.25 -0.121 10.056 -17.16 0.103 103 1 665.70 -0.087 10.048 -12.18 0.105 201 202 453.37 0.268 10.526 92.01 0.017 202 203 709.85 0.075 10.051 10.51 0.104 203 3 537.24 0.060 10.056 8.53 0.103 2000 102 125.05 -0.188 10.138 -28.72 0.089 2001 201 425.95 0.048 10.063 6.82 0.102 Adjusted stations Adjusted stations Error ellipse confidence level at 0.950 Station X Y Sx Sy Su Sv t ================================================================================ 1 2,477,236.78 420,351.57 26.138 27.582 95.745 40.382 137.21° 2 2,477,500.02 419,949.06 18.862 32.883 95.031 41.414 158.93° 3 2,477,835.61 420,206.18 22.972 41.726 124.575 38.036 155.07° 4 2,478,007.59 420,410.17 28.839 47.264 145.573 41.617 151.30° 5 2,477,631.63 420,566.15 32.636 36.320 127.266 40.399 138.75° 6 2,477,667.20 420,320.89 26.259 37.620 118.995 39.741 147.78° 102 2,476,455.42 419,742.35 9.931 6.023 27.726 15.494 75.95° 103 2,476,731.25 419,918.44 14.445 13.310 40.980 34.721 120.10° 201 2,476,576.61 419,589.00 8.329 9.077 27.541 19.397 37.62° 202 2,476,948.76 419,330.06 12.410 16.494 46.100 32.569 16.98° 203 2,477,465.47 419,816.79 15.649 30.287 85.777 36.510 163.29° Adjusted Distance Observations Adjusted Distance Observations Station Station Occupied Sighted Distance V S Std.Res. REFERENCES 1493 Blunder Detection Techniques in Adjustment Computations Blunder Detection Techniques in Adjustment Computations APPENDIX (A) Blunder detection example ============================ Number of Control Stations = 2 Number of Unknown Stations =11 Number of Distance observations = 19 Number of Angle observations = 17 APPENDIX (A) Blunder detection example ============================ Number of Control Stations = 2 Number of Unknown Stations =11 Number of Distance observations = 19 Number of Angle observations = 17 Initial approximations for unknown stations Initial approximations for unknown stations Control Stations Initial approximations for unknown stations Control Stations Station X Y 1 2 477 233.88 420 353.62 2 2 477 497.99 419 951.98 3 2 477 832.67 420 210.17 4 2 478 023.86 420 438.88 5 2 477 630.64 420 567.44 6 2 477 665.36 420 323.31 102 2 476 454.17 419 743.39 103 2 476 728.88 419 919.69 201 2 476 576.25 419 589.24 202 2 476 948.76 419 331.29 203 2 477 463.90 419 819.56 Station X Y 2000 2 476 334.60 419 710.09 2001 2 476 297.98 419 266.82 Distance Observations Station Occupied Station Sighted Distance S 1 3 615.74 0.02 1 2 480.71 0.02 3 1 615.74 0.02 3 4 298.10 0.02 3 6 201.98 0.02 3 5 410.44 0.02 3 2 422.70 0.02 5 2 629.58 0.02 5 1 450.67 0.02 5 6 246.61 0.02 5 4 397.89 0.02 5 3 410.46 0.02 102 103 327.37 0.02 103 1 665.79 0.02 201 202 453.10 0.02 202 203 709.78 0.02 203 3 537.18 0.02 2000 102 125.24 0.02 Distance Observations 1494 Volume 13 June 2007 Journal of Engineering Journal o Volume 13 June 2007 Number 2 2001 201 425.90 0.02 Angle Observations Station Back sighted Station Occupied Station Fore sighted Angle S 2 1 3 316° 48' 00.5" 6.3" 2 3 4 167° 32' 28.0" 14.5" 2 3 6 71° 42' 51.5" 15.1" 2 3 5 98° 09' 36.5" 10.3" 2 3 1 51° 07' 11.0" 7.2" 203 3 2 8° 59' 56.0" 6.5" 2 5 3 318° 20' 54.5" 7.0" 1 5 3 268° 49' 32.5" 9.8" 6 5 3 338° 36' 38.5" 10.7" 3 5 4 324° 17' 44.0" 8.1" 2000 102 103 162° 58' 16.0" 28.9" 102 103 1 172° 01' 43.0" 11.8" 2001 201 202 263° 54' 18.7" 9.7" 201 202 203 101° 49' 55.0" 8.1" 202 203 3 176° 49' 10.0" 8.4" 102 2000 2001 109° 10' 54.0" 25.5" 2000 2001 201 36° 04' 26.2" 7.4" Jour Volume 13 June 2007 Number 2 2001 201 425.90 0.02 Angle Observations Station Back sighted Station Occupied Station Fore sighted Angle S 2 1 3 316° 48' 00.5" 6.3" 2 3 4 167° 32' 28.0" 14.5" 2 3 6 71° 42' 51.5" 15.1" 2 3 5 98° 09' 36.5" 10.3" 2 3 1 51° 07' 11.0" 7.2" 203 3 2 8° 59' 56.0" 6.5" 2 5 3 318° 20' 54.5" 7.0" 1 5 3 268° 49' 32.5" 9.8" 6 5 3 338° 36' 38.5" 10.7" 3 5 4 324° 17' 44.0" 8.1" 2000 102 103 162° 58' 16.0" 28.9" 102 103 1 172° 01' 43.0" 11.8" 2001 201 202 263° 54' 18.7" 9.7" 201 202 203 101° 49' 55.0" 8.1" 202 203 3 176° 49' 10.0" 8.4" 102 2000 2001 109° 10' 54.0" 25.5" 2000 2001 201 36° 04' 26.2" 7.4" 1495 Blunder Detection Techniques in Adjustment Computations Blunder Detection Techniques in Adjustment Computations SOLUTION Red.# ======================================================================= 1 3 616.23 0.495 5.356 26.05 0.746 1 2 480.95 0.243 5.921 13.29 0.689 3 1 616.23 0.495 5.356 26.05 0.746 3 4 266.81 -31.287 6.525 -1802.59 0.622* 3 6 203.77 1.789 6.889 106.83 0.579 3 5 413.76 3.317 5.059 171.47 0.773 3 2 422.77 0.069 5.459 3.67 0.736 5 2 630.97 1.394 6.012 76.87 0.679 5 1 449.39 -1.281 7.192 -79.16 0.541 5 6 247.83 1.225 7.100 74.85 0.553 5 4 407.04 9.153 7.819 614.84 0.458 5 3 413.76 3.297 5.059 170.43 0.773 102 103 327.25 -0.121 10.056 -17.16 0.103 103 1 665.70 -0.087 10.048 -12.18 0.105 201 202 453.37 0.268 10.526 92.01 0.017 202 203 709.85 0.075 10.051 10.51 0.104 203 3 537.24 0.060 10.056 8.53 0.103 2000 102 125.05 -0.188 10.138 -28.72 0.089 2001 201 425.95 0.048 10.063 6.82 0.102 1496 Volume 13 June 2007 Number 2 Volume 13 June 2007 Journal of Engineering Adjusted Angle Observations 2 1 3 316°49'53.8" 113.32" 2334.36 28.07 0.411 2 3 4 167°35'29.5" 181.54" 5764.13 22.07 0.322 2 3 6 71°43'01.5" 10.01" 5660.17 1.05 0.397 2 3 5 97°55'08.5" -868.02" 2793.81 -101.88 0.684 2 3 1 51°06'18.6" -52.42" 2462.81 -10.32 0.498 203 3 2 8°59'35.6" -20.36" 3049.99 -13.36 0.055 2 5 3 318°25'19.8" 265.26" 1871.66 45.52 0.693 1 5 3 268°58'58.7" 566.22" 3246.9 79.45 0.529 6 5 3 338°42'49.1" 370.61" 4309.5 62.85 0.304 3 5 4 322°04'20.0" -8004.04" 3222.81 -1745.41 0.321* 2000 102 103 162°23'46.9" -2069.09" 10624.86 -110.49 0.420 102 103 1 171°57'47.3" -235.73" 5605.07 -112.46 0.032 2001 201 202 263°58'29.6" 250.90" 4536.27 104.48 0.061 201 202 203 101°52'55.9" 180.90" 3608.54 58.03 0.148 202 203 3 176°50'15.3" 65.26" 3819.12 23.14 0.113 102 2000 2001 109°40'20.8" 1766.77" 9348.51 106.51 0.423 2000 2001 201 36°07'53.9" 207.69" 3440.52 104.46 0.072 Station Back Sighted Station Occupied Station Fore Sighted Angle V S Std. Res. Red. # Adjusted Angle Observations Adjustment Statistics Iterations = 4 Redundancies = 14 Reference Variance = 232,981.728 Reference So = ±482.7 Failed to pass X² test at 95.0% significance level! X² lower value = 5.63 X² upper value = 26.12 Possible blunder in observations with Std.Res. > 1,588 Convergence! Possible blunder in observations with Std.Res. > 1,588 Convergence! SOLUTION Then the problem be as following:- Then the problem be as following:- Number of Control Stations = 2 Number of Unknown Stations = 11 Number of Distance observations = 18 (It was 19) Number of Angle observations = 17 After that readjustment will be done. After that readjustment will be done. 1497

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